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India is credited with carrying out the most successful experiments in modern agriculture between 1968 and 1978 leading to a record 131 m tons of food production in 1978-79. This was coupled with a 30 per cent increase in yields per unit of farmland since 1947. The spectacular gains achieved in food production in India, and a few other countries, came to be recognized as ‘Green Revolution.’ The revolution arrived just in time when a resurgent and independent India was facing population explosion and food-grains were getting ever scarcer. A perfect blend of policy, governmental support and technological innovation proved decisive in achieving the much needed quantum jump in food production. In just thirty years, since independence, India had ensured that a repeat of 1943 famine would never ever haunt the country. The magical transformation of India’s status from a food importing country to a food exporting country was largely possible due to:

Gregor Mendel was an Augustinian monk in Brno, Czechoslovakia, who was curious to find out why ‘like begets like.’ Unable to answer this question using honey bees he turned to the humble garden peas for answers. In a series of experiments with peas of different types he discovered the ‘particulate theory of inheritance’ and postulated the ‘law of segregation’ of characters. His ‘lost’ ideas (since 1865) were only re-discovered in 1895 and the word ‘gene’ was coined much later! Little had he imagined that his ideas which came to be known as ‘Mendelian Genetics’ would one day revolutionize agriculture. Farmers and animal keepers had been developing ‘varieties’ and ‘breeds’ for centuries but the Science of Genetics forever changed the way plant and animal varieties are bred. The new high yielding varieties developed in the 1950s exploited hybrid vigour to boost crop yields all over the world. Sadly, militarism dominated the world during the first half of the 20^th century and delayed the ‘revolution’ in agriculture. In fact the food security achieved by this agrarian revolution might just have prevented a third world war, who knows?!

Green Revolution exploited the existing scientific knowledge in the developed world and effectively leveraged to develop high yielding varieties, improving irrigation and use of chemicals for plant protection that boosted crop yields in the developing world.

The technology for developing high yielding varieties that would respond to inputs and overcome lodging had been perfected elsewhere in the world leading to the now well known dwarf wheat and rice varieties. These varieties were introduced into India and crossed with indigenous cultivars to produce ‘miracle seeds’ suited to Indian conditions. This experience was quickly applied to other food-grain crops such as corn and millets with great success.

The crop area under HYV varieties increased from seven per cent to 22 per cent of the total cultivated area between 1968 and 1978 alone. In fact more than 70 per cent of the wheat area, 35 per cent of the rice area and 20 per cent of the millet and corn crop, used the HYV seeds during these years.

Technological innovation has been so successful that cereal production in the country increased almost three folds between 1965 and 1985.

The spectacular success of Green Revolution that saw a 250 per cent increase in food production at the global level in the last three decades now appears to be seriously threatened. The food and nutritional security that was almost in our grip is nearly certain to slip out of our hands. Several factors are responsible for this which were nicely summed by Dr. Norman Borlaug in his Nobel address ‘…we are dealing with two opposing forces, the scientific power of food production and the biological power of human reproduction….’ It would also be difficult to repeat the success of green revolution since the new technology will have the handicap of addressing the issues of sustainability and environmental impacts.

Nevertheless, the need for finding technological solutions to drive the second green revolution is long overdue and the following factors should precipitate action for a second green revolution:

Even with current trends of increasing productivity, the crisis with food security is expected to reach a critical phase by 2020 when the world population is expected to reach a whopping ten billion hungry mouths. The science of biotechnology built on science of genetics might help us achieve food security just in time.

Biotechnology, by its broadest definition, is perhaps as old as settled agriculture; whenever the inhabitants of Harappa and Mohen-Jo-Daro made curds or yoghurt they were perhaps using biotechnology. However, just as the science of ‘Mendelian Genetics’ revolutionized breeding of plants and animals, the developments in science and technology of molecular genetics have brought in the second transformation in agriculture, human health and industry (see box below).

The rediscovery of Mendelian laws of inheritance opened the floodgates to new ideas in biology. In the century after Mendel, the ‘gene’ was defined, genes were identified as the blueprint of life, gene structure was elucidated, its mechanisms of action discovered, they were even synthesized, ‘spliced’ and ‘amplified’, different genes were whipped up in lab pans and recombined to produce novel genetic material. The entire evolutionary processes that took millions of years could now be replicated on lab benches in a matter of a few years or even months. The physicists came to the biologist’s party in 20th Century and helped develop the science of Genetics into ‘Molecular Biology’ and its cousin ‘Biotechnology.’ If the twentieth century was the century of Physics the 21^st Century belongs to Biology. And India should join the historic changes sweeping across the field of biotechnology initiating proactive measures, just as it had done with Green Revolution.

The science of biotechnology has the potential to improve all facets of agricultural productivity from improving yields through resistance to pestilence, improved nutritional quality and better shelf-life of farm produce. For the first time in human history science has enabled moving ‘traits’, ‘proteins’ or ‘genes’ across organisms- a process that has been naturally occurring but at a very slow pace over millions of years. Molecular breeding and transgenics emerged as steering avenues to improve the traits of our interest in focal crops. These advanced tools of agricultural biotechnology have today opened up the possibility of ‘breeding by design’ to improve crops with specific quality traits in much shorter time than would be required by conventional breeding tools. It is important here to reiterate that biotechnological tools are unlikely to contribute towards increasing the yield potential of the crops but it has the potential to reduce the whopping gap that exists between the potential and the realized yields by mitigating the many stresses that limit the productivity of the crops.

In light of rapid advances that are sweeping the field of biotechnology and keeping in view the needs of the state and the country the University of Agricultural Sciences, GKVK, Bangalore proposes to establish a CENTRE FOR AGRICULTURAL BIOTECHNOLOGY at the university to harness the potential of biotechnology for achieving food, nutritional and health security for the country. The idea of strengthening the molecular biology activities at UAS, Bangalore as a focused programme is in tune with Karnataka Agriculture Policy 2006 that lays emphasis on ‘Investment in biotechnology research and extension will be stepped up, agricultural universities will endeavor to establish their brand name in the seed and technology sector.’

Increasing population has effectively blunted the benefits of Green Revolution. The yields of many crops have remained stagnated over the last decade. Much of this is a product of limitations to the biological productivity on the one hand and the opposing abiotic and biotic stresses that are curtailing the realization of the full potency in productivity of many crops. An estimated 30 % of the productivity is lost to only insect pests and diseases would add to the problem. With nearly 64 % of the cultivable land area under rainfed conditions, the perennial drought has remained a challenge to beat. All these add to the woes of food and nutritional security of the teeming millions of the State. Beating this Malthusian paradigm has been made a possibility by the powers of Biotechnology. Drought tolerance, salt tolerance, improved nutritional status, insect and disease resistance and much more have all been made achievable by the powers of biotechnological innovations. India, barring the case of cotton, is yet to harness the myriad potentials of biotechnology for its farming community. But the first moves have been made and it is only the question of sustaining the efforts to launch itself on the pedestal of Agricultural Biotechnology.

There are three cardinal reasons why an integrated centre for agricultural biotechnology is essential for boosting agricultural productivity that has been seriously compromised for the first time since the green revolution. The full potential of biotechnology is providing a thrust to improving crop productivity can be realized through:

Further, progress in this direction is critically dependent on a continuous process of enhancing our skills and knowledge in agricultural biotechnology through Innovation, the knowledge thus generated should be carried forward and developed into products through Translation and also ensure that the skewed distribution of knowledge is corrected through Human Resource Development for indigenizing and sustaining the pace of research in the field of agricultural biotechnology in the country. The proposed centre would provide the necessary infrastructure and the institutional support required for achieving these goals.

With 74 per cent the cultivable area in the State being prone to drought, year after year, the UAS team is putting its best foot forward in reducing the impact of drought on the dryland crops. Having championed the science of “drought physiology”, crops like groundnut, finger millet and tomato have been targeted for development of drought and dehydration tolerant transgenic lines.

Second major approach has been to develop insect resistant transgenic groundnut, field bean, pigeon pea, and cotton. Many of these events are in a fairly advanced stage of development and closing in on to the level of field trials. Similarly, fungal disease tolerant transgenic groundnuts are also in the pipeline. Potential of transgenic technology for salt tolerance, herbicide tolerance and improved nutritional quality are also being addressed in groundnut and finger millet.

With nearly 20 transformation events in various stages of development, the University has chalked up programmes to expand the work to mitigate more of the stresses that are limiting the crop productivity.

In fact the University has already made significant forays in to the field of agri-biotech and initial experimentation, has been highly successful. The progress achieved has enhanced the prospects of exploiting the transgenic technology in a couple of crops in the immediate future. The collaboration forged with other institutions both within and outside the country has opened new opportunities for R & D. The University therefore aims to expand the Agricultural Biotechnology work to a higher level, through improvement of infrastructural facility and by seeking funding. Initial efforts towards this end have already led to the development of many successful grant proposals. However the work envisaged needs much greater support than what can be realized through short-term grant proposals and this is expected to be realized through this proposal.

Considering the importance of biotechnology, the University of Agricultural Sciences, Bangalore, winner of the Sardar Patel Best Agricultural University award (2002) has initiated several research programmes in agri-biotech. The University which is a pioneer in introducing the country’s first Sunflower and Rice hybrid varieties has made significant progress in the area of agri-biotech too. The University has been in the forefront of biotechnological research and developed protocols and tools for molecular breeding, development of transgenic crops and functional genomics and bio-informatics. The development of transgenics for tolerance to drought and salinity in finger-millet, groundnut and insect resistant transgenics in pigeon pea, field-bean and groundnut are in a fairly advanced stage.

The scientists of the University have also developed the necessary tools for marker assisted selection of crops to develop varieties for abiotic and biotic stress tolerance in rice, groundnut, field bean and finger-millet. The University possesses the necessary expertise and the basic infrastructure for pursuing agri-biotech research at the cutting-edge to solve the problem of stagnating agricultural productivity. The University has been successful in attracting competitive grants for time-bound projects both through the Central and Stage

Governments in diverse disciplines such as horticulture, entomology, crop physiology, microbiology, biotechnology, post-harvest technology and plant pathology. However, the diverse expertise and the multiple research questions pursued by the university faculty need to be integrated to address specific problems facing the farming sector. In this context a comprehensive strategy has been formulated to integrate innovative research, product development and enhancement of human resource skills in agri-biotech in form of a Centre for Agricultural Biotechnology at UAS, Bangalore.

Many first successes in the field of Agricultural Biotechnology, especially the development of transgenics to mitigate drought, salt, insect and fungal disease tolerance has been made possible by the generous gift of gene constructs from many individuals and institutions. These successes prove the strength of collaborative efforts in taking the powers of technology to the doorsteps of the millions.

Scientists from IISc, Bangalore, IARI, New Delhi, ICRISAT, Hyderabad, ICGEB, New Delhi, NBRI, Lucknow, University of Delhi (south campus) and Central University, Hyderabad have contributed for the success of these programmes. University looks forward to expand

The Centre for Agricultural Biotechnology proposed at UAS, Bangalore is visualized as an integrated centre for innovative research that would facilitate development of new knowledge, carry forward existing tools and knowledge to develop biotech products through translational research and undertake human resource development by imparting training in state-of-art skills in agricultural biotechnology.

‘Integrate innovative and translational research to realize the potential of biotechnology to overcome stagnating agricultural productivity.’

The centre will strive to realize its vision of overcoming stagnating agricultural productivity through specific short term long term objectives. There exists a huge gap between the potential yield of crops and currently realized yields. The gap is mainly attributed to the biotic (pests and diseases) and abiotic (drought) stress on crops. The immediate thrust of the center therefore would be to harness the existing skills and tools of biotechnology to significantly close this gap in productivity and meet the target of 4 per cent growth of the agriculture sector set out by the National Development Council. The long-term objectives of the center would be to ensure continuous innovation and human resource development for sustaining developments in agricultural biotechnology.

The objectives of the centre encompass specific set of tasks that are distinct and non-overlapping but also need to critically integrate with each other to realize the overall vision. The first objective mainly concerns development of new knowledge, tools and techniques – Innovation; the second objective concerns the development of products from the new knowledge – Translation; and the third objective lays emphasis on outreach activities for both scientists and stake holders-Human Resource Development. The structure of the proposed center will thus have three sub-units viz., the Biotech Innovation Centre, the Translational Research Centre and the Human Resource Development Centre (see accompanying figure) with each unit having its specific set of goals.

The proposed structure of Centre for Agricultural Biotechnology with its three sub-cetnres or units.

The Biotech Innovation Centre (BIC) has the primary goal of networking scientists already working in the University to undertake intensive research towards exploration of novel genes and develop techniques and tools for facilitating “Breeding by Design”. The specific tasks of the Biotech Innovation Centre will be to:

These specific tasks, which cover a very wide array of research activities, would require state-of-art molecular biology laboratory infrastructure. Establishment of such an infrastructure would require huge investment. Therefore the innovation centre is proposed to be established by building on the existing basic infrastructure available within the university in different departments.

The innovation centre would integrate the research activities being carried out by the faculty in their respective labs. The innovation centre would achieve this by ‘functionally’ networking the university faculty both within the university and with other scientists outside the university. Thus the BIC would primarily be a VIRTUAL CENTRE networking and facilitating the functioning of a large number of scientists in the identified thematic areas of innovation in Agricultural Biotechnology.

Further, keeping the National Development Commission’s policy on agriculture, the centre would strive to pursue the following thematic areas of research with a view to enhance the productivity of the agriculture sector:

In essence the BIC would provide raw materials in the form of tools, techniques and information for many fields of research besides developing the technology on its own. Much of these innovations may need to be translated into utilizable products and therefore, BIC is expected to serve as the feeder for Translational Research Centre.

To answer why innovation should drive the engine of progress in the agriculture sector, we should perhaps recall how the success of the first green revolution was achieved. The success of green revolution during the early 1970’s was achieved primarily by the successful co-operation between scientists cutting across national boundaries. The scientific knowledge, skills, techniques and resources in the form of ‘dwarf gene’ carrying races were freely exchanged to develop hybrids or the so called ‘miracle seeds.’ The progress and development achieved since has led to ‘globalization’ and IPR regimes and biodiversity laws. Ironically ‘globalization’ does not itself to collaboration and easy exchange of knowledge and material anymore!

The agricultural research in India today cannot ignore basic research with the hope that we can continue to borrow scientific knowledge and resources as we did in the pre-globalization era. If Indian agriculture has to usher in the second green ‘revolution’ it will have to come from the fire-power of indigenous efforts in all spheres of basic, strategic and applied research. Japan which is synonymous with technological excellence has 2.85 times more scientists than men in armed forces, in comparison the number of scientists in India is only 13 per cent of the total strength of the armed forces – a clear indication of where the firepower lies in achieving scientific excellence. Therefore the key to success in improving the productivity of Indian agriculture would depend on how effectively we adopt the INNOVATION MANTRA.

7.2 Translational Research Centre It has been increasingly realized that the slow progress for generating commercially viable transgenics as stipulated by biosafety guidelines, is the lack of interface from the development of primary transfromants to the commercially viable transgenic product. The university and many other institutions have the necessary intellectual capacity to create such transgenic or recombinant products but often lack the resources, skills, and infrastructure to advance their research results to the next level of development. In fact one of the biggest challenges in the development of the biotech products is the ability to convert the putative products into full fledged practically and commercially viable products. Limited success in the conversion of putative products into viable ones is largely due to the high infrastructural needs to meet the requirements of genotyping and phenotyping of the products. This proposal aims to fill this gap by establishing a Translational Research Centre’ (TRC) to facilitate the translation of technologies for the development of commercially viable transgenics/ products. It will form the interface between innovation and commercially viable product development and establish the necessary infrastructure for meeting the demand for biotech products.

Further, despite good infrastructure many of the proposed functions can not be met independently. Therefore, the TRC will also be responsible for forging collaborative and contractual research programmes considering the private sector also. Public-private partnership is expected to enhance the rate of progress in development of viable Biotech products. Lastly, the TRC will be responsible for transfer of technology developed.

Establishment of a TRC at the University is justified by the fact that the University has already made significant strides in the field of biotechnology and several products are under evaluation and advancement. At present, the University has to its credit, drought, salt, insect and fungal disease tolerant crop strains under advancement. Nearly 20 transgenic products (see Appendix in later sections) in various crops need to be advanced through several generations and the establishment of TRC would augur well even for harnessing short term benefits to the farming community.

While the BIC and TRC will cater to the research needs for improving the productivity in the near future, manpower development is equally important for sustaining the benefits of biotechnology in the long run. Thus the Centre for Agricultural Biotechnology at UAS, Bangalore will specifically address the HRD needs in the area of agri-biotech to meet the crucial requirement of continuing education and outreach for the public and private sector and also the stake holder viz. the farmers and other interest groups. The primary aim of the proposed Biotechnology Education and Training Centre (BETC) would be to impart short and long term training to develop human resources in the field of Agricultural Biotechnology for the country.

The University of Agricultural Sciences, currently offers short term courses on various aspects of techniques and tools in biotechnology through its’ Department of Biotechnology. Further a four year undergraduate programme leading to B.Sc. (Agri. Biotech) has also been started from the current year. In the years to come, sustaining these efforts would require a strong infrastructural facility and good funding. Further, the intended graduate level course in Crop Physiology and Molecular biology along with the post graduate course in biosafety require additional support. It is hoped that the establishment of an exclusive educational centre would meet the human resource requirements for all academic activities agri-biotech.

In addition to the above mentioned activities, a number of research programmes that are already in operation and those that would start in the near future would provide immense opportunity for post- graduate students to have a first hand experience in Agri-biotech research skills and tools. Thus the centre would greatly facilitate the development of high quality human resource for Agri-biotech needs of the country.

Unprecedented opportunities available for the modification of crop plants by biotechnological means have greatly enhanced the chances of mitigating serious hurdles in improving crop productivity. Two important approaches viz., molecular breeding and transgenics together provide a wide repertoire of tools to counter challenges of crop productivity. It is hoped that, in the long run, biotechnological interventions would help alleviate problems of stagnating agricultural productivity, rural poverty and nutritional security.

Biotic stresses such as pests and diseases are known to substantially reduce yields to the tune of nearly 30 to 35% in most corps leading to as much as an estimated annual loss of Rs. 29,000 crores (summary in the accompanying table). As a result, many crops such as cotton, cabbage, tomato, pigeon pea receive very heavy doses of insecticides. However, in many dry land crops, the uncertainty associated with rains make the application of insecticides a very unviable strategy and as a result, most farmers fail to apply the required measures of pest management. Nevertheless, the Government agencies have been making enormous efforts to promote pest management measures to manage these crop pests

and disease. But yet the losses continue to occur. Seed borne solutions should put an end to these problems and some solutions for many crops are in the realm of possibility.

An estimated 112 lakh ha of area covering nearly 74 per cent under semi-arid tracts. The entire area is prone to repeated drought besides facing innumerable pest and disease problems. The drought related losses can be as immense as 60 per cent of the total yields in almost every crop grown in this area. However by developing transgenics and improving desired traits for Biotic and Abiotic stress tolerance together can substantially mitigate the drought and enhance the productivity of important dry land crops. We anticipate a benefit of close to 20 per cent for every farmer who adopts this technology in drought prone conditions.

Area, production and expected loss due to insect pest damage in different crops in Karnataka

Biotechnological tools help develop such seed borne solutions for many of the crop productivity problems. Keeping this potential in view, the University of Agricultural Sciences, Bangalore has launched several modest initiatives to harness the benefits of both molecular breeding and transgenics to tackle specific constraints in the dry land crops. In addition, the work on functional genomics envisages developing many potential genes for use in mitigating the problems of drought.

Significant strides already made in these areas targeting the dry land crops are expected to provide viable solutions to manage several problems. Products in pipe line would help reduce:

Thus the major thrusts imparted in targeting the dry land crops would help realize tremendous economic benefits even at as low as 20 per cent adoption rates. Leaving aside the huge losses caused by drought, benefits of insect tolerant target crops alone would help realize a reduction in losses to the tune of Rs. 1500 crores per annum. Other incorporated traits such as abiotic stress would further enhance these benefits. In addition, the proposed work programme expects to touch upon nutritional security needs, besides expanding the work on insect pest, diseases is expected to provide large scale tangible economic benefits in the first place. Further, the reduction in cultivation operations, health benefits, reduction in the reliance on vagrant monsoon add many intangible short and long term benefits.

The country has only recently begun to reap the benefits of long term investments in public education since independence. Thus the role of the proposed centre for agricultural biotechnology in the development of human resources is expected to meet the manpower needs of the public and private Agri-biotech sector in the near future and help develop long term technological capabilities. Establishment of Centre for Agricultural Biotechnology at the University of Agricultural Sciences, Bangalore as proposed above is expected to realize the dream and nutritionally and economically secure nation by facilitating the 4 per cent growth of agri-sector as envisaged by the National Development Council.

Being aware of the potentials of biotechnology, the University, with all the constraints of infrastructure and funding, has initiated several modest programmes to exploit the technology for the benefit of the dry land farming community of the state.

These programmes have greatly helped improve the technical capabilities of the researchers involved, besides understanding the limitations to progress. Significant successes with these initiatives has provided a platform and the confidence for the team at UASB to harness the tools of biotechnology and served as an impetus to set larger goals.

The scientists of the University have been successful in obtaining competitive grants from national and international funding received over the years provided the required support. The experience gained and the successes met have created a need for seeking larger funding to take forward the successful events to their logical end. DBT and DST have supported the faculty research through several short-term projects in the area of biotechnology. Similarly the DST has recognized the strength of the University faculty by extending the largest number of infrastructure grants for teaching and research under the FIST programme for any agricultural university.

Early work was also significantly progressed due to contributions from many scientists from across the country and out side. As a result inter-institutional collaboration in these areas of research has also grown over the years. Collaborative research is expected to only strengthen further with the launch of new programmes of research aimed at tackling the problems of agricultural productivity.

The expertise of faculty in diverse disciplines, collaboration with national and international institution and the facilities available for field trials across the state are the major strengths of the University in agricultural biotechnology that justify the strengthening of the research programmes and infrastructure in the form of a centre for agricultural biotechnology.

Ever since the production of revolutionary genetically modified oil eating bug by Dr. Anand Chakrabarti in the early 1980’s biotechnological innovations have swept the field of medicine and agriculture in many countries. Particularly in agriculture, transgenic technology has provided strong seed borne solutions to manage the problems of both abiotic and biotic stresses experienced by many crops. Cold, insect, disease and herbicide tolerant transgenic crops have swamped the fields of USA, Argentina, China, Canada in particular. Even in India, nearly 5.6 m ha of the cotton area is under transgenic varieties. However in molecular marker assisted selection protocols, the breeding has made progress in developing rice blast and bacterial blight resistant transgenic varieties. Similarly quantitative trait loci associated with deep root morphology has helped develop stable and high performing Aerobic rice that have already occupied substantial area in the country. Thus the molecular breeding is contributing for the development of local technology for mitigating many stress tolerant varieties of crops, while only the success of the transgenic cotton serves as a model for transgenic technology. However, there is no matching innovation to come by in other crops. This is largely due to the fact that India was a late starter in aiming to exploit the benefits of the novel technology and the transgenic cotton owes its existence in the country to a borrowed technology.

While starting late is not a sin in itself, the approach to develop the basic tools to harness the power of the technology has remained at its infancy. Trait specific gene identification, cloning and functional genomics in molecular breeding approaches call for greater attention and investment in terms of funds, man power and time. Lack of strong thrust in the development of these basic skills and tools of biotechnology has resulted in the poor development of application oriented products in the country. In the recent past, however, there is a realization to meet this gap in knowledge and investment. These new trends would augur well for the “breeding by design” programmes in developing varieties with novel traits.

Any “breeding by design” programme requires a strong back up biotechnological tools and techniques. At the University of Agricultural Sciences, Bangalore a number of programmes have been launched with a view to breed the crops with design. Substantial successes with the programmes attempted has only led to the idea of more attempts to be made in the near future. As a result, it has become imperative that efforts be made to sustain the progress with a strong back up efforts in the development of novel tools and techniques relevant to the programmes envisaged. The first steps towards this end have begun and a number of parallel programmes are currently running in the University. In order to consolidate these isolated efforts and to expand on these activities, it is necessary that the scientists working on various aspects of gene search and functional genomics, molecular breeding and transgenic technology be brought together under a single umbrella. Biotech Innovation Centre has been proposed with a view to meet this requirement.

Biotech Innovation Centre (BIC) has the primary goal of networking the scientists to undertake intensive research towards exploration of novel genes and develop techniques and tools for adoption to facilitate and undertake “Breeding by Design”.

A number of studies are addressing the issues relevant to the proposed objectives of the centre. The researchers involved in these activities are being pursued scattered across various Departments of the University. Some of these works are being carried out in collaboration with scientists from many other institutions. There is a need now to consolidate the efforts being made by these various units and to facilitate their functioning in an effective way. The proposed BIC would thus primarily be a VIRTUAL CENTRE aiming at networking and facilitating the functioning of a large number of scientists in the identified thematic areas of Agricultural Biotechnology. The centre therefore has been designed to incorporate these ideas such that the functions and the thematic areas are suitably merged to develop a strong programme in “Breeding by Design”, such that the innovations made will be transmitted to Translational Research Centre. The figure below provides the gist of the idea of centre.

Figure 1: A pictorial depiction of the structure of the proposed Agri-biotech Innovation Centre as a virtual centre.

Further, keeping the National Development Commission’s policy, the centre would strive to pursue the following thematic areas of research on a mission mode with a view to enhance the growth rate in Agriculture Sector.

As much as 74 % of the cropped area in the State is under rainfed conditions. Consequently, the agricultural productivity is subject to vagaries of monsoon. Incessant and periodical drought has an over riding impact on the agricultural out put. Thus drought tolerant crop varieties suitable to these large areas would be a boon for the farming community of the state. As a result the major thrust of the present programme is to alleviate the impact of drought in rainfed crops especially groundnut, sunflower, redgram, chickpea, field bean and finger millet. These crops have also been shown to be good sources of a large number of transcriptomes suitable for drought management. Therefore a two pronged approach of using both molecular breeding through QTL mapping and marker assisted selection and then to use known and documented effective drought tolerance genes in the development of suitable transgenics should help substantially improve the yield situations in years of drought in these selected crops.

While drought generally takes the major toll of yields in these rain fed crops, in years of plenty, it is the biotic stresses that take an upper hand. Both insect and diseases especially the fungal and viral diseases have remained a perennial threat for the production of many of these crops. An estimated 30 to 35 % loss is exclusively attributed to these stresses. Therfore efforts at mitigating them would go a long way in the stabilization of yields in rainfed crops. Therefore, biotic stress tolerance would be the next most targeted set of programmes of the Biotech Innovation Centre. Both molecular breeding through MAS would be the priority approach for the management of some of the important fungal and viral disease in selected pulse crops. Such would be the approach in these crops wherever strong sources of resistance have already been identified. Pioneering and strong pulse breeding programme, with large many accessions to screen for disease resistance supported by a multidisciplinary team of scientists tomeet the many challenges of the work will provide a good platform for taking up molecular breeding for biotic stress tolerance. Thus the University is extremely well equipped for meeting the basic requirements for taking up this line of work. A number of important diseases such as Fusarium wilt in redgram and chickpea, sterility mosaic virus n redgram, yellow mosaic of horse gram and green gram and rust in cowpea are the primary targets at the centre. Work has already been initiated on these lines and the QTL mapping for selected traits is in progress.

An alternative approach would be to use the transgenic technology for mitigating the insect and disease resistance problems. A number of important problems that can be potentially managed with the transgenic technology have been identified in the important oil seeds and pulses of the state. Requisite genes for the purpose are also available with the investigators. Experimental approaches of working with as many as 8 crops and nearly 15 gene-problem-crop- variety combinations have yielded successful events that are being advanced. More however, needs t be done and a number of challenges remain to be attempted. These will be the targets that are planned to be addressed at the proposed Biotech Innovation Centre.

Breeding by design for quality traits of crop plants such as Fe, Zn, oil and vitamin A and to improve the post harvest shelf life

A strong base in crop physiology, human nutrition and horticulture have led the scientists of the University in understanding and appreciating the various limitations of the food crops in terms of the quality traits such as nutritional levels and shelf life of many vegetables and also the food crops. Thus the scientists have identified some of these important multifaceted problems of crop plants. It is proposed that through primarily transgenic approach, and to some extent MAS breeding these problems would be approached at the BIC.

Microbial biotechnology to prospect the rich microbial biota of the State for green energy and novel trait specific genes

A number of successful programmes at the University in harnessing the power of the potent microbes have helped the University to develop a strong base in microbial biotechnology. Nitrogen fixing bacteria, PSBs, Bacillus thuringiensis, fermentation processes, EPN associated symbiotic bacteria and VA mycorrhizae are a few of these programmes have been amoang the on going programmes at various departments of the University covering the Departments of Agricultural Microbiology, Biotechnology, Plant Pathology and Entomology. However much of this work needs to be strengthened through molecular approaches to improve the traits of the desired microbes, develop the products, or even to source them for trait specific viable quality genes. Thus these would be the targeted works in the proposed BIC, so that the on going works would be greatly strengthened to aim at product oriented research.

Exploiting the potential of Bio-farming in the production of useful pharmaceuticals

An important ongoing programme at the University is the biofarming. Considerable progress has been made in exploiting the ability of plants to produce human and other vaccines. Pproudction of manyother pharmaceuticals such as hepatitis B vaccine, anti rabies vaccine and insulin in the plants has been initiated with variable successes. Strong infrastructural facility and development of a multidisciplinary tems to handle such difficult problems would help make beteer progress in this direction. The BIC it is hoped that would provide the right set up for building on the reasonably good foundation laid in the field of biofarming especially of the pharmaceuticals

Many perennial plant species of economic importance among the forestry and horticultural plants are not easily amenable for development of varieties. The University has made efforts to identify a large number of such plant species and the required variants need to be mass produced. A robust and high throughput method for mass multiplication would be required for this purpose. Development of micropropagation/ tissue culture technology would be a handy tool for mass production of these genetically valuable plant material. Secondly, many plants that are primarily propagated by vegetative means provide somaclonal variants of high quality in tissue culture systems. In order to create and multiply such somaclonal variants of better quality traits this technology is of great importance. Thus tissue culture research needs to be strengthened at the University and the BIC would have this as one of the responsibilities.

Project 1: Development of gene constructs to improve drought tolerance of dry land crops.

Sub-project 1: Cloning and characterization of genes for improving drought traits from drought adapted crop plant- Groundnut.

Sub-project 2: Cloning stress specific promoters from stress adapted species.

Sub-project 3: Gene prospecting and functional genomics in finger millet for abiotic stress tolerance.

Project 2: Identification of QTL for relevant drought tolerance traits and their introgression to improve productivity under water limited conditions

Sub- project 1: Association of genetic diversity with phenotype to identify QTLs conditioning drought tolerance traits in groundnut.

Sub- project 2: Improved drought tolerance and productivity in sunflower through molecular breeding.

Sub- project 3: Introgression of relevant drought tolerant traits through molecular

Sub-project-4: Identification of molecular markers linked to seed yield and drought resistance in sesame (Sesamum indicum L.)

Sub-project 5: Identifying molecular markers associated with root morphology in Pigeon pea and Sunflower under drought and well watered condition.

Project 3: To improve drought tolerance by developing transgenics expressing validated stress genes.

Sub-project 1: To improve drought tolerance in groundnut by developing transgenics expressing validated transcription factors.

Project 4: To improve specific traits for enhancing productivity of aerobic rice cultures through molecular breeding and transgenic approaches

Sub- project 1: Identification of QTLs conditioning drought tolerance traits using trait specific Doubled Haploid mapping populations and evolution of trait introgressed lines by marker assisted selection.

Sub-project 2: Over expression of C₄ genes in rice to improve productivity under water limited conditions: Carbon isotope ratios as an accurate selection criterion of putative transformants.

Sub-project 3: DNA marker assisted identification of weed competitive and high

Project 1:Targeting genes for ion homeostasis and salt tolerance in Rice and other crops in coastal regions: Over expression of genes for compartmentation and proton gradient could significantly enhance salt tolerance

Project 2:Enhancing phosphorus uptake in Pigeonpea (Cajanus cajan(L.) Millsp) under P deficient conditions: A transgenic approach involving genes encoding citrate synthase.

Project 1: Development of SSR markers for Fusarium wilt and sterility mosaic

Project 2: Use of molecular markers and marker assisted breeding for the development

Project 3: Development of SSR markers and marker assisted selection for mungbean

Project 4: Development and utilization of DNA based molecular markers for biotic and

Project 6 : Marker assisted back cross breeding for Fusarium wilt (Fusarium species)

Project 7: Development of multiple disease resistance to tomato leaf curl virus, bacterial

Project 8: DNA marker assisted introgressions of sheath rot, bacterial leaf blight and

Project 9: Development of multiple disease resistance in TyLCV bacterial wilt and nematodes in tomato by using MAS

Project 1: Development of pod borer resistant transgenic BRG-2 and BRG-4 varieties of

Project 4: Development of defoliators and capsule borer resistant castor lines

Project 5: Development of sunflower necrosis disease resistant transgenic lines

Breeding by design for quality traits of crop plants such as Fe, Zn, oil and vitamin A and to improve the post harvest shelf life.

Project 1: Biofortification of Zn-dense pigeon pea ( Cajanus cajan) and finger millet (Eelusine coracona)-Molecular breeding and Transgenic approach.

Project 2: Development of Quantitative Trait Loci (Qtl) and Marker Assisted Selection (Mas) to improve the Zinc and Iron content in grains of Rice (Oryza Sativa)

Project 1: Isolation of novel antifungal gene and expression of the gene to increase the post harvest shelf life of Tomato.

Project 2: Nutritional Improvement and Enhancement of Shelf life of Tomato, Onion and Potato by Lactic Acid Bacteria

Exploiting the potential of bio-farming in the production of useful pharmaceuticals

Project 1: Cloning and expression of Challengevirus standard strain (CVS) Rabies glycoprotein gene in Pichia pastoris and plants and immunization of mice with the recombinant rabies glycoprotein

Project 2: Plant expression of Hepatitis-B antigen and targeting molecules for immunization in mice

Project 3: Transformation of crop plants with a gene encoding GAD 65, a major auto antigen in Insulin Dependent Diabetes mellitus via Agrobacterium

Microbial Biotechnology to prospect the rich microbial biota of the State of green energy and novel trait specific genes

Project 2: Microbial stains for higher production of alcohol using crop residues.

Project 3: Functional heterogeneity and genetic diversity of Saccharomyces cerevisiae, isolated from the naturally fermenting fruits of Western-Ghats of Karnataka

B. Microbial systems as source for genes for nutrient acquisition and insecticidal toxin proteins:

Project 1: Microbial systems as source for genes for nutrient acquisition and insecticidal toxin proteins:

Project 2: Application of AM fungi of different agroclimatic zones of Karnataka on plant growth and molecular characterization of their phosphate transporter genes

Project 3: Cloning and characterization of phosphate solubilizing gene/s to improve phosphorus uptake of crops through transgenic approach.

Project 4: Isolation and biocontrol application of novel toxin complex genes from Photorhabdus luminescens

Project 5: Exploration of novel Bt genes for development of transgenic plants

Project 1: Belowground biodiversity in sustainable agricultural systems

Project 2: Isolation of Beneficial Microflora from Weed Rhizosphere to improve

nutrient acquisition in crop plants.

Project 1: Production of plants of high value crops such as Banana cvs Nanjangud

Project 2: Crop improvement via in vitro mutagenesis. Banana cv. Nanjangud Rasabale

Project 3: Conservation of endangered Forest species more particularly of medicinal and

aromatic species which are listed in the Red Book, particularly species relevant to

A significant reduction in yield leading to financial losses is encountered annually due to several abiotic stresses experienced by crop plants. An estimate of yield reduction and loss in revenue is given in the flow chart below. The challenge of feeding 10 billion by 2020 can only be met by developing suitable technologies for increasing the productivity of arid and semi-arid farming systems. The key to enhancing productivity under dry land agriculture lies in simultaneously mitigating the abiotic and biotic stress through trait improvement of cultivars as a seed based technology. This can be achieved through application of biotechnological tools rendering crop cultivars resistant to major abiotic stresses.

It is apparent from these figures that drought stress is the most overriding constraint in achieving potential productivity of crop plants. The percent drought prone area in the state of Karnataka is the highest (78%) as compared to 64% in Rajasthan and Gujarat. Globally, the area hit by severe

drought has increased by nearly 12% in 1970 to as high as 30% by early 2000 (NCAR J. of Hydrometeorology, Dec. 2004). Since an over whelming 70% of cropped area comes under water limited rain-fed conditions, improving the ability of plants to tolerate drought stress is crucial for agricultural production. From this context mitigating drought by crop improvement as well as saving irrigation water by producing more with less water is the global agenda which is most pertinent to the Indian scenario.

Oilseeds and pulses constitute nearly 30% of the food basket. But unfortunately, 90% of the area under these crops come under water limited rain-fed conditions. The following table illustrates the loss in productivity and revenue because of the various abiotic stresses experienced by these crops under dry land conditions.

In this scenario, it has to be emphasized that rice is a major consumer of water. It is estimated that for every kg of rice grain, the plant requires upto 5000 L of water. With rice being the major staple cereal of over 70% of populations, concerted efforts need to e made in two directions (a) improve the water use efficiency of rice crop and (b) to evolve aerobic rice cultivation practices with suitable cultivars to save irrigation water, without any loss in productivity.

Predicted increase in productivity through improvement in water use efficiency

With an average annual rainfall of about 800 mm and about 45% of this water available for transpiration, it is estimated that a 0.1 unit (g.kg^-1) increase in WUE can enhance productivity by 0.324 t.ha^-1.

Thus, it is imperative to have a concerted and intensive program to improve Water use efficiency of rain-fed crops both through modern biotechnological approaches as well as conventional breeding.

Two other stresses that also deserve technological intervention are Salinity and nutritional stresses. For example, in Karnataka, 4.04 lakh ha is salt affected out of which 59% is outside canal command and 21% is coastal. Similarly, Phosphorus, zinc and iron deficiency are common in most places resulting in major loss in productivity.

Global research efforts as well as our own findings have provided leads in identifying drought tolerance traits for target environments. Evidences have convincingly suggested that besides cellular level drought tolerance the plant traits associated with water relation (like water mining, water use efficiency and water conservation) have paramount significance. Although the underlying molecular mechanisms of traits associated with water relations are poorly understood, it is well known that these traits are multi-gene controlled and are quantitatively inherited. Therefore identifying genotypes having superior alleles for these traits is the only option and hence it becomes essential to identify “donor” genotypes for these traits. The success in identifying such trait donor genotypes entirely depends on our ability for accurately phenotyping these traits in a high throughput manner. Such approaches would also form the basis for identifying QTLs that can be subsequently used for trait introgression onto an elite genetic background. The application of modern biological and biotechnological tools are expected to provide the requisite solutions to achieve the goal of developing cultivars with improved productivity under water limited conditions. This warrants the development of a comprehensive road map to initially identify relevant drought tolerance traits and genomic sources for these traits (genes or genotypes) and finally strategize approaches to introgress these traits, into a single elite genetic back ground.

The fullest advantage of these quantitative traits is exploited only when these traits are brought under a single genetic background with reasonably superior intrinsic tolerance at cellular level. Therefore, it is equally important to clone and characterize genes that regulate stress tolerance at cellular level. Recent advances in Molecular and genomic analysis has facilitated the discovery of novel genes and gene promoters that drive specific pathways related to drought tolerance in plants. It has been opined that gene sources from known stress adapted species will be more efficient and hence drought adapted species such as Finger millet and groundnut would form the potential platform for prospecting stress responsive genes. This necessitates the profiling of stress transcriptome of such species and their functional characterization to identify genes for translational genomics.

Development of superior crop plants with novel drought specific promoters and genes warrants the discovery of efficient transformation and high throughput stress screening protocols. Standardized protocols are available for transforming recalcitrant dry land crop species such as pigeon pea, groundnut, cotton etc. The in-planta transformation approach provides a suitable strategy to deploy relevant gene/gene constructs in developing drought tolerant transgenic plants.

Therefore the aim of this program is to identify genotypes with specific drought traits and genes and promoters that regulate drought tolerance mechanisms and adapt molecular breeding and transgenic tools to introgress these traits into a single genetic background.

Program centers around FOUR major interrelated areas for an orchestrated attempt to mitigate drought.

ii. Saved water is available to alleviate drought at critical stages of important dryland crops like pigeon pea, Groundnut, Ragi and Sunflower.

1. Development of gene constructs to improve drought tolerance of dry land crops

2. Identification of QTL for relevant drought tolerance traits and their introgression to improve productivity under water limited conditions

3. To improve drought tolerance by developing transgenics expressing validated stress genes.

4. To improve specific traits for enhancing productivity of aerobic rice cultures through molecular breeding and transgenic approaches.

Crop	Approach	Gene/traits
Groundnut	Molecular breeding Transgenics	WUE and root traits NAC, DREB, Helicase,
Pigeon pea	Molecular breeding Transgenics	Root traits, WUE P Acquisition (Nutrient stress)
Finger millet	Functional genomics	Stress transcriptome.
Rice	Molecular breeding Transgenics	WUE and Root traits C₄ gene expression
Sunflower	Molecular breeding	WUE and Root traits
Sesame	Molecular breeding	Drought tolerance

The expertise of the faculty at UAS Bangalore is reflected in the concerted programs developed in different directions by various teams.

1. Stable isotope discrimination (D¹³C and D¹⁸O) to quantify water use efficiency (WUE), traits associated with it and genetic analysis of these traits using molecular markers - A National facility for WUE studies: DST/DBT, Rs. 2.2 Crores

2. Center of excellence (CoE) program to develop drought tolerant crop varieties by biotechnological approaches DBT; 4.5 Crores

3. Integrated Center for Drought Research and Management-Genetic Engineering for Developing Crop Plants Resistant to Abiotic Stresses (Niche area of excellence): ICAR, Rs. 2.79 crores

4. Marker assisted participatory plant breeding for drought resistance in rice: Rockefeller foundation. Rs. 2 Crores

5. Breeding for drought tolrenaxe and molecular mapping in Finger Millet: Mc. Knight foundation. Rs. 4 Crores

Scientists have published large number of papers in high impact journals like

PNAS, Euphytica, Planta, JXB, Ann of Applied Biology, Crop Science, Ann of Botany, TAG, Biologia Plantarum,

Faculty have adequate expertise for high tech molecular biology and biotechnology research work. Several scientists have also visited leading overseas laboratories to get trained in cutting edge research lines in contemporary plant biology. Specialized training facilities for other scientists has been established at the University with the assistance of Kirkhouse Trust, UK.

Development of gene constructs to improve drought tolerance of dry land crops.

Drought stress tolerance is a complex phenomenon ranging from cellular functions to whole plant physiology to morphological traits. Thus, a concerted approach of introducing novel stress tolerance genes under the control of stress specific promoters into an elite agronomic background would render a crop cultivar tolerant and perform better under drought stress. Therefore, over expressing novel stress responsive genes in crop genotypes with superior inherent traits such as better root characteristics, high water use efficiency, water conservation through improved epicuticular wax load etc., would be the most desirable approach t improve producitivyt under rainfed conditions.

Cloning and characterization of genes for improving drought traits from drought adapted crop plant- Groundnut.

Rationale: It has been proved that drought related genes from stress adapted species are unique and have novel regulatory mechanisms that are required for tolerance. Groudnut (Arachis hypogea) is one of the major crops in drought prone areas and has efficient stress tolerance mechanisms. Information is available on the relevance some of the stress genes as evidenced in model systems, which can form a basis to clone the homologs/orthologs from groundnut. The stress genes cloned can be effectively used to improve drought tolerance of susceptible crops such as sunflower thorough transgenic approaches.

Rationale: Discovery and validation of stress responsive genes associated with cellular tolerance from stress- adapted species is one of the major focuses in recent years. Stress gene promoters play a crucial role in regulation of gene expression. There are several experimental evidences to show that highly stress responsive genes are driven by efficient stress inducible promoters as seen in adapted crops. Highly stress inducible, native promoters can only bring about required tolerance under field condition. It is equally important to clone stress inducible promoters from the same species in which we are interested for improving stress tolerance, and such promoters can act as efficient native promoters to drive specific stress responsive genes under stressful environments.

Gene prospecting and functional genomics in finger millet for abiotic stress tolerance.

Rationale: Developing genomic resource from highly drought adapted crops like finger millet and exploiting the resources (genes and gain-in-function mutants) is the only sound scientific strategy to combine desirable characters for improving crop productivity in a sustainable manner. Since finger millet has a close synteny with rice, a stable food in the world, it can serve as a source of relevant genes to improve abiotic stress tolerance of rice. It may be possible to improve the productivity of rice that grows aerobically and under upland conditions using the genomic information generated from Finger millet by translational Genomics. Therefore comprehensive approach to develop genomic resources in finger millet will unravel the basic mechanisms of abiotic stress adaptation, which is the need of the hour.

II. Identification of QTL for relevant drought tolerance traits and their introgression to improve productivity under water limited conditions

Rationale: The physiological traits that have relevance for improving drought tolerance such as WUE and water mining traits associated with roots are multi-gene regulated and are quantitatively inherited. Since introgressing more than one trait seems to be essential for achieving an overall improvement in drought tolerance and crop productivity, a marker assisted molecular breeding strategy appears like the only potential option. However, the success of this strategy is almost entirely dependant on the accuracy of phenotyping for the traits of interest. The stable isotope ratios of carbon and oxygen have emerged as powerful options for an accurate and high throughput phenotyping for WUE, transpiration rate and root traits among large number of germplasm as well as mapping populations.

DNA markers flanking QTL regions are conventionally identified by genotyping and phenotyping a mapping population obtained by crossing genotypes that are contrasting for the trait of interest. More recently, a Linkage Disequilibrium (LD) based association mapping has emerged as a potential alternative to identifying marker trait association.

Association of genetic diversity with phenotype to identify QTLs conditioning drought tolerance traits in groundnut.

Generation of a large number of data points representing the whole genome and genotyping of a diverse reference collection may offer several folds polymorphic data points that can be applied for undertaking LD-based association studies. The availability of the diverse reference collection of groundnut and the DArT facility being established at ICRISAT, as a part of the Centre of Excellence (CoE) and several hundred SSRs (~1000) developed and assembled at ICRISAT and UAS form the basis of assessing genetic polymorphism to undertake association studies.

Improved drought tolerance and productivity in sunflower through molecular breeding

Introgression of drought tolerance traits such as Root traits, high Water use efficiency, intrinsic tolerance at cellular level and moisture conservation associated with water conservation strategies onto an elite genetic background will substantially improve tolerance to water deficit conditions. The robust phenotyping strategy based on stable isotope signatures forms the basis for identification of QTLs conditioning traits relevant to drought tolerance.

· To evaluate sunflower inbreds for variability in WUE, root traits and intrinsic tolerance and identification of contrasts to develop mapping populations.

Introgression of relevant drought tolerance traits through molecular breeding to develop superior pigeon pea genotypes

Pigeonpea is an important pulse crop, predominantly grown under raifed conditions. Under drought situations, though it can sustain its growth, productivity is drastically reduced . Hence, there is a need to identify /develop genotypes with lesser reduction in yield under moisture stress conditions. Therefore, this project envisages to achieve this objective by introgressing relevant traits associated with water acquisition, WUE and desired response to VPD by employing molecular breeding strategies

Identification of molecular markers linked to seed yield and drought resistance in sesame (Sesamum indicum L.)

Principal Investigator (PI) : Dr.E.GANGAPPA, Dept. of Genetics & Plant Breeding

Sesame is predominantly grown under rainfed conditions. Frequent and prolonged dry spells are a major limiting factor for lower productivity. Root traits, among others contribute significantly to drought resistance and our earlier studies on sesame have indicated significant genetic differences among the selected sesame genotypes for root length measured at different intervals. Further root length had a positive association with seed yield; suggesting the utility of roots traits, especially root length as a surrogate trait for genetic enhancement of sesame for drought tolerance. Being difficult for routine phenotyping for root traits in large germplasm collections and segregating populations, molecular markers linked to root traits would be handy for accelerated sesame genetic improvement through marker-assisted selection.

3. Introgression of root traits QTLs into elite agronomic background through Molecular marker-assisted backcross breeding

4. Evaluation of superior introgressed lines for seed yield under field conditions

Identifying molecular markers associated with root morphology in Pigeon pea, and Sunflower under drought and well watered condition.

Roots play an important role in determining if the crop is successful in combating the stresses that are common in the farmers’ fields. Extensive studies on root morphology of rice, finger millet, sorghum and maize has indicted shown immense quantity of genetic variation for root morphology and whole plant morphology. The data on root morphology has already helped in development of drought tolerant lines. These deep rooted drought tolerant lines have shown the ability to save water up to 50 % in rice cultivation.

III. To improve drought tolerance by developing transgenics expressing validated stress genes.

The development of transgenic crops for improving the stress tolerance has made tremendous progress since the first transgenic tobacco resistant to Tobacco Horn worm was developed I the late 80’s. Since then, a large umber of transgenic crops with tolerance to various biotic and abiotic stresses including herbicides have been developed in a varieties of crops ranging from cereals, pulses, vegetables, fruit crops and cotton with over 100 million ha of transgenic crops world wide. A recent estimate put the number of transgenics developed to impart tolerance to abiotic stresses like salinity, drought, cold etc., around 250. However, many of these transgenics are developed in model plant system and field evaluation of crop plants is lacking. But recent developments in functional characterization of stress transcriptome are likely to provide useful leads.

The team at UASB has achieved a major breakthrough by developing transgenics through in-planta transformation technique. This forms the basis for realization of transgenics in difficult to transform recalcitrant species such as pigeon pea, castor and groundnut. The team will capitalize on this breakthrough for developing transgenics tolerant to abiotic stresses in selected crop species

· Identify candidate genes by validating up-stream regulatoery genes for acquired stress tolerance and genes coding for inherent traits associated with water uptake and conservation.

· By analyzing the stress subtracted c-DNA from stress adapted species, Arabidopsis/rice full length homologs will be accessed and their relevance validated in model systems.

To improve drought tolerance in groundnut by developing transgenics expressing validated transcription factors.

Rationale: Groundnut is the major oilseed crop in India and extensive grown under rain-fed conditions. Drought stress at any stage reduces growth growth and productivity. Development of transgenics crops for drought resistance by over xpressing transcription factors like NAC, DREB, Helicase etc will improve the drought resistance capacity of the crop.

IV. To improve specific traits for enhancing productivity of aerobic rice cultures through molecular breeding and transgenic approaches

Rice, under the irrigated systems consumes tremendous amounts of water. With the receding water availability, it has become essential to save irrigation water. Cultivation of rice under aerobic systems is expected to result in saving substantial amounts of water. However, it is imperative that rice cultivars are evolved that are suitable for cultivation under aerobic conditions without any substantial reduction in productivity.

Irrigated paddy requires intensive land preparation and standing water rendering this crop a phenomenally high water user per unit productivity. Aerobic paddy could now be grown akin to arable crops like wheat, maize, sorghum or any millet and substantially save irrigation water. A suitable agronomy can be developed for aerobic cultivation provided appropriate cultivars are evolved.

Molecular markers associated with quantitative trait loci with roots, osmotic adjustment and grain yield have been used to facilitate this task. We have also designed a set of candidate-gene markers for screening drought resistance, grain yield, root characters and silicon content. All the above mentioned set of markers has been judiciously employed in a marker-assisted selection program. Eight new rice genotypes (ARB 1 – 8) identified through a MAS program are currently undergoing extensive multi-location field trials all over the state of Karnataka and some sites in the country since kharif 2005. The yield levels are significantly higher under low-moisture stress and comparable to irrigated rices under well watered conditions. The results of two years of trials across six locations under three hydrological conditions in each location are presented in table1. The ARB lines have outyielded the checks (national and International) by two to three times). The third year trials are being conducted during Kharif 2007.

Identification of QTLs conditioning drought tolerance traits using trait specific Doubled Haploid mapping populations and evolution of trait introgressed lines by marker assisted selection.

The major constraint in identifying QTLs for relevant traits is the time taken for developing trait specific mapping populations. Double haploidization of the anthers derived plants tremendously hastens the development of “immortal” mapping populations. This approach can also be adopted to introgress desirable trait QTLs in to a cultivated genetic background.

Over expression of C₄ genes in rice to improve productivity under water limited conditions: Carbon isotope ratios as an accurate selection criterion of putative transformants.

The C₄ species through the CO₂ concentrating mechanism have naturally evolved a superior ability for drought tolerance as well as growth rates and productivity. Thus expressing the C₄ genes in a C₃ plant like Rice would substantially increase drought tolerance and hence increase productivity with limited water resources. This strategy is expected to save substantial amounts of irrigation water.

1. Construction of a gene expressing cassette of a few relevant C₄ genes and tranformation of rice.

DNA marker Assisted Identification of Weed competitive and High growth vigoured and short duration genotypes of aerobic rice for water scarce areas of Karnataka

Principle Investigator: Dr Shailaja Hittalmani, MAS Lab, Department of genetics and Plant Breeding, UAS, GKVK.

Co-Principle Investigator: Dr H V Nanjappa, Weed Agronomist, Department of Agronomy, UAS GKVK

With all the advantages of aerobic rice cultivation of direct seeding, cost savings, water savings, low methane production, low pest and disease incidence, and retaining of good soil structure as against puddled cultivation, Aerobic rice stands to have a major problem with weed control. Controlling weeds during the crop growth period remains a major problem in aerobic rice cultivation. Many-a-times chemical weed control is necessary to prevent yield loss, causing soil pollution and environmental pollution apart from excess cost to farmers. The plants suffer due to lack of initial vigour and slow growth and in vegetative stage, thus affecting tillering capability. Hence, breeding genotypes that have initial high seedling vigour and weed competiveness helps to overcome this problem as well as saves the cost of the weedicides and maintain good crop stand in the field. Detection of DNA markers for high seedling and plant vigour and fast growing genotypes with early duration-maturity types helps to overcome the problem. The University of Agriculture Sciences, Bangalore in its pioneering effort has developed aerobic rice varieties suitable for various soil types and ecosystems of Karnataka.

Germplasm screening and Backcross breeding for developing introgressions lines: time saving and rapid process to develop stable genotypes

Marker Assisted screening and phenotype confirmation based on performance infield.

1.DNA markers associated with plant vigour, weed competiveness and early duration linked to high yield.

3. Water saving Aerobic varieties with farmers’ friendly cultivation and high yield.

Targeting genes for ion homeostasis and salt tolerance in Rice and other crops in coastal regions: Over expression of genes for compartmentation and proton gradient could significantly enhance salt tolerance”

Over 800 million hectares of land area is salt affected (6% of world’s land area). In Karnataka the area under salinity is 4.04 lakh ha out of which 1/5^th (21%) is coastal, 59% is outside canals and 20% is canal command. Restoring ion Homeostasis in Plants disturbed by Salt stress is crucial to avoid Cellular Damage and Nutrient deficiency

1) To develop transgenics for salt tolerance by over expressing functional and regulatory genes involved in ionic homeostasis.

2) To test the transgenics with single and pyramided genes separately to determine the degree of enhanced tolerance at field /greenhouse level.

Approaches and Deliverables

Enhancing phosphorus uptake in Pigeonpea (Cajanus cajan(L.) Millsp) under P deficient conditions: A transgenic approach involving genes encoding citrate synthase.

Pigeon pea (Cajanus cajan(L.) Millsp) is one of the important pulse crops of India. Most of soils are acidic in nature and availability of phosphorus (P) in these soils is lower than in the other soils. Organic acids are known to improve availability P to the plants. So over expressing genes encoding enzymes involved in organic acid biosynthesis in P starved plants increases exudation and solubilization of bound P in the soil.

Objectives

4. To assess the performance of transgenic lines by measuring CS activity and Quantification of citrate through HPLC

Approaches and deliverables

1. Aarati Karaba, Shital Dixit, Raffaella Greco, Asaph Aharoni, Kurniawan R Trijatmiko, Nayelli Marsch-Martinez, Arjun Krishnan, Karaba N. Nataraja, Makarla Udayakumar& Andy Pereira 2007, Improvement of water use efficiency in rice by expression of HARDY an Arabidopsis drought and salt tolerance gene , PNAS (USA), (in Press).

2. Nadaradjan, S., Impa. S. M., Shashidhar G. Parsi, Sheshshayee, M. S., Prasad, T. G., and Udayakumar, M., 2007. Molecular mapping of QTLs associated with whole plant Water Use Efficiency (WUE). Paper communicated to Rice Genetics News Letter. Vol 34:

3. S.M. Impa, S. Nadaradjan, M.S. Sheshshayee, T.G. Prasad, M. Udayakumar and Shailaja Hittalmani¹ (2007) Identification of markers for Mean transpiration rate and oxygen isotope enrichment (δ¹⁸O) in recombinant inbred lines of Rice. Rice Genetics News Letter. Vol 34:

4. L. Krishnamurthy¹, V. Vadez^1*, M. Jyotsna Devi¹, R. Serraj², S.N. Nigam¹, M.S. Sheshshayee³, S. Chandra¹, A. Rupakula¹ (2007).Variation in transpiration efficiency and its related traits in a groundnut (Arachis hypogaea L.) mapping population. J. Agron Crop Sci, (In Press)

5. R. Hema, M. Senthil Kumar, S. Shiva Kumar, P. Chandrasekhara Reddy, M. Udaya Kumar 2007 Chlamydomonas rainhardtic, a mudel system for functional validation of abiotic stress responsive genes. Planta (in Press).

6. Bindhu Madhava , T.G. Prasad, M.K. Joshi and N. Sharma, 2007, Assessing the availability in intrinsic water use, mesophyll and carbonization efficiencies of Gibberdun treated tea (camellia Dinensid L) accession using gas exchange approach Current Science 93(3): 291-292.

7. Philippe Monneveux, Madavalam S. Sheshshayee, Javed Akhter, Jean-Marcel Ribaut Using carbon isotope discrimination to select maize (Zea mays L.) inbred lines and hybrids for drought tolerance. Plant Science, 2007. (In Press).

8. Sheshshayee M.S^*1, Bindumadhava H¹,Ramesh R², Prasad T.G¹ & Udayakumar M. Relationship between oxygen isotope enrichment (D¹⁸O) in leaf water (or biomass) and stomatal conductance. J. Isotopes in Environment and Health, 2007 (In Press).

9. Prabuddha, H. R., Manjunatha, K., Venuprasad, R., Vinod, M. S., Jureifa. J. H. and Shashidhar. H. E. 2007. Identification of Isogenic Lines and Near-Isogenic Lines: An innovative approach, validated for root and shoot morphological characters in a mapping population of rice (Oryza sativa L.). Euphytica, Ref. No: EUPH1103 in print

10. Senthil kumar, M., Udayakumar, M. 2006. High throughput Virus induced gene silencing approach to assess the functional relevance of a moisture stress induced cDNA homologous to lea4. J. Expt. Bot. 57:2291-2302.

11. Senthil kumar, M., Govind, G., Kang, L Mysore, K.S, Udayakumar, M. 2006. Functional characterization of Nicotiana benthamiana homologous of pea nut water deficit-induced genes by virus-induced gene silencing (VIGS). Planta. DOI (0-007/500425-006-0367-0).

12. Senthil kumar, M., Kumar G., Srikanthababu, V., Udayakumar, M. 2006. Assessment of variability in acquired thermotolerance: A potential option to study genotypic response and the relevance of stress genes. J. Plant Physiology.

13. S. C. Misra, R. Randive, V.S. Rao, M. S. Sheshshayee, R. Serraj, P. Monneveux. 2006. Relationship between Carbon Isotope Discrimination, Ash Content and Grain Yield in Wheat in the Peninsular Zone of India. J. Agron. Crop Sci.

14. M. S. Sheshshayee, H. Bindumadhava, Nageswara Rao, Rachaputi^,T.G. Prasad, M. Udayakumar, G. C. Wright and S. N. Nigam. 2006. Leaf chlorophyll concentration relates to Transpiration Efficiency in Peanut. Ann. Appl. Biol. 148: 7-10.

15. Nethra P, Nataraja KN, Rama N and Udaya Kumar M, 2006, Standardization of environmental conditions for induction and retention of posttranscriptional gene silencing using tobacco rattle virus vector, Current Science, 90(3): 431-435.

19. Kumar. S., Ramesh. R., Sardesai, S and Sheshshayee, M.S. 2005. Effect of incubation time and substrate concentration on N-uptake by phytoplanktons in the Bay of Bengal. Biogeosciences. 2: 1331-1352.

20. Sheshashayee M. S., Bindumadhava H., Ramesh, R., Prasad, T. G., Udayakumar, M, & Lakshminarayana M. R. 2005, Oxygen isotope enrichment as a time averaged measure of Transpiration .J. Exp Botany. 56: 3033-3039.

21. S. M. Impa, S. Nadaradjan, P. Boominathan, G. Shashidhar, H. Bindumadhava, and M.S. Sheshshayee. 2005. Carbon isotope discrimination accurately reflects variability in WUE measured at a whole plant level in rice (Oryza sativa L.). Crop Science. 45: 2517-2522.

22. Sathik M, Nataraja KN Molly-Thomas and James J., 2005, An efficient method for the isolation of good quality total RNA from bark tissues of mature Hevea brasiliensis trees. J. Rubber Research 8(3): 182-189

23. Kumar. S., Ramesh. R., Bhosle. N.B., Sardesai, S and Sheshshayee, M. S. 2004. Natural isotopic composition of nitrogen in suspended particulate matter in the Bay of Bengal. Biogeosciences. 1: 63-70

24. Kumar. S., Ramesh, R., Sardesai, S and Sheshshayee, M. S. 2004. High new production in the Bay of Bengal: Possible causes and implications. Geophysical Resaerch Letters. 31: L18304.

25. Jagadeesh G Nataraja KN and Udaya Kumar M 2004, Shockwaves can enhance bacterial transformation with plasmid DNA. Curr. Sci., 87:734-735.

26. Mohan Rao. G., Lakshmikanth Reddy, Kulkarni R.S, Lalitha Reddy. S.S and Ramsh.S. 2004. Stability analysis of sunflower hybrids through non-parametric model. Helia, 27:59-66

27. Annadana S., Schipper B., Beekwilder J., Outchkourov N., Udayakumar M. and Jougsma M. A., 2003. Cloning, functional expression in Pichia pastens and purification of potato cystatin and multistatin. J. Biosci. Bioengineering. 95(2):118-123.

28. Senthil Kumar M., V. Srikanthbabu, B. Mohan Raju, Ganeshkumar, N. Shivaprakash and M. Udayakumar. 2003. Screening of inbred lines to develop Thermotolerant sunflower hybrid through temperature induction response (TIR) technique: A novel approach by exploiting residual variability. J. Exp. Bot. 54: No. 392. 2569-2578.

29. Debabrata Ray, M.S.Sheshshayee, Kakoli Mukhapadhya, H. Bindumadhava, T.G. Prasad, and M. Udaya Kumar. (2003). High nitrogen use efficiency in rice genotypes is associated with higher net photosynthetic rate at lower Rubisco content. Biologia Plantarum, 46(2): 251-256

30. M.S. Sheshshayee., H. Bindu Madhava, , A.G. Shankar, T.G. Prasad and M. Udaya Kumar. 2003. Breeding strategies to enhance Water Use Efficiency for crop improvement. J. Plant Biology, 30(2): 253-268.

34. Srikanth Babu, V. Ganeshkumar, Krishnaprasad B. T., Gopalakrishna, R. Savitha, M. Udayakumar, M. 2002. Identification of pea genotypes with enhanced thermotolerance using temperature induction response technique (TIR). J Plant Physiol, 159/5: 535-545.

35. Annadana, S., Beekwilder M. J., Kujipers, G., Outchkourov, N., Pereira, A., Udayakumar, M., Dejong, J. and Jongsma, M. A., 2002. Cloning of the Chrysanthemum EUPI promoter and comparative expression on florets and leaves of Dendrotherma grandiflora. Transgenic Research. 11:437-445.

36. Seetharam Annadana, WimRademaker, Ludmila Mlynarova, Jande Jong, Makarla Udayakumar and Jan Peter Nap. 2002. The potato Lhca3.St.1 promoter confers high and stable transgenic expression in chrysanthemum in contrast to CaMV-based promoters. Molecular Breeding. 8: 335-344.

37. Aarati Pradyumna, Krishnaprasad, B. T., Ganeshkumar, Savitha, M., Gopalakrishna, R., Ramamohan, G. and Udaya Kumar, M. 2002, Expression of an ABA responsive 21 KDa proteins in fingermillet (Eleusina coracana) under stress and its relevance in stress tolerance. Plant Science. 164: 25-34.

38. Seetharam Annadana, Kujipers, G., Visser, P.B., deKogel, W.J., Outchkourov, N., Beekwilder, M.J., Schipper, B., Udayakumar, M. and Jongsma, M.A. 2002. Effect of potato multicystatin expressed in florets of chrysanthemum on western flower thrips Frakiliniella occidentalis. Acta Horticulturae. 572: 121-129.

39. Holbrook, N. M., Sashidhar, V.R., James, R.A, and Munns, R. 2002. Stomatal Control in tomato with ABA-deficient roots: Response of grafted plants to soil drying. J. Exp. Botany (UK). 53: 1503-1514

41. Hittalmani, S., H. E. Shashidhar, P. G. Bagali, N. Huang, J. S. Sidhu, V. P Singh, & G. S. Khush, Molecular mapping of quantitative traits loci for plant growth, yield and yield related traits across three diverse locations in a double haploid rice population Euphytica, (2002), 125 (2): 207 – 214

42. Viswanath, K.P., Bhushana,H.O., Aftab Hussain,I.S., Ashok., and Girish, G. (2002). Genetic analysis of transpiration efficiency and other physiological traits in cowpea (Vigna unguiculata L. Walp.). Journal of plant Biology 29, 13 – 22

Team Leaders: Drs. Shailaja Hittalmani, Dr. H.E. Shashidhar and ByreGowda

Department of Genetics and Plant Breeding, College of Agriculture, UAS, Bangalore

Molecular markers which have proved to be very valuable for genetic studies and crop improvement endeavor in major cereals like rice, has a potential to make an immense impact in other crops too. They can be a valuable tool to address complex problems associated with decrease in productivity of Pulses, Oilseeds and Millets where there are chronic problems associated with biotic and abiotic stresses. Being cultivated in less-than-optimal agronomic conditions and with lesser monitory and non-monitory inputs, they manifest great losses (upto 100% in yield) when challenged by stresses.

Molecular tools will help discern the genetic mechanism associated with disease and pest resistance in the host. These tools will also help quantify the genetic variability among the pathogenic races or biotypes. The influence of the environment on the development and progression of the stress will also be possible when the studies are conducted over years and seasons. These data on molecular profiling will enable selection of the right parents prior to initiating the breeding program, identifying the most effective allele to incorporate and pyramid and also enable development and deployment of long lasting field resistance management mechanisms.

The project involves application of MAB tools and techniques to supplement efforts of Plant Breeders to improve pulses and oilseed crops for stress resistance and thus impact productivity. Distant hybridization and introgression of desirable alleles from this exotic gene pool will be a possibility. Markers associated with traits of interest, near-isogenic lines that are developed for each important trait will auger well for the crops being improved.

Where mapping populations are available, it would be ideal to map quantitative trait loci associated with the important traits. The information would help in discerning the actual genetic loci, the number of

1. Development of crop specific and broad spectrum molecular markers for pulses and oilseeds and standardization of reaction conditions

2. Development of molecular profiles of the active gene pool of pulses and oilseeds being used by breeders and mine alleles associated with traits using association analysis

Molecular marker investigations related to all these projects will be done in a central facility to maximize cross linking of tools and techniques and prevent duplications in investment and maintenance. The phenotyping facilities will also be shared to improve deliverables.

Development of SSR markers for Fusarium wilt and sterility mosaic disease resistance and pyramiding of genes for multiple resistance in pigeonpea

Principal Investigator: Dr, M, Byre Gowda, Principal Scientist (Plant Breeding), AICRP on Pigeonpea

Pigeonpea is the major pulse crop in India grown under diverse agro-ecological and cropping systems. India accounts for about 90 per cent of world production and acreage, producing about 2.21 million tonnes from an area of 3.38 million hectares with the productivity of 653 kg/ha. In Karnataka, it occupies an area of 5.8 lakh hectares with annual production of 2.6 lakh tonnes with an average productivity of 476 kg/ha (Anonymous, 2004).

The two most devastating diseases of pigeonpea are wilt and sterility mosaic disease (SMD).. These diseases are being severe in southern parts of the country. The yield loss depends on the stage at which the diseases occur, it can approach 100% when wilt and SMD occurs at the flowering stage, about 70 per cent when it occurs at podding stage. The annual pigeonpea crop loss from these diseases in India alone has been estimated at 205,000 tones of grains (Kannaiyan et al. 1984).

Conventional breeding methods are being deployed to address the problems in these crops at a slow pace. The experience of breeders reveal that, conventional breeding as a means of genetic improvement for yield increase is often limited by the narrow genetic base, long breeding cycle and out crossing problems in pigeonpea. Wilt and SMD are the most serious diseases because of their wide spread and destructive nature. The breeding and selecting process for developing a new high yielding variety with resistance to these diseases is an arduous process that may take up to 8-10 years. Using suitable DNA markers for selection will help to identify the right genotype that are resistant. Development of markers to identify wilt and SMD resistance in pigeonpea and deploying them through marker-aided selection in breeding program would fasten the process of developing resistant lines. Despite the great advances in genomic technology observed in several crop species, crop specific markers are limited in these crops and till date only 10 sequence tagged microsatellite Site (STMS) primers are available in pigeonpea, (Burns et al., 2001). Few efforts were made in the past to map the resistant genes for wilt disease of pigeonpea (Bonn, 2006 Kotresh et al. 2006,). Bonn (2006) tested 220 soybean primers in pigeonpea. Nine of the markers developed were polymorphic in Fusarium wilt recombinant Inbred Lines (RIL) mapping population.. Recent reports indicated that 30 pigeonpea specific SSR primers were developed at ICRISAT (personal communication).

5. Development of trait introgression lines resistant by crossing wilt and SMD resistant donor parents with agronomically elite cultivars.

1. Development of SSRs specific to pigeonpea , construction of genetic map for wilt and SMD resistance

2. Superior genotypes with resistance to wilt and SMD will be developed through the integration of conventional breeding and MAS approach.

Byre Gowda, M., Mahadevu, P., Venkatesha, S.C., Ramanjini Gowda., Mohan Rao., Ellis, N., and Knox, M.R., 2005. Molecular markers for genetic diversity and the selection of genetic mapping lines for Lablab purpureus In: 4^th International Food Legumes Research conference, October, 18-22, 2005, held at New Delhi,

Venkatesha, S.C., Byre Gowda, M., Mahadevu, P., Mohan Rao, A., Kim, D.J. Ellis, N., and Knox, M.R., Transferability of PCR- based markers from a range of legume species for genetic studies of Lablab purpureus (in press)

Bagali P. G., Shailaja Hittalmani, Srinivasachari R., Shedaksheri Y. G. and Shashidhar H. E. 1998, Identification of DNA markers linked to partial resistance for blast disease in rice across four locations. Developments in Plant pathology; Advances in Rice blast Disease, Edited by D. Tarreau, M. H. Lebrun, N. J. Talbot and J. L. Notteghem. Pp 34 – 42 Proceedings of the 2^nd Int’l rice Blast Conference, 4-8 August 1998, Montpellier, France, Kluwer Academic publishers.

Prashanth G. Bagali, Shailaja Hittalmani and Shashidhar, H.E., 1998. Evaluation of indica x japonica doubled haploid population of Rice (Oryza sativa L.) for field resistance to leaf blast disease. Oryza 35: 34-39

Shailaja Hittalmani, Srinivasachary, Prashanth G. Bagali and H.E. Shashidhar, 1999. Genetic Markers associated with Leaf and Neck Blast Resistance in rice across locations. Euphytica 2000.

Girishkumar K., Srinivasachary, Shashidhar H.E. and Shailaja Hittalmani 2004. Mapping quantitative trait loci for resistance to leaf blast disease in rice. Ind. J plant Protection 32: (1) 84-87

Girishkumar K., Srinivasachary, Shashidhar H.E. and Shailaja Hittalmani 2004. Mapping quantitative trait loci for resistance to leaf blast disease in rice. Ind. J plant Protection 32: (1) 84-8

Use of Molecular Markers and Marker Assisted breeding for the development of yellow mosaic virus resistant Horsegram varieties

Principal Investigator: Dr. K.P. Viswanatha, Professor (Plant Breeding), AICRP on Arid Legumes, UAS,GKVK,Bangalore-65.

Horsegram (Macrotyloma uniflorrum (Lam.) Verdc) (Syn. Dolichos biflorus L.) commonly known as “Kulthi” is a traditional tropical grain legume, well known for its hardiness and adaptability to poor soil, adverse climatic conditions that are unsuitable for most other crops. Horsegram is extensively grown in peninsular India and cultivated up to 5000 feet elevation of Himachal Pradesh and Nepal. It is also grown in on hilly slopes of North eastern regions, hilly districts of Uttaranchal, tribal hilly regions of Orissa and Madhya Pradesh. Karnataka has the largest area under Horsegram compared to other states. In Karnataka Horsegram stands second with respect to acreage among all pulses occupying 3.14 lakh hectares of the total cropped area with a production of 1.75 lakh tones, (Anonymous, 2003). It is considered as “Poor Man’s Pulse”, cultivated and consumed by poor and marginal farmers. It is a cheap source of protein, vitamins, calcium and iron. Horsegram is a rich source of Urease. Seeds are well known for its medicinal (Diuretic) properties. Since ancient times it is being used for patients suffering from urinary and kidney problems, known to have anti-calcifying properties which dissolves kidney stones in human beings. It is being grown on very poor and marginal land with least care and almost minimum input except seed. Farmers are broadcasting only traditional or local varieties without any inter cultivations. As a result the productivity and the production of this important food legume are very poor compared to its potential yield.

Like any other pulses, this crop also suffers most due to biotic and abiotic stresses. Among biotic stresses, diseases play a major role in Horsegram. Common diseases seen on Horsegram are Yellow Mosaic Virus, Powdery Mildew, Dry Root rot, Dieback, Root rot, Rust, Anthracnose and Leaf spot. Yellow Mosaic on Horsegram has been reported to be prevalent in Southern India (Williams et al., 1968; Muniyappa et al., 1975) resulting enormous losses with respect to yield and quality.

There are only two improved varieties viz., BGM-1(KBH-1) and PHG-9 released and recommended for cultivation in the state. All traditional varieties and released varieties are susceptible for YMV and as a result, farmers are not able to get even the minimum yield in this crop. In Bangalore centre of All India Coordinated Research Project on Arid legumes, we have screened 700 genotypes obtained from NBPGR, New Delhi and Trissur along with local collections against YMV disease.

2. Hybridization and development of F₂ mapping population for identification of the molecular markers linked to Yellow Mosaic Virus.

3. Transfer of YMV resistant genes to agronomically superior but susceptible varieties through MAS breeding.

4. Development of RILs from F₂generation, forwarding F5 to F6 generation through Single Seed Descent (SSD) method.

1.Identification of resistance resources for powdery mildew and YMV through filed and glass house screening

2. Identification of markers linked to HYMV and linkage analysis and development of pure lines

1. 700 germplasm lines of Horsegram were evaluated at field in three seasons in field condition using spreader row technique and in glass house for one season. Five resistant lines were identified as resistant to YMV.

2. Hybridization programme was initiated with agronomically superior genotypes which are susceptible to Yellow Mosaic Virus disease (PHG-9, BGM – 1 and Tcr-04) as female parents were crossed with yellow mosaic virus resistant genotypes (DPI 2278, AK- 21, AK-26, AK-38 and Tcr-512) in a separate crossing block during December, 2006.

4. Sowing of these F₁s along with their parents was take up during summer 2007 and F₁generation was evaluated for all the characters including disease resistance.

K.T. Rangaswamy., V.S. Seshadri., M. Byregowda., J. Chandraprakash and K.P.Viswanatha., 1997., Chemical Control of Powdery Mildew in Horsegram, Curr. Res. 26 : 228 –229.

Viswanatha, K.P., Balaraju, M. K. Jayashree and P. Arunachalam, 1999,Genetic Analysis of Some Physiological Traits in Cowpea (Vigna unguiculata (L.) Walp.), Mysore J. Agri. Sci.,33: 234-236.

Shankar, M. A., M. C. Devaiah, K. N. Ravi and K. P.Viswanatha, 1999, Relative performance of different mulberry varieties when intercropped with pulses and seeds under rainfed condition, In: The proceeding of the XVIII^thInternational Sericultural commission congress, held at Cairo-Egypt during October 12-16, 1999: 23-26.

Shankaralingappa, B.C., B. Shivaraj and K.P.Viswanatha, 2000,Interaction effect of phosphorus and sulphur on uptake of nitrogen, phosphorus, potassium and sulphur by cowpea, Karnataka J. Agric. Sci., 13(2): 295-298.

Bhushan, H.O., K.P. Viswanatha, P. Arunachalam and G. K. Halesh, 2000, Heterosis in cowpea(Vigna unguiculata (L.) Walp.) for seed yield and its attributes, Crop Research,19(2):277-280.

Ramanagoudar, H., K.P. Viswanatha, S. Subramanya and C.K. Babu, 2000, Efficiency of non chemical seed treatments against pulse beetle (Callobruches chinensis L.) in horse gram (Macrotyloma uniflorum lam. Verdic.,) during storage. Bulletin of grain technology, 33 (1): 54-58.

Bhushana, H.O., K.P. Viswanatha, H.C. Lohithaswa and K. Dhanaraj, 1998, Combining ability for water use efficiency and related components in cowpea (Vigna unguiculata (L.) Walp.), Karnataka J. Agric. Sci., 14(1):23-26.

Jayashree, P. Hiremath, Sunanda Sharan and K.P. Viswanatha, 2001, Chemical composition and functional properties of some important genotypes of Horsegram Macrotyloma uniflorum Lam Verdc., Karnataka J. Agric. Sci., 14:943

Viswanatha K.P., M.K. Jayashree and P.Arunachalam, 2001, Heterosis and inbreeding depression in physiological traits in cowpea(Vigna uniguiculata (L.) Walp.), Forage Res.,27 (2):pp.83-86.

Viswanatha K.P., H.O. Bhushana, I.S. Aftab Hussain, Ashok and G. Girish, 2002, Genetic analysis for transpiration efficiency and other physiological traits in cowpea (Vigna unguiculata (L.) Walp.), J.Plant Biol., 29(1): 13-22.

Rudresh, N.S., K.P.Viswanatha and Shailaja Hittalamaini, 2006 “Molecular diversity of Cowpea [Vigna unguiculata (L.) Walp.] using RAPD”, J. Arid Legumes 3(2): 52-56.

Development of SSR markers and marker assisted Selection for Mungbean yellow mosaic virus and Powdery mildew in green gram and blackgram

Principal Investigator: Dr. D. L. Savitramma, Professor, Department of Genetics and Plant Breeding, College of Agriculture, GKVK, Bangalore

Co PI : Dr. H. E. Shashidhar, Professor, and Head, Department of Genetics and Plant Breeding, College of Agriculture, GKVK, Bangalore

Mungbean (Vigna radiata L.Wilczek) is an important food grain legume in India. It ranks fifth among over ten different food legumes in India. It contains 22% - 28% total protein, mostly water-soluble and easily digestible. The global annual production of mungbean is estimated at 2.9 x 10⁶ tons from 5.7 x 10⁶ ha with an average yield of 0.5 t / ha. India accounts for about 60% of the world's mungbean area; harvests 47% of the world’s production. India is the largest grower in terms of area but harvests only 0.4 t/ha. In Karnataka mungbean is grown in an area of 5.24 lakh ha with a production of 0.82 lakh tones with a productivity of 257 kgs per ha.

Black gram is one of the important pulse crop, grown throughout the country. It contains vegetable protein and supplements to cereal based diet. It contains about 26 per cent protein. It is also used as nutritive fodder. In Karnataka it is grown in an area of 1,32,511 ha with 1,62,28 tonnes with a productivity of 129 kgs per ha.

From among the serious diseases mungbean yellow mosaic virus, powdery mildew and Cercospora leaf spot are the critical yield limiting factors. The mungbean yellow mosaic virus (MYMV) and powdery mildew are the serious and most devastating biotic stresses in Indian subcontinent and also in Karnataka causing yield losses up to 70 per cent. The requirement of protein in Indian diet has to be met through pulses especially for the vegetarians like mungbean which has low flautelence and also like blackgram.

Mungbean Yellow Mosaic Virus is the most destructive disease and can cause yield losses even to the tune of 100%. Use of resistant genotypes is the most effective alternative to mitigate this yield loss. Development of resistant varieties is the most appropriate approach to control the disease. Backcrossing is the main breeding method used to transfer resistance to the diseases. Progress in breeding is hampered because direct selection after inoculation is difficult to assay on individual plants if infection is less. In such circumstances, indirect selection using molecular markers linked to resistant genes should be an effective approach with lab screening and the associated markers for the trait helps in tracking right genotypes precisely and the associated markers for the trait helps in

tracking the genes responsible for resistance in F_1’s, F₂’s, backcross populations and in germplasm lines..

Microsatellites have attracted scientific attention because they have been shown to be part of or linked to some genes of agronomic interest. The traditional and most simple procedure of microsatellite isolation involves the cloning of small genomic DNA fragments and the screening of clones through by colony hybridisation with repeat containing probes. To increase the chances of success, the use of enriched libraries was proposed based on selective hybridization (Karagyozov et al. 1993; Billotte et al. 19996) have been the most successful.

Marker-assisted selection shortens the time needed to fix resistance to yellow mosaic virus and powdery mildew in segregating populations. Markers tightly linked to genes of interest increase the efficiency and accuracy of selection. Present project proposal is aimed at developing molecular markers (SSR) and to construct a genetic linkage map as a basic requirement towards initiating marker assisted selection technique in mungbean and black gram for MYMV and powdery mildew resistance. These SSR markers will also help future generation of breeders to address the various problems faced by mungbean crop.

Conventional breeding methods have been employed in the past to solve some of these problems at a slow pace. The use of molecular marker technology can help accelerating mungbean improvement process through the marker-assisted selection technique. From among the markers, Microsatellites offer several advantages, they are highly reproducible, highly polymorphic, PCR-based and readily portable within a species. Conventional breeding methods are being deployed to address the problems in these crops at a slow pace. The breeding and selecting process for developing a new high yielding variety with resistance to these diseases is an arduous process that may take up to 8-10 years. Multi environment testing will always be required to confirm that the identified phenotype indeed have desired agronomic characters combined with resistance. However, in current practice a significant amount of effort is devoted to testing selections that simply do not have the genetic potential. Hence, tools for determining the genotypes of the experimental lines increases the efficiency of the selection process.

Using suitable DNA markers for selection will help to identify the right genotype that are resistant. Development of markers to identify YMV resistance in blackgram and mungbean and deploying them through marker-aided selection in breeding program would fasten the process of developing resistant lines. Despite the great advances in genomic technology observed in several crop species, crop specific markers are limited in these crops and till date in greengram 93 SSR markers were developed by Gwag et al.,(2007) but only 7 were found to be polymorphic.

The present project proposal is aimed at developing molecular markers and to construct a genetic linkage map as a basic requirement towards initiating marker assisted selection to develop resistant varieties in blackgram and mungbean. The closely linked markers identified will be used in enhancing the breeding process and precision selection for identifying resistant genotypes and trait introgression. The SSR markers developed will be specific to the crops and the diseases and will also help future generation of breeders to address the various problems faced by these crops.

In this regard, present investigation is aimed at development of SSR markers and characterization of mungbean and blackgram cultivars for Yellow mosaic virus and powdery mildew resistance through marker assisted selection.

1. Screening for MYMV and Powdery mildew resistance and Molecular profiling of wild species, mutant types and cultivated varieties in green gram and blackgram.

2. Development of SSR markers in Mungbean and Black gram for MYMV and Powdery Mildew resistance

3. Generation of mapping population and identify molecular markers tightly linked to MYMV and powdery mildew resistance by adopting Bulked segregant analysis.

4. Adopt fast track generation advancement strategies to develop a breeding population for traits of interest and validation of newly developed SSR Markers. And Mapping QTL’s adopting Single Marker Analysis for yield and resistance to MYMV.

Methodology

1. Screening for MYMV and Powdery mildew resistance and Molecular profiling of wild species, mutant types and cultivated varieties in green gram and blackgram.

a. Field Screening : Greengram and black gram germplasm of around five hundred which include wild species and cultivated varieties will be screened for resistance to Mungbean Yellow Mosaic Virus (MYMV) and powdery mildew under natural field condition during early kharif and summer seasons by sowing spreader rows which are highly susceptible to MYMV (Chinamung and a highly susceptible local variety in greengram) in randomized complete block design and morphological characterization will be done along with scoring for MYMV and powdery mildew.

b. Glass house screening for MYMV :Glass house screening will be done for field resistant genotypes in glass house for mungbean and blackgram seedlings 7 to 10 day old will be inoculated individually with 10-15 viruliferous whiteflies. Genotypes which are free from yellow mosaic symptoms will be tested by using polymerase chain reaction to detect MYMV if any. Based on the differences for MYMV resistance from both cultivated and wild species genotypes will be selected for hybridization work.

c. Molecular Profiling : SSR markers developed in crops like cowpea, blackgram, pigeonpea, blackgram and mungbean (heterologous probes) will be used to study polymorphism among both resistant and susceptible genotypes to increase the impact. The genetic distance of these selected genotypes will be determined by assessing DNA polymorphism using already existing microsatellite markers. A large number of primers (two to three hundred or more) will be screened for the determination of DNA polymorphism. Genetic distance will be assessed comparing the Microsatellite profiles using the Gel Compar software.

d. Hybridization :Most contrasting genotypes for MYMV resistance and also for powdery mildew will be identified and used in hybridization programme. The crosses will include Resistant X Susceptible (used for mapping population) wild species X susceptible cultivated varieties, and Pusa baisaki mutant (Resistant in greengram) X Cultivated susceptible varieties. Their reciprocal crosses will also be done to identify the genes of interest in cytoplasm apart from the crosses already made. The F₁ plants will be evaluated for combining ability and heterosis and advanced to generate useful breeding material. The resistant X susceptible crosses in greengram include Chinamung x BL-849, PDM-84178 x OBGG 11, Tap-7 x KM-1883, PS-16 x KM 1883, Pusa baisaki x OBGG 11, PDM 84-178 x PMB 43, Tap-7 x KM 1883. PBM x BL-849, Pusa baisaki x Vigna umbellata for MYMV resistance.

2. Development of SSR markers in Mungbean and Black gram for MYMV and Powdery

The traditional and most simple procedure of microsatellite isolation involves the cloning of small genomic DNA fragments and the screening of clones through by colony hybridisation with repeat containing probes. To increase the chances of success, the use of enriched libraries based on selective hybridization (Karagyozov et al. 1993; Billotte et al. 1999; Edwards et al. 1996) will be used.

The genomic DNA will be cut into fragments using restriction enzyme and these fragments will then be inserted into suitable cloning vector and then bacteria. The size of the DNA fragments will be monitored by separating a 10 μl aliquot of the restricted DNA on a agarose gel. The DNA fragments of approximately 500 base pairs will be selected as these will be small enough to sequence easily, yet still retain a high probability of having enough DNA flanking the Microsatellites that primers can be designed.

b. Ligating linkers to DNA fragments, Dynabead enrichment for Microsatellite containinig DNA fragments, PCR recovery of enriched DNA, Ligating enriched DNA into Plasmid, Transforming plasmid DNA , PCR and storing positive colonies:

Positive and selected clones from the enriched library will be purified for DNA sequencing. Sequences containing at least 6 di-nucleotide repeats and 4 tri-nucleotide (or larger) repeats or larger will be selected as a microsatellite. Primers will be designed for each SSR locus using Primer 3 web interface program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).

All sequences from the enriched library will be clustered together and low complexity DNA and simple sequence repeats will be masked with Repeat Masker software (http://www.repeatmasker.org/cgi-bin/WEBRepeatmasker). Non-redundant sequences will be used for searching various databases. The basic local alignment search tool (tBLASTx) family of programs will be used to compare all sequences with public databases of Arabidopsis thaliana, Medicago truncatula and Glycine max L. Markers

will be screened using both resistant and susceptible genotypes which will be used in hybridization to allow easy evaluation of polymorphism between them. Primers which show consistently different banding patterns in two parents in atleast two amplification events will be used to genotype RIL’s and NIL’s.

3. Generation of mapping population and identify molecular markers tightly linked to MYMV and powdery mildew resistance by adopting Bulked segregant analysis.

· F_1’s together with parents will be evaluated in glass house by inoculating with viruliferous white flies as explained earlier. Screening will be done for the traits like susceptibility and resistance to MYMV and powdery mildew. Selected F₁crosses with good combining ability will be advanced to F₂ generation which is the mapping population in glass house.

· By using mapping population genotyping will be done for MYMV and powdery mildew resistance. DNA pools will be created from bulks of resistant and susceptible plants in mapping population. SSR markers (heterologous probes) from other pulse crops like cowpea, Pigeonpea, Blackgram will be used for bulked segregant analysis (BSA). Identify markers tightly linked to MYMV and powdery mildew resistance. Phenotyping will be done in F₃ generation (grown in glass house) which helps in validation of markers associated with MYMV and powdery mildew resistance.

· Every generation is grown in glass house so that we can study the inheritance pattern of the MYMV disease, powdery mildew and gene action and also can select resistant genotypes with good agronomic background.

· Newly developed and existing SSR markers will be used for bulked segregant analysis (BSA) (Michelmore et al. 1991) for the susceptible and resistant DNA pools. Based on genotypic and phenotypic analysis of F₂mapping population and F₃ respectively, a linkage map will be constructed by using MAPMAKER Software (Lander et al .1987). Transgressive segregants that show the desirable traits will be identified and advanced.

· Adoption of fast-track generation-advancement strategies by taking up three generations advancement per year to get recombinant inbred lines (RIL,s) upto F₇ generation. Single plants will be selected for one or a combination of the above mentioned traits with MYMV and powdery mildew resistance will be forwarded in plant to row progeny and in each generation selection of resistant plants with higher yield will be continued by MAS.

· Development of backcross introgression lines with resistance to MYMV and powdery mildew : F₁'s will be backcrossed to agronomically superior genotype to get desirable alleles with disease resistance at a faster rate with marker assisted selection in each advancing generation in glass house by using the vector white fly

for MYMV resistance and for powdery mildew in field. Backcrossing will have to be done for six generations for the production of NILs. RIL’s and NIL’s will be used for mapping by using polymorphic SSR markers and newly developed SSR Markers.

· Validation of SSR markers in the backcross introgression lines / any other population : The marker identified by mapping and by BSA in mapping population will be checked in germplasm lines (Around 400) for wider applicability of the linked marker(s) for Marker Assisted Selection.

· Selection will be made during development of introgression lines (Back crossing) by using markers combined with conventional selection methodology.

· SMA consists of analyzing one marker at a time by a single software package Q Gene (Nelson, 1997) for fast and interactive viewing of marker, map, and trait data on several scales and from several aspects will be used. The QTL and the genes will be tracked and confirmed by selected SSR markers associated with traits already identified by mapping. We will be using RIL’s and NIL,s for SMA.

Ψ Development of high yielding new varieties with MYMV and powdery mildew resistance.

Ψ Development of SSR markers for MYMV and powdery mildew resistance specific to mungbean and blackgram for future application.

Ψ Using already existing and newly developed Simple Sequence Repeat (SSR) markers in marker assisted selection for MYMV and Powdery mildew resistance.

Ψ Genetics of resistance to MYMV and Powdery mildew and nature of genes will be assessed.

With the success of the present project the production of greengram level is expected to be increased by 40% ( from 0.82 lakh tonnes to 1.15 lakh tonnes), which will further help the farmers to earn an increased returns of about 8,200 lakh rupees per annum ( from 20,500 lakhs to 28,700 lakhs).

The production levels of blackgram is expected to increase by 0.25 lakh tonnes from the present level of 0.16 lakh tonnes which will earn around 40% higher returns of about 5,376 lakh rupees per annum.

Publications related to the proposal by Principal Investigator (Dr. D.L. Savithramma)

Savithramma, D.L. and Marappa, N. 2007, Screening for multiple disease resistant sources for mungbean yellow mosaic virus in mungbean. Article submitted to Indian Journal of Virology.

Marappa, N. and Savithramma, D.L., 2007, Inheritance of resistance to mungbean yellow mosaic in seven crosses of mungbean (Vigna radiata L. Wilczek). Article submitted to Euphytica.

Marappa, N. and Savithramma, D.L., 2007, Genetic variability studies in mungbean. Article submitted to Crop Research Hissar.

Rekha D., Savithramma D.L. and Shankar A.G. 2006. Molecular markers for Water Use efficiency and Estimation of Genetic variability in ten elite genotypes of groundnut for growth and yield related characters. Indian J.Plant Physiol.(Accepted for publication).

Publications related to the proposal of Co- investigator ( Dr. H.E. Shashidhar)

Vinod, M. S., Naveen Sharma, Manjunatha, K., Adnan Kanbar, Prakash, N.B. and Shashidhar, H. E. 2006. Candidate Gene approach for drought tolerance and improved productivity in rice (Oryza sativa L.). J. Biosci. 31(1): 69-74.

Adnan Kanbar, Janamatti, M., Sudheer, E, Vinod, M. S. and Shashidhar, H. E. 2006. Mapping QTLs underlying seedling vigor traits in rice (Oryza sativa L.) Current Science , 90 (1): 24-26.

Shashidhar, H. E., Vinod, M. S., Sudhir, Naveen Sharma, G.V. and Krishnamurthy, K. 2005. Markers linked to grain yield using bulked segregant analysis approach in rice (Oryza sativa L.) Rice Genetics Newslette, 22: 69-71.

Chaitra, J., Vinod, M. S., Sharma, N., Hittalmani, S. and Shashidhar, H.E. 2005. Validation of markers linked to maximum root length in rice (Oryza sativa L.) Current Science 90 (6): 835-838.

Steele, K. A., Price A., Shashidhar H. E., Witcombe J. R,.2005, Marker-assisted selection to introgress rice QTLs controlling root traits and aroma into an Indian upland rice variety, Theor Appl. Genet.: 383.

Development and utilization of DNA based molecular markers for bioticstress tolerance in chickpea (Cicer arietinum L)

Principal Investigator: Ms. M. K. Jaishree, Assistant Professor, AICRP on Chickpea, Department of Genetics and Plant Breeding

Co PI : Dr. C. S. Police Patil, Professor, AICRP on Chickpea, Department of Genetics and Plant Breeding

Chickpea (Cicer arietinum L) is a self pollinated diploid (2n=16) annual grain legume crop. It is the third important grain legume crop of the dry areas of Indian subcontinent, North Africa and west Asia an important of dietary protein. Chickpea also plays major role in biological nitrogen fixation there by contributing to crop rotation and sustaining soil productivity. However, the crop suffers due to biotic and abiotic stress and consequently its productivity has remained historically low & unstable. Despite major efforts at conventional breeding the gap between the potential yield (3-5 t/ha) and realizable yield (world average 0.7t/ha) still remains large. Hence the challenge now is to use the recently developed molecular marker technique to improve efficiency of chickpea breeding. Molecular markers have shown great potential in accelerating plant breeding especially through construction of genetic linkage maps which are useful for map based cloning of important genes and for marker assisted selection of agronomical important traits. In addition this DNA marker also provide new insight in to genome analysis, help in germplasm characterization phylogenitic analysis and genetic diagnostic. With a view of exploiting molecular marker for bringing about improvement in chickpea, a project is proposed.

Marker assisted selection can hasten crop improvement for traits that are difficult or inconvenient to score directly. Intensive efforts have been made to identify marker for fusarium wilt resistance in chickpea- linkage analyses indicated that one of the three genes for resistance to race1 and one of the two genes for resistance to race 4 and genes for resistance to race 5 were in the same linkage group, while genes for resistance to race 0 was not linked to those genes (Ratna parkhe et al 1998 Tekeoglu et al 2000 b) these linked fusarium wilt resistance genes have been assigned to linkage group VI of cicer genome. A distance of 5 cM was estimated between the genes for resistance toe race 1 and race 4 (Tullu et al 1998) and 11 cM between the genes for resistance to race 4 and 5 (Winter et al 1999). Two RAPD worker (CS- 27700 and UBC-170550) were maped at the distance of 9 cM and one ISSR markers (UBC 855500) at a distance of 5 cM from the gene for resistance to race 4. An allele specific prmer (SAP) developed from seed 27 primer RAPD marker 27700 was located between the genes for to race 4 and race 5 with a distance of 7 and 4 cm, respectively (waiter et al 1999) Recently one resistance gene analogs (RGA) has also been maped to this linkage group.

Chickpea wilt caused by Fusarium oxysporum f.sp. ciceris is one of the major yield limiting factors in chickpea. The disease causes 10-90% yield losses annually in chickpea. Eight physiological races of the pathogen (0, 1A, 1B/C,2 ,3, 4, 5 and 6) are reported so far whereas additional races are suspected from India. The distribution pattern of these races in different parts of the world indicates regional specificity for their occurrence leading to the perception that F. oxysporum f. sp. Ciceris evolved independently in different regions. Pathogen isolates also exhibit differences in disease symptoms. Races 0 and 1B/C cause yellowing syndrome whereas 1A,2,3,4,5 and 6 lead to wilting syndrome. Genetics of resistance to two races (1B/C and 6) is yet to be determined, however, for other races resistance is governed either by monogenes or oligogenes. The individual genes of oligogenic resistance mechanism delay onset of disease symptoms, a phenomenon called as late wilting. Slow wilting, i.e., slow development of disease after onset of disease symptoms also occurs in reaction to pathogen; however, its genetics are not known mapping of wilt resistance genes in chickpea is difficult because of minimal polymorphism;however, it has been facilitated to great extent by the development of sequence tagged microsatellite site (STMS) markers that have revealed significant\nt interspecific and intraspecific polymorphism markers linked to six genes governing resistance to six races (0, 1A,2,3,4 and 5) of the pathogen have been identified and their position on chickpea linkage maps elucidated. These genes lie in two separate clusters on two different chickpea linkage groups. While the gene for resistance to race 0 is situated on LG 5 of Winter et al. (Theoretical and Applied Genetics 101:1155-1163, 2000) those governing resistance to races 1A,2,3,4 and 5 spanned a region of 8.2 cM on LG 2. The cluster of five resistance genes was further subdivided into two sub clusters of 2.8 cM and 2.0cM, respectively. Map-based cloning can be used to isolate the six genes mapped so far; however, the region containing these genes needs additional markers to facilitate their isolation Cloning of wilt resistance genes is desirable to study their evolution, mechanisms of resistance and their exploitation in wilt resistance breeding and wilt management.

Pod borer (Helicoverpa armigera Hubner) is the most devastating insect pest of chickpea and annual global losses are estimated at $ 500 million (Ryan 1997) considerable program has been made on development of screening methods (Sharma at al 2003) and to identify sources of resistance is in the cultivated and wild cicer species. ICC 506 appears to be the best source of resistance available in the cultivated species. Over six species ICC 506 showed a mean of 9% of damage as against 30% in the popular variety Annigeri. ICC-506 is highly susceptible to fusarium wilt. The development of breeding material with high level of wilt resistance and a level of Helicoverpa resistance similar to ICC 506 has been a challenging task.

Drought, particularly terminal drought, it the most important abiotic stress for chickpea as it is grown rainfed, in post-rainy season on residual soil moisture. The crop often experiences progressively increasing drought stress during the reproductive phase,

resulting in early senescence and reduction in pod and seed development. Two genetic options being employed in chickpea for drought management are escape and tolerance.

Efforts have been made to identify drought tolerant germplasm and the traits contributing to drought tolerance. Some promising drought tolerant lines are ICC 4958, ICC 5680 and ICC 10448. high root mass was found responsible for drought tolerance in ICC 4958, whiule smaller leaf area was the most important drought trait in ICC 5680 and ICC 10448 (Sexena, 2003). The large root trait helps in greater extraction of water available in soil, and the smaller leaf area reduces transpiration loss of water. ICC 4958 was found to have 30% more root volume than the popular variety Annigeri (Saxena et al., 1993) Lines with greater degree of drought tolerance have been developed by combining large root traits of ICC 4958 with fewer pinnules trait of ICC 5680 (Saxena, 2003). A recent screening of the mini-core collection has identified several other lines with large root traits (Krishnamurthy et al., 2003)

Ψ Two RAPD markers for two QTL’s conferring ascochyta blight (AB) Resistance has been identified

Ψ One RAPD marker (OPAC 04/1200) linked to major QTL for resistance to (AB) has been identified.

Ψ Fusarium wilt (FW) race 3 controlled by single gene (foc-3) linked to SSR markers TA 96 and TA 27 and STMS marker CS 27A has been identified.

At present yield loss due to wilt is 10-90% due to Helicoverpa armigera, pod borer is 20%, and due to drought is 90%

Ψ Introgress the ICC 506 pod borer resistant characters in to Annigere-1 through MAS

Ψ Introgress iCC 4958 root system and ICC 5680 leaf area smaller leaf area in to Annigere-1 through MAS

K.P, Viswanath, M.K, Jayashree and P. Arunachalam 2001 Heterosis and inbreeding depression in physiological trait of cowpea[V. unguiculata (L) Walpers], Forage research 27 (2)83-86

K.P.Viswanath, M.K.Jayashree 2001, In heritance of three qualitative characters in cowpea [V. unguiculata (L) Walpers], Forage research 27 (1) 11-13

K.P.Viswanath, Balaraj, M.K.Jayashree and P. Arunachalam -1999 Genetic analysis of some physiological traits in cowpea [V. unguiculata (L) Walpers] Mys. J. Agri-33, 234-236

P. Arunachalam, K.P. Vishwanata, K.K.Chakravarthy, A Manjunath & M.K.Jayashree -2002 Efficiancy of breeding methods in early segregating generation in [V. unguiculata (L) Walpers],Ind. J.Genat 62(3) 228-231

Principal Investigator : Dr. M. S. UMA, Associate Professor, Department of Genetics and Plant Breeding, College of Agriculture, GKVK, Bangalore

Major prerequisites for a disease resistance breeding programme are, the availability of stable resistant sources to cowpea rust. A national cowpea germplasm collection held by NBPGR, many local/landraces, indigenous and exotic accessions have been found to show resistance to rust and other biotic stresses. Although, a large number of resistant germplasm lines have been identified, they possess undesirable agronomic attributes making them commercially unacceptable.

Several genotypes (76 local /landraces) have been identified as rust resistance from the screening experiment of NBPGR cowpea collections (225 landraces/local genotypes) at UAS. The promising genotypes will be used as donors in the breeding programme for upgrading of popular varieties.

Rapid development of resistant varieties using the popular variety as a base will be carried out by the integration of DNA marker assisted selection in backcross breeding programme. After identifying the linked markers for rust resistance, those markers identified will be mapped using RIL mapping populations.

3) To identify polymorphic SSR markers for parents and F2 & Backcross populations

Marker Assisted back cross breeding for Fusarium wilt (Fusninm species) resistance and tagging of markers linked to other economically important characters in Bambara groundnut (Vigna subterranere L.)

Principal Investigator: Dr. R. Nandini, Department of Genetics and Plant Breeding

Co PI: Dr. H .E. Shashidhar Department of Genetics and Plant Breeding, College of Agriculture, GKVK, Bangalore 560065

Bambara groundnut (Vigna subterranean L.) is an important food and nutritional legume crop. It has a great potential in India especially in semi arid dry farming zones which constitutes about 65-75 per cent of the total arable land in the country. It has a potential as a supplementary food and pulse crop in India, especially for tribal backward population. Seed is a rich source of protein (16-25%) which is superior in quality to that of cowpea, groundnut and pigeon pea because of the high proportion of Lysine and methionine. It is rich in corn with 5.48 mg per 100 g as compared to most Legumes (2-10 mg). Its energy value is 367-414 K cal/100 g.

Although the crop is resistant to many pests and diseases, it is highly susceptible to fusarium wilt and wilting of Bambara groundnut occurs at the seedling stage of the crop. No information exists on the selection or breeding of resistant lines or the sue of germplasm for crop improvement, because practically no work seems to have been undertaken on Bambara groundnut. Nor have been any individual research findings available on resistance potential to fusarium wilt although there are several reports depicting the losses of Bambara groundnut due to fusarium wilt. Hence, the main objective of the proposed programme is:

Development of multiple disease resistance to Tomato Leaf Curl Virus, Bacterial wilt and nematode in Tomato by using molecular marker assisted selection.

Principal Investigator : Dr. R. S. Kulkarni, Professor, Department of Genetics and Plant Breeding, College of Agriculture, GKVK, Bangalore 560065

The production of tomato in many tropical and subtropical countries is hampered mainly by Tomato Leaf Curl Virus, Bacterial wilt and nematode. Use of wild germplasm for transferring disease resistance to elite breeding lines is well proven phenomenon in conventional breeding programs. But because of high linkage drag the program requires many generations of selection which is time consuming and laborious. DNA marker technology has been used in plant breeding program since as early as 1990 and has proved helpful for the rapid and efficient transfer of useful traits into agronomically desirable varieties and hybrids. Markers linked to disease/pest resistance loci can now be used for marker-assisted selection (MAS) programs, thus allowing introgression of several resistance genes to the same genotype which is popularly known as ‘gene pyramiding’. In the proposed investigation the molecular marker technology will be used to introgress disease resistance genes from wild germplasm to elite breeding lines.

Molecular marker assisted selection helps to screen the segregating material not only for the presence of introgressed disease resistance gene but also for the background genes which are inherited from the agronomically superior parent. Thus it helps to identify the segregating progeny which contain only the resistance gene from the wild donor parent and maximum possible genomic content from agronomically superior parent and this phenomenon is exploited for increasing the efficiency of selection and reducing the time required for the intended breeding program.

D. Current status: Breeding material has been evaluated for multiple disease resistance and a few promising breeding lines have been identified.

DNA Marker Assisted Introgressions of sheath rot, bacterial leaf blight and blast resistance into elite Rice Varieties of Karnataka

Principal Investigator: Dr Shailaja Hittalmani, Professor, Department of Genetics and Plant Breeding, College of Agriculture, GKVK, Bangalore

Rice Blast, (Magnaporthe grisea), sheath rot (Sarocladium oryzae) and Bacterial Leaf Blight (Xanthomonas oryzae) are three important diseases of rice that can individually and together cause upto 80 of grain yield loss and cause low quality fodder. Of the three, rice blast affects at seedling, vegetative and reproductive stages. Neck last can be more severe than leaf or grain blast. Neck blast together with sheath rot causes 100 sterility in grain preventing the translocation into the spikelet. Bacterial leaf blast affects photosynthetic areas and reduces the yield drastically and produce partial grain filling and low quality fodder yield. DNA markers were first used to map blast resistance major genes and QTLs and bacterial leafblight resistance genes at IRRI. Sheath rot resistance genes were identified and mapped at UAS with both for QTLs and major genes operating in the region. There are six major genes that are important for various races operating and Xa 21 is the most strongest of them, about 30 genes in blast and 5 major genes and 9 QTLs in sheath rot are identifed. To ether they are impart reasonable good resistance in rice. Since markers are known and mapped for all the three important disease it is possible to

incorporate ans select the resistance plant with prcsison and rapidly by integrating them in conventional breeding program,

Methodology:

Blast resistance genes Pi- 1+2 for blast,(neck and leaf) BLB- Xa 21 for Bacterial leaf blight and IR 50 for neck blast resistance. Moroberekan is resistant to sheath rot and blast. Three way crosses will be carried out the F1s will be crossed further to combine all the three resistance genes. DNA flanking markers tightly associated will used to select for resistance. In our earlier work we have mapped sheath rot and blast in IR64. BPT Sona Mahsuri will be used as Recurrent parent as it is the most markertable and acceptable variety for the masses. The F2 generation plants with three gene products in various combinations will be raised and introgressed lines in different combinations will be screened with markers to obtain homozygote and heterozygote for genes of interest as shown by the markers.

The plants selected for resistance and good plant phenotype will be further screened for background to in Sona Mahsuri. Search for superior transgresssant with resistance will be used for further backcross and also for advancement as new breeding line:

2. Raise F1 with the three genes combination the F1 and check for the resistance genes.

4. Marker assisted selection for the three genes in BC generation for Target genes and background will be made

BPT rice variety with blast, blacterial leaf blight and Sheath rot resistance.

1.Two and three gene combinations for multiple resistance in superior agronomic variety, BPT Sona Mahsuri.

Development of Genomic Resources (SSR Markers), and mapping and validation of QTLs conferring wilt resistance in Chili/hot pepper (Capsicum annuum L.)

Principal Investigator: Dr. A. Mohan Rao, Associate Professor of Plant Breeding

Hot pepper (Chilli) is one of the major vegetable/spice crops of Karnataka. The production environment ranges from irrigated to rainfed and tropical to subtropical areas in the region. Productivity of chilli in major pepper growing regions of Southern India, specifically Karnataka is affected by a host of biotic constraints. Fungal and bacterial wilts cause significant yield and quality losses up to 60 per cent. Wilt, a soil borne disease in chilli is caused by several groups of pathogens. It becomes important to document the severity of this disease and increase our understanding etiology, pathogenicity, and genetic relatedness of representative isolates from major chilli growing regions of Karnataka.

Host plant resistance (HPR) can play a major role in wilt management and considerably reduce the dependency on chemical control & environmental & health hazards associated with their use. Conventional breeding for HPR has not been successful owing to the high degree of pathogenic variability and the occurrence of disease at any of the growth stages during crop growth period, which hinders in the effective screening for resistance under field conditions. The knowledge on the pathogen variability, its virulence and interaction with the host plant and the inheritance of HPR and the availability of large-scale and cost-effective hot spot/field/greenhouse screening technique and resistance sources would enhance the pace and efficiency of breeding hot pepper for wilt resistance. DNA markers are known to detect and measure the variability and identify different wilt causing pathogen complex. Molecular marker-assisted selection would be handy and complement the conventional approaches for introgression of resistant genes from resistant sources in the elite agronomic background.

Among the many factors responsible for the stagnation of yields of pulses and oilseeds in the country is the problem of biotic stresses by insect pests, diseases and weeds. Attempts elsewhere in the world to mitigate these problems with the application of biotechnological tools has met with great success and transgenic in insect resistant cotton is the evidence to see in India. As a result there is an increasing realization among the Agricultural Scienctists in the country to explore for strong biotechnological tools both to enhance the system of breeding for specific traits using MAS and also to incorporate new traits through transgenic technology. As a consequence, a large number of programmes to utilize MAS in breeding and transgenics to develop stress tolerant crops have begun. The University of Agricultural Sciences, Bangalore has led this work from front by developing many programmes through national and international funding.

Initial works to develop semi irrigated rice, fungal leaf and neck blast tolerant finer millet have been the examples of great success in molecular breeding using MAS procedures. Similarly, a number of genes available with various scientists across the country and out side have been out sourced and a massive programme in the development of transgenics to mitigate many biotic stress problems have been attempted. So much so that today, as many as 15 different successful events are under advancement. Insect, fungi, virus, and herbicide resistant crop varieties have been attempted. Many more that are not easily amenable for management through transgenic approach are being attempted to solve through molecular breeding approach using the MAS procedure. But many more remain to be tackled. Strong back ground knowledge in plant breeding, thorough understanding of the basics of the biotechnological tools, availability of the required genes, expertise in the procedures of MAS, have all led to the success of the many programmes launched. Headway made through these efforts has provided a great impetus for the University to explore the possibility of expanding the work on these lines. Therefore, the current programme on development of biotic stress tolerant crop plants using both molecular breeding and transgenic approach is being proposed as part of the work under Biotech Innovation Centre.

Dr. A.R.V. Kumar, Dr. K.T. Rangaswamy, Dr. P. Chandrashekara Reddy, Dr. Rohini Srivathsa, and Dr. K. Chandrashekara

Among the many factors responsible for the stagnation of yields of pulses and oilseeds in the country is the problem of biotic stress by insect pests and diseases. Attempts elsewhere in the world to mitigate these problems with transgenic approach have met with tremendous success. As a result today the area under transgenic crops has risen enormously in different countries. Transgenic approaches have given excellent control against pest problems and herbicide resistance. However, several options for management of diseases especially against viruses and to some extent against fungi have revealed the potency and the products are envisaged to be in the market soon. One of the classic successes of this approach in India is that of cotton against Lepidopteran pests. The rampant growth of area under transgenic cotton is a testimony for utility, economy and the farmer acceptance of the technology. Thus the potency of the technology has been proven and the platform is ripe for more experimentation and adoption of the transgenic technology on a large scale.

Apart from drought, most of the field crops suffer from heavy damage due to biotic stresses leading to whopping losses (see introduction). Insect pests and diseases caused by fungi and viruses are responsible for nearly 30 % losses. An estimated annual losses for these crops is Rs. 29,000 crores. Major oil seeds such as groundnut and sunflower experience an annual loss of nearly Rs. 462 crores in the state of Karnataka. Pigeon pea, chick pea and field bean together experience losses to the tune of over Rs. 427 crores in the state. In India, field crops, barring cotton and vegetables have largely remained as the domains of the public sector research areas, primarily because of the investment constraints of the cash starved dry land farmers. As a result the onus is on these agencies such as State Agriculture Universities to develop management strategies for biotic stresses in these crops. Thus the current programme under the Biotechnology Innovation Centre envisages developing transgenic varieties to mitigate some of the major problems of these crops in particular and dry land field crops in general. Insect pests and the diseases caused by fungi and viruses in major oil seed and pulse crops of the state are the primary targets.

A transgenic technology for adoption requires, a) an identified target crop and a problem b) availability of effective gene/s, c) a protocol for transformation of the crop with the desirable gene and the d) methodology to screen, evaluate and advance the transgenic line to a stable high performing line. At the University of Agricultural Sciences, the attempts made since the last few years have demonstrated the potency to undertake all these steps and to develop insect and fungal resistant lines of pulses and oil seeds.

A) Over the last decade the yields of many pulse and oil seed crops has been a point of concern due to stagnation. A major chunk of this problem is represented by the insect pests and diseases. Among the many insect pests that attack the pulses and oil seeds are the lepidopteran pests that cause up to 30 % damage in productivity of many of these crops. For example among the oil seed crops groundnut suffers from damage due to RHHC (Amsacta

albistriga), tobacco caterpillar (Spodoptera litura) and leaf miner (Aproaerema modicella) and sunflower suffers from Helicoverpa armigera and tobacco caterpillar. These are the two most important oilseed crops of the state and their productivity has been greatly affected due to these pests. The amount of damage by these pests can reach upto near 100 % in specific cases and on an average is estimated to be around 30-35 % yield loss amounting to an annual total of Rs. 212 crores in the state due to insect pests alone in groundnut. Similarly, the annual economic loss in Sunflower can go up to Rs. 250 crores in the state. Casor is another crop that suffers from many insect pests including both defoliators such as Achoea janata and tobacco caterpillar and the capsule borer Dichocrocis punctiferalis. Again the castor is a highly marginalized crop that receives poor attention from the farmers. As a result, the losses caused particularly by capsule borer can reduce the yields by over 50 %.

All the main pulse crops of the state such as redgram, chickpea and field bean suffer heavy damage due to Helicoverpa armigera. Redgram further suffers from other lepidopetran pests such as Maruca testulalis, Sphenarchis cafer, Lampoedes boeticus, and Cydia critica. Although the various State Agricultural Universities have developed effective packages of measures for managing these pests, the losses continue to occur as the adoption levels of the technology are extremely low. On the whole, from insect pests alone, the estimated losses in the state are Rs. 334 crores in Redgram, Rs. 81 crores in Bengal gram and Rs. 12 crores in field bean. One of the main causes for these low adoption rates is the marginalization of the crops. Many of these crops are generally grown by poor, dry land farmers who suffer from investment crisis. Poor cash investment abilities are also dictated by the future vagaries of the monsoon so that the problem remains unmanaged even in good rain fall years. Therefore, these crops are ideal target crops with their array of Lepidopteran pests to explore for transgenic approach in mitigating the insect pest problems.

Apart from insect pests, all these crops also suffer from the ravages of many fungal and viral diseases. Among them, a host of fungal diseases that attack groundnut, such as Cercospora, Alternaria, Rhizoctonia and Fusarium are of prime importance in groundnut. Further, the Alternaria leaf spot is also equally important in sunflower. In pulses, the Fusarium wilt is the major problem affecting both redgram and bengalgram. During years of heavy rainfall, the crop gets completely devastated in large tracts of the state due to this disease alone. Considering the viral diseases, groundnut bud necrosis a few years ago was responsible for complete loss of the crop in the main dry land groundnut belt of Karnataka and Andhra Pradesh. Sunflower necrosis disease remains a nagging and perennial problem with isolated crops suffering near 100 % damage due to the disease. Viral diseases are now amenable for management using coat protein genes.

Thus in essence, the two important oilseed crops, groundnut and sunflower and the three important pulse crops, redgram, chickpea and field bean are all amenable for target specific transgenic approach for mitigating both insect pest and disease problems.

B) Considering the gene, the University has access to many insecticidal, fungicidal and viral coat protein genes which can be effectively deployed in crop plants to combat insect pests, fungal and viral diseases.

C) Keeping these in mind and the urgent needs for developing seed borne solutions to reduce the cost of management, intensive efforts are being made at the University of Agricultural Sciences, Bangalore to develop transgenic varieties resistant to various diseases and insect pests. Most of these crops are not easily amenable for conventional transformation.

However, the efforts made so far using the Agrobacterium mediated in planta transformation techniques have yielded fruitful viable transgenics. The methodologies for transformation of groundnut, sunflower, redgram, chickpea, fieldbean, castor, cotton and even rice have been standardized and verified at the University. Viable transgenics with high expression of insect resistant genes have been developed and some of them advanced through three generations.

D) Protocols for advancement of the putative transgenics through successive verification for quality agronomic traits along with the intended novel trait have been the hall mark of these studies in progress. The PCR based identification of the transgene, characterization of the expression levels through bio-assays, ELISA for ascertaining the expression levels have all been followed through successive generations to confirm the transgenic nature of the selected lines for advancement. Further, the Southern blots and RTPCR techniques have been used to confirm the copy numbers and to identify the stable high expression transgenics.

Thus the above clearly suggest the capability of the team at University of Agricultural Sciences, Bangalore in developing, testing and advancing the transgenic lines suitable for mitigating the target specific problems in all the crops listed above. Some of these works are in quite an advanced stage and need to be taken forward. Some others, however, are to be restarted or initiated afresh to meet the challenges of field problems. Keeping these in view the present proposal is being developed with the following objectives of developing novel target specific transgenics to mitigate the biotic stresses in selected field crops

Development of novel transgenics to mitigate some of the serious problems of selected crops is planned as an activity of the Bitechnology Innovation Centre. In specific, the development of transgenics is aimed at to tackle insect, disease and herbicide tolerance in oilseeds and pulses. The following list provides the targeted crop and the problem to be alleviated using transgenic approach.

Table 1 : List of crops and the problems proposed to be tackled using transgenic approach at the Biotechnology innovation centre.

1. Development of pod borer resistant transgenic BRG-2 and BRG-4 varieties of redgram

Cry1AabcF is a synthetic gene developed at IARI, New Delhi, that has been found to be effective against a number of Lepidopteran pests such as Diamondback moth (Plutella xylostella), Pod borer (Helicoverpa armigera), red headed hairy caterpillar (Amsacta albistriga) and tobacco caterpillar (Spodoptera ltura). The gene is currently available for use at University of Agricultural Sciences, Bangalore for purposes of development of transgenics targeting Lepidopteran pests. A number of crops have already been developed using this gene and are in various stages of testing and advancement. One among them is the TTB-7 variety of redgram that needs to be advanced further to select for stable high expression lines. However, BRG-2 and BRG-4 are recent newly developed varieties of redgram that are bold seeded and high yielding with consistent five seeds per pod. These varieties have attracted the attention of the local farming community in the southern parts of Karnataka and are in great demand leading to near replacement of the conventional HY3C and TTB-7 varieties that were popular earlier. Development of podborer resistant lines of these genetic backgrounds will be of great value to the farming community. Thus, it is planned that transgenics be developed with the genetic background of BRG-2 and BRG-4, the leading local varieties of redgram.

The approach to the development of these transgenics would be the one that has already been tested to develop insect resistant TTB-7 variety. The Agrobacterium mediated in planta technique would be followed for development of these transgenics also (See Appendix 1). The requisite binary vector plasmid containing the marker npt-II (Kanamycin resistant) and Cry1AabcF genes is available and has been cloned into Agrobacterium tumefasciens.

The transgenics, once developed, would be transferred to the Biotechnology Translational Research Centre for further development of the products.

Dr. K. Chandrashekara, Dr. Rohini Srivathsa, Dr. A.R.V. Kumar and Dr. Jayashree

Chickpea is grown in nearly 5.00 lakh hectares of Karnataka. However the yields are extremely poor at just around 600 kg/ha. Much of this is problem of low yields is largely due to the problem of pod borer, Helicoverpa armigera. Although a comprehensive management package for this pest is in place, most farmers do not take up any crop protection measures to manage the problem. Therefore in order develop a seed borne solution to manage this problem, it is proposed to develop insect resistant chickpea. Annigeri-1 is a conventional variety that is grown widely in the state. However, the recently released Vishal has potential to yield much higher than the conventional Annigeri-1 variety. Considering this situation, it is proposed that both Annigeri-1 and Vishal be transformed to obtain insect resistant stable transgenic lines.

The gene to be used and the methodological approach are similar to that of redgram mentioned above with suitable modifications to meet the needs of chick pea crop.

Head borer, Helicoverpa armigera is the major insect pest of sunflower causing heavy damage to the yield in both dryland and irrigated crops. The Cry1AabcF gene has been found to be highly effective against H. armigera and therefore would be an ideal tool to be incorporated into sunflower for combating this pest. Hence, it is proposed that under the programme a suitable hybrid would be selected for transformation with the said gene. The methodology would be suitably modified to suit the sunflower.

Castor suffers from a number of lepidopteran pests. Among them the capsule borer (Dichocrocis punctiferalis) is the major pest to cause sever economic damage. Semilooper (Achoea janata), tobacco caterpillar (Spodoptera litura), redheaded hairy caterpillar (Amsacta albistriga) and Bihar hairy caterpillar (Spilosoma obliqua) are the other important defoliators that affect the crop. Therefore, this crop is being targeted for transformation with the said gene. The protocols would be similar to the redgram in planta transformation with suitable modifications to suit the crop. However, as the major crop damage to castor is due to the capsule borer, development of transgenics with high levels of expression in the reproductive parts is uncertain by the current approaches being followed. Therefore, it is less likely that the conventional gene and the promoters would be of great use in eliciting the sufficient levels of expression of the toxic protein in the reproductive parts. Therefore, two novel constructs Cry1Ec and a Bt VIP gene along with suitable promoters are planned to be pyramided into a single genetic background. Both these genes are to be accessed from NBRI and ICGEB, New Delhi.

Dr. K.T. Rangaswamy, Dr. Rohini Srivathsa, Dr. H.S. Savithri and Dr. T.G Prasad

Peanut bud necrosis virus transmitted by Thrips palmi is extremely difficult to manage due to very low threshold population requirement for the spread of the disease. As a result, the potentially the problem can cause havoc in major groundnut belts of the state. Early infection can wipe out the yields while late infections can cause up to 70 % yield loss.

In the recent past, several attempts to manage virus diseases using viral coat protein mediated resistance has attracted considerable attention. Papaya ring spot virus and watermelon mosaic virus resistance are the successful examples to this effect. N and Nss gene constructs required for the purpose are currently available in binary vectors for development of groundnut transgenics. These have been procured from Dr. H.S. Savithri, IISc, Bangalore. Testing of the putative transgenics can be done with RNA-i mediated approach. Therefore, the present work is being proposed.

The approaches to transformation would be the same as employed for insecticidal genes.

It is envisaged that the herbicide resistance in crop plants would help reduce the problem of crop –weed competition and help also reuce the labour input, obtaining which is both a social and economic problem. EPSPS gene mediated glyphosate resistance has been well documented and largest area under transgenic crops is currently controlled by the glyphosate resistant transgenic crops. This also serves a s a testimony to suggest the importance of the

trait in mitigating several crucial cultivational problems. The necessary EPSPS construct is available in a binary vector and the in planta transformation protocol can be efficiently employed to transfer the gene to groundnut varieties. Similarly, the team also has the construct IgrA available for this work. It has been the experience of the team that the two genes independently are capable of providing significant tolerance to glyphosate, but the two together would enhance the tolerance levels to a relatively high level so that the use of herbicide can be tolerated even at a little higher level. Therefore, it is proposed that pyramided transgenic lines with both these genes be created in different genetic backgrounds for better performance of the crop under field conditions. Therefore an attempt would be made to develop glyphosate resistant K-134variety of groundnut for use especially under irrigated conditions, with two genes pyramided into each of them.

Tomato is one of the major vegetable crops of the state that experiences high damage due to

Weed competition. Recently the University has released three varieties of tomato that are tolerant to Tomato Leaf Curl Mosaic. The present proposal envisages developing herbicide tolerant Vybhav and Nandi lines of tomato using the pyramided IgrA and EPSPS genes.

All the developed hybrids tested through one generation would be transferred to the translational research centre for furthering the selected events to the level of deliverable products.

Thematic area: Breeding by design for quality traits of crop plants such as Fe, Zn, oil and vitamin A and to improve the post harvest shelf life.

Similar to elsewhere in the world, Indian agriculture faces the challenge of delivering safe, high quality and health promoting food as well as bio-products in an economical, environmentally sensitive, and sustainable manner. People of both rural and urban areas of our country predominately consume cereals as major diet (which provides 80% of the energy & some amount of other nutrients except vitamin D and B₁₂), but they are deficient in several essential micronutrients from human nutrition point of view. Especially diets of poor income groups are deficient in several nutrients, namely Fe, Zn, Vitamin A and Iodine. This is essentially because the prime source of all the nutrients for humans and animals comes from agricultural products.

Several elements are reported to be below critical level in the soil around the globe. Among the micronutrients, Fe and Zinc are found to be deficient for plant growth and productivity. Wherein plants are unable to take Fe from rhizosphere because of its presence in unavailable form, though 3 percent of earth crust is Fe, where as Zn is generally deficient in soils. It is reported that, 25 -30 percent of cropped area is deficient globally. 60-70 per cent cropped area is deficient in India and 70% cropped area in Karnataka especially children, pregnant women and lactating women are the target population.

The deficiency of these elements alone causes several disorders in humans, which is accounting to trillion dollars annually on health exchequer, globally. This is a serious concern in developing countries including India and more than 300 million people live with one dollar a day in India.

World Bank (Anonymous, 1994) reported that 5% GNP is lost every year due to low intake of Fe, Vit A and I in south -Asia alone. In addition to this Zn is having much wider ramification on human health, because more than 39% of global population are not getting adequate amount of Zn from health perspective. Supplements program for this region alone for one nutrient (Fe) itself would cost 500 million dollars over 10 years, on the other hand investments on plant breeding and biotechnological research are for lower. According to one estimate it would cost 10 million dollars in over 7-8 years to develop Zn dense varieties of wheat and rice, the leading staple food crops of the world. There will be enormous economic benefits of the biofortification strategy and returns expected would be 4.9 billion dollars on a total cost of 42 million dollars (Bouis, 2002).

Though several interventions namely, supplementation, fortification and diversification are advocated they are neither economically feasible nor can sustain in the long run. Existing plant genetic resources and current breeding methods alone are insufficient for understanding the mechanisms underlying important traits and for catalyzing a major leap in yield, sustainability and quality improvement. Hence, biofortification seems to be a sustainable approach and providing the required amount of Zn and Fe through natural

food for both humans and livestock looks a best plausible approach. And care also should be taken not to compromise yield and profitability while breeding or developing nutritionally enhanced cultivars.

Pulses are an excellent source of dietary protein for millions of people, especially in the developing world. But, the per capita availability of pulses is declined from 64 gm/ capita/day (1951-56) to less than 30 gm/capita/day as against the WHO recommendation of 80gm/capita/day.

Our laboratory in India and Agri-Food and Agri Canada have already initiated work in pulses in this direction. And Also a collaborative program is envisaged in collaboration with KVL Copenhagen, Denmark to develop low phytic acid (lpa) mutants to develop the varieties with low phytic acid content in the grains, because phytic acid is one of the major anti-nutritional factors (ANF) that reduces the bio availability of these micronutrients from human nutrition perspective. To assess the bio availability of these micronutrients, a program has been initiated with National Institute of Nutrition (NIN), Hyderabad using Caco-2 cells in the laboratory and using piglet and rat model system and at our laboratory. Based on these initial efforts, in this proposal, we seek to undertake a marker assisted selection (MAS) breeding and biotechnological approaches and bio availability studies to address these important issues. Recently, GOI and HarvestPlus (the international consortium) with global alliance of research institutions that have come together to breed and disseminate crops for better nutrition signed an MOU to develop and disseminate micronutrient- dense crop variety to reduce the micronutrient malnutrition among at – risk populations of India.

We analysed 49 different food grains, vegetables and fruits that are commonly consumed on a daily basis to meet the caloric requirement, for Zn and Fe. The data suggests that the population is not in a position to meet the RDA requirement of 15mg/day Zn and 12-15mg/day Fe (as per WHO). The situation is still worst with rural and poor people, because more than 300million people live on a dollar per day in India. And the children, pregnant women and lactating women are at greater risk.

The recent advent of molecular tools and their application in wheat and barley strongly suggests that it is possible to improve Fe and Zn in food grains compared to several other conventional interventions, biofortification of food crops seems to be a best plausible approach.

The major research emphasis at the Department of Crop Physiology, University of Agricultural Sciences, Bangalore, India has been to understand the mechanism and physiology of the genetic variability in nutrient uptake, translocation and biochemical utilization of P, Ca, Fe and Zn. Over the last couple of years, we have assessed the genetic differences in nutrient uptake and physiological processes associated with large number of crop plants such as pigeon pea, Rice, finger millet, etc.

Shashidhar et al., (2006) have reported that it is possible to improve productivity in crop species using candidate gene approach. Even grain yield can be enhanced by identified markers using bulk segregant analysis (Shashidhar et al., 2005). However, the similar progress has not been made in pulses.

QTL approach of simultaneous introgression and evaluating the derivatives using anchored molecular markers will help to identify the derivatives with high Zinc.

Development of QTLs controlling the useful variability can be used for screening the additional germplasm lines for gene discovery using advanced functional genomics.

Our group has strong expertise in assessing the genetic variability for Zn in several food crops. We are already addressing this issues in finger millet (DST, GOI funded project), in Pigeonpea (DBT, GOI funded project) and Rice and in several commonly consumed food grains.

Our group also conducted bioavailability studies by fortifying the feed with Zinc. The plasma Zinc (Fig.a) and gain in body weight (Fig.b) was phenomenal in both piglets and rats. Histopathological studies also indicated there were no toxic lesions in the vital organs (Fig. c) of the body.

Though the fortification studies strongly suggest it is possible to overcome the diet deficiency of humans through fortification. These interventions including supplementation and diversifications are not economically sustainable and feasible in the long run. Our study also suggests that without reducing ANF’s (eg. Phytic acid), a moderate increase in grain Zn content will be sufficient to address this problem. Hence, we strongly believe biofortification of food grains is a sustainable and cost effective approach in the long run to overcome the dietary deficiency of micronutrients from human nutrition perspective.

A. Title: Biofortification of Zn-dense pigeon pea ( Cajanus cajan) and finger millet (Eelusine coracona)-Molecular breeding and Transgenic approach.

PI: Dr A.G SHANKAR, Professor Department of Crop Physiology UAS, GKVK Bangalore-65

Rationale: After moisture stress, one of the major stresses limiting the crop productivity is nutrient deficiency stress. Several elements are reported to be below the critical level in the soil, of them Zn & Fe are found to be deficient for plant productivity. This in turn has led to less content grains & short in supply to meet the human dietary requirement. The RDA of 15mg/day is not met, when the population has solely dependent on staple food crops to meet all the nutrient requirements.

According to a recent survey conducted by the National Statistic Bureau, malnutrition caused by deficiency of Zn alone results in a loss of US $ 1.3 billion annually, accounting for 2-3% of gross domestic product (GDP).

Hypothesis: Crop biofortification is a food based strategy for reducing micronutrient deficiencies in a sustainable way among the target population. The genetic variability exists in germplasm lines of pigeon pea and finger millet for Zinc content in grains and can be exploited either through MAS breeding or transgenic approach.

Current Status: The zinc content in 48 different food grains that are commonly consumed and the quantity of different food grains that are consumed on daily basis to meet the calorie requirement by different age groups strongly suggests that only 45-55% of RDA requirement of Zn is met on a daily basis.

Objectives: The overall objective is to biofortify the pigeon pea and finger millet grains with high Zn. The specific objectives are:

2. Effect the crossing b/n high and low, using the F2 mapping population develop QTL.

4. Development of transgenics over expressing Zn transporters to the grain in finger millet ruling variety (GPU-28)

5. Standardization of bioavailability protocols using Caco-2 cells and piglet model system and quantifying the bioavailability status.

B. Title:Development of Quantitative Trait Loci (Qtl) and Marker Assisted Selection (Mas) to Improve the Zinc and Iron content in grains of Rice (Oryza Sativa)

PI: NAGARATHNA,T. K., Asst. Professor, Department of Crop Physiology UAS, GKVK Bangalore-65

Zinc and Iron are the two most deficient elements from the point of plants, human and animal health perspective. Recently it has been reported that zinc and iron deficiency in humans is the most wide spread nutritional disorder next to Vitamin A and B. Populations that depend on grains and legumes as staple foods consume diets low in Zn and Fe. According to recent survey, 49% of global population do not meet their daily intake of Zn (15mg/day/adult) and 33 % of the population for Fe (10-15 mg/day/adult), since they depend on plant foods for the supply. Bioavailability of these micronutrients also reduces to a greater extent due to the presence of antinutritional factors like phytic acid, a major form of phosphorus storage in cereals and legumes. Especially people in developing countries are at risk, since plant food is their major diet.

Rice is a vital staple food for more than half of the world’s population, primarily for poor people living Asia. Because of the high consumption of rice in developing countries, increasing its nutritional value can lead to significant positive health outcomes. Therefore, the major objective of the proposal is to develop superior biofortified rice varieties by developing transgenics. To do this, the genetic variation in Zn and Fe content will be assessed initially in diversified germplasm lines/varieties of rice and Zn and Fe transporters will be outsourced for developing transgenics.

To test the hypothesis, Genetic variability in rice germplasm lines for Zn and Fe content, in nearly 450 lines /varieties, Z and Fe content have already been estimated. Effective crossings have been made between high and low types. F2 Population is transplanted in Kharif 2007.

P.I: Dr Shailaja Hittalmani, Geneticist, Co- PIs: Dr Ramakrishna Parama, Soil Scientist, Dr Sunanada Sharan , Food and Nurtritonist

India is one of the country where cereals are the staple food for vast majority of the population. Except for wheat all other cereals esp. Rice and other millets are all low in protein and rich in carbohydrates. Major micronutrient deficiency disorders concern protein, iron, zinc, vitamin A and iodine. These deficiencies are especially severe in our country. It is reported very year approximately 2.0 billion women and children suffering from protein deficiency. Rice Protein deficiency refers to both quantity and quality esp the composition of essential amino acids apart from total protein. Daily intake of rice of 100 g of rice provides only 8-10 percent of the requirement of essential amino acids that are required. If rice protein is increased by about 25 percent the supply can be enhanced..

Most rice varieties that are bred till now are concentrated on yield enhancement or disease and pest resistance. Nutrient enhancement though talked about is yet to be achieved. Rice the diet of most Asian and half the Indian population contains on an average 7 percent protein. Hence r=mainly rice eaters suffer from protein deficiency. Hence increasing rice varieties with protein is a challenge to breeders as it is a quantitative character as well as associated with several negative characters. It is possible to break this linkage through developing recombinants. Most local lines and land races in rice have varying level of protein with low yield and inferior or non acceptable grain quality. By developing recombinants of high yield and elite popular varieties and local genotypes it is now possible to develop genotypes with acceptable grain quality, reasonably high yield and also with enhance total protein upto 1-12 percent as against the existing 7 percent. This would considerably reduce the protein deficiency by rice consumers. Hence breeding high protein rice varieties will help to overcome protein deficiency to some extent

We have already identified donor rice genotype from local rice with high protein and made crosses with argonomically superior rice variety. The F2 and the F3 genotypes as well as back cross genotypes have been generated.

1. Analysis of 2000 F2 segregating plants and F3 families for recombinants for high protein containing genotypes and superior yield.

2. Amino Acid profiling of selected high yielding and superior grain quality genotypes for lysine rich genotypes.

3. Bulk-segregant analysis of F2 DNA for DNA markers linked to increased total protein and lysine rich genotypes.

4. Developing BC3 genotypes with total high protein and superior in yield and lysine content using linked markers.

1. High protein genotypes with total high protein (11-12 percent), good grain quality and high grain yield.

5. Near isogenic Introgression lines (NIILs)for high protein in superior Agronomic background.

6. Recombinant inbred lines carrying QTLs for total high protein and grain quality

Dissection of components of gene action and identification of candidate genes governing grain micronutrient contents in finger millet (Eleusine coracana Geartn)

Principal Investigator: Dr. S. Ramesh,, Assistant Professor of Plant Breeding,,Bio-fuel Park, ARS, Madenur- 573220

Co-Principal Investigators: 1. Dr. Jayarame Gowda, Geneticist (Small Millets), Project Coordinating cell All India Coordinated Small Millets Improvement Project,GKVK, Bangalore, Dr. A. Mohan Rao, Associate Professor of Plant Breeding Department of Genetics and Plant Breeding, GKVK, Bangalore

Micronutrient malnutrition, primarily the result of intake of diets poor in bio-available vitamins and minerals [Iron (Fe) and Zinc (Zn)] causing blindness and anemia (even death) especially among women and pre-school children, is prevent in south-east Asian countries including India (Underwood 2000). In most parts of these regions, people have poor purchasing power and access to diets rich in these two vital minerals (Fe and Zn). However, in these regions coarse cereals such as sorghum, pearl millet and finger millet

are consumed in large quantities on a daily basis. In Karnataka, especially in southern parts, finger millet is cultivated and consumed as a staple food by millions of people.

The introduction of finger millet varieties selected and/or bred for increased Fe and Zn contents through crop breeding approach will complement the existing approaches (intake of fortified foods, supplemented foods, pills etc.) to combat micronutrient malnutrition. The crop breeding approach unlike others would help avoid dependency on behavioral changes in target population while implementing programs to combat micronutrient malnutrition (Welch and Graham 2002).

The availability of genetic variability, knowledge on inheritance pattern and identification of candidate genes controlling grain Fe and Zn contents are pre-requisites for developing micronutrient-dense finger millet cultivars. Several studies have been initiated in staple coarse cereal crops such as sorghum (Reddy et al. 2005) and pearl millet (Velu et al. 2006) in this direction. However, such studies in finger millet are scanty.

Horticultural crops not only provide us with nutritional and healthy foods, but also generate a cash income to growers. Appropriate production practices, careful harvesting and proper packaging, storage and transport all contribute to the good produce quality. Once a crop is harvested it is impossible to improve its quality. The horticultural crops, because of their high moisture content are inherently more liable to deteriorate especially under tropical conditions. Moreover, they are biologically active and carry out transpiration, respiration, ripening and other biochemical activities, which deteriorate the quality of the produce.

Losses during post harvest operations due to improper storage and handling are enormous and can range from 10-40 percent. Post harvest losses can occur in the field, in packing areas, in storage, during transportation and in the wholesale and retail market. Severe losses occur because of poor facilities, lack of know-how, poor management, market dysfunction or simply the carelessness of farmers. Proper storage conditions, temperature and humidity are needed to lengthen the storage life and maintain quality once the crop has been cooled to the optimum storage temperature.

In a vast country such as India, transportation of fruits and vegetables across long distances requires extensive periods of storage. Unfortunately, most fruits and vegetables have a very short shelf life that leads to extensive losses during storage as well as transportation due to damages through over ripening. Due to absence of proper storage and marketing facilities, farmers are forced to sell their produces at throwaway prices. Sometimes farmers do not even get the two way transportation cost, so they would rather dumb their produce near the market area than bearing the transportation cost required for taking the produce back.

With the advent of biotechnological techniques resulting in transformation of crop plants with novel genes or gene constructs, avenues have been opened for reducing post-harvest losses and for improving the quality of horticultural crops. The main focus here is to prevent and minimize losses due to over-ripening, physical damage, chemical injury and pathological decay.

The biotechnological methods for delayed ripening can be adopted to extend the shelf life of perishable horticultural crops. Genetic manipulation for long life, diseases and environmental-stress resistant cultivars will help the poor farmers to get higher price with reduced post harvest losses. The estimated area and production of tomato for India are about 3, 50,000 hectares and 53, 00,000 tons respectively.

A. Title: Isolation of novel antifungal gene and expression of the gene to increase the post harvest shelf life of Tomato.

Co-PI: Dr.K.M.Harini Kumar, Associate professor, Department of Biotechnology

Tomato is the world’s largest vegetable crop and known as protective food both because of its special nutritive value and also because of its wide spread production.. As it is short duration crop and gives high yield, it is important from economic point of view and hence area under its cultivation is increasing day by day.

In Karnataka tomato is grown in large area and there is glut in the market during June –July. Farmers are under loss as they can not preserve the fruits for long time due to spoilage by fungal and bacterial diseases. The phenomenon of suicides amongst farmers in Karnataka has been a recurrent theme in the agricultural sector due to crop losses by pests, diseases, price fall and post harvest losses.

Most of the genes used for transformation of plants for disease and pest resistance are all imported. These genes have patented by the organizations or scientists. Hence there is a need to isolate our own genes for disease and pest resistance. We have preliminary data to show that one of the horticultural crop, high degree of resistance to fungal diseases were recorded even after keeping the crop produce for several months and the other variety is highly susceptible. Hence we are isolating this gene by subtractive hybridization.

The endohydralase chitinase catalyzes the hydrolysis of chitin a beta 1,4, linked polymer of N-acetyl-D- glucose amine and Glucanase catalyzes the hydrolysis of beta 1,-3 glucan. There by causing the death of the pathogen.

In India, no transgenic plants for increasing the shelf life of Horticultural crops have been introduced by transgenic technology. In other countries it is demonstrated that the constitutive co-expression of tobacco Chitinase and B-1,3-glucanase genes in tomato plants confers higher levels of resistance to a fungal pathogens that either gene alone, indicating a synergistic interaction between the two enzymes in vivo.

E. Objectives: -

I. Collaborating institutions/Scientists: ICRISAT, Hyderabad, IIHR, Hesaraghatta, Bangalore

B. Title: Nutritional Improvement and Enhancement of Shelf life of Tomato, Onion and Potato by Lactic Acid Bacteria

PI: Dr. Suvarna, V. Chavannavar, Associate Professor, Dept. of Agril. Microbiology

Co-PI: Dr. D, Radhakrishna, Professor and Head, Dept. of Agril. Microbiology

Annual post harvest losses in tomato, potato and onion range between 40 -50 % of the produce. This loss can be reduced considerably by using Lactic acid bacteria (LAB) for preservation. The rationale behind exploiting LAB in bio-preservation involves multifold benefits like –1. Value addition to the product by their probiotic properties like vitamin production, reducing the antinutritional factors, production of antimicrobial compounds etc., 2. Ecofriendly Approach by avoiding chemical preservatives 2. Less expensive.

Nutritional improvement of vegetables and their products especially vitamins and minerals can be achieved using LAB for fermentation. Lactic acid bacteria can also be

exploited in bio-preservation to increase the shelf life of these vegetables, as these organisms are known to produce large number of antimicrobial compounds.

Generally, 40 – 50 % of the produce gets wasted in India due to the post harvest spoilage due to the lack of proper facilities for preservation of vegetables like tomato, potato and onion. Even if preservation is attempted, it is chemical preservation. Chemical preservation is becoming an obsolete technology due to the demerits associated with chemicals. Recent trend in the market is to opt for organic products. Hence, the need for bio-preservation is felt. Bio-preservation using cider vinegar is becoming popular. Use of fermented fruit juices is also becoming popular among the public who are health conscious. Bio-preservation may compensate for 20 - 25 % of reduction in annual post harvest loss. At the same time lactic acid bacteria can be exploited for improvement of the nutritional status of the society, as they are known to enhance the nutritional value of the vegetable or their products. Employing lactic acid bacteria either in fermentation or in preservation can easily attain bioavailability of minerals especially Fe and Zn. The major health problem associated with women and children. is prevalence of anemia due to Fe deficiency. This situation can be easily managed by trapping the sources that can assist in bioavailablity of minerals and lactic acid bacteria happens to be a promising source.

1. Isolation, characterization and identification of lactic acid bacteria from vegetables viz, tomato, potato and onion collected from different places of Karnataka.

2. Standardization of formulations of lactic acid bacteria for improvement of shelf life studies of vegetables.

4. To analyze for the nutrients like vitamin B, C and minerals Fe and Zn in the vegetables and their fermented products.

5 To evaluate the technology in terms of shelf life, economics and nutrient contents of the product.

Sunflower (Helianthus annuus L.) is one of the oil seed crop species and Sunflower acreage is in increasing trend in India. Commercially available sunflower varieties contain from 39 to 49% oil in the seed. The oil accounts for 80% of the value of the sunflower crop, as contrasted with soybean which derives most of its value from the meal. Sunflower oil is generally considered a premium oil because of its light color, high level of unsaturated fatty acids and lack of linolenic acid, bland flavor and high smoke points. The primary fatty acids in the oil are oleic and linoleic (typically 90% unsaturated fatty acids), with the remainder consisting of palmitic and stearic saturated fatty acids. Oleic acid is a monounsaturated omega-9 fatty acid found in various animal and vegetable sources. It has the formula C₁₈H₃₄O₂ (or CH₃(CH₂)₇CH=CH(CH₂)₇COOH). The saturated form of this acid is stearic acid. The primary use is as a salad and cooking oil or in margarine. In India sunflower is the preferred and the most commonly used oil. OLEIC ACID (a monounsaturated fatty acid) content of oilseedshas important implications for product performance and consumerhealth.

High oleic sunflower oil (over 80% oleic acid) was developed commercially in 1985 and has higher oxidated stability than conventional oil. It has expanded the application of sunflower oils for frying purposes, tends to enhance shelf life of snacks, and could be used as an ingredient of infant formulas requiring stability. Oil from genetically modified sunflowers has increased stability against oxidation, giving them a longer shelf life, as well as having an improved health profile, suggests new research.

"Genetic modification of sunflower oil, to decrease linoleic acid and increase oleic acid, could increase the oxidative stability during storage and deep fat-frying, as well as improve the health benefits,"

By reducing the linolenic acid content the shelf life can be extended and numerous studies in the literature show that trans fatty acids raise serum levels of LDL-cholesterol, reduce levels of HDL-cholesterol, can promote inflammation, can cause endothelial dysfunction, and influence other risk factors for cardiovascular diseases (CVD).

The most frequent fatty acid composition in sunflower oil is: 55-65% of linoleic acid, 20-30% of oleic acid, and the remainder including other fatty acids, primarily palmitic and stearic.

Inheritance of high oleic acid content has been studied. The efforts made in the past for enhancement of quality oilseeds through conventional plant breeding and crop husbandry techniques have been quite successful. It is now being increasingly felt that, in certain cases, modern molecular biological approach may prove to be more effective and rapid in delivering the desirable results. The approach here is Marker Assisted Breeding (MAS) for the polygenic traits or those traits for which the metabolic pathways and the candidate gene are yet to be discerned.

The objectives of this study were to examine a set of sunflower genotypes (including

CMS, R and Inbred lines) for mode of inheritance, combining ability and gene effects controlling the contents of oleic and linoleic acids. And to develop sunflower variety with high oleic acid content in oil.

Sunflower presents an array of oil quality improvement aspects to be dealt with the biotechnological interventions that have been identified for sunflower for enhancement of quality are described below.

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Adnan Kanbar, Chandrashekara, M., Manjunatha, K., Vinod, M. S., Hittalmani, S., Janamatti, M. and Shashidhar H. E. 2004. Molecular markers for root morphological traits under low-moisture stress using transgressant backcross of rice (Oryza sativa L.) Indian Journal of Genetics & Plant Breeding 64 (3): 185-188.

Adnan Kanbar, Janamatti, M., Sudheer, E, Vinod, M. S. and Shashidhar, H. E. 2006. Mapping QTLs underlying seedling vigor traits in rice (Oryza sativa L.) Current Science 90 (1): 24-26.

Shankar A. G., Geetha K. N., Yamunarani B. R. and Nagarathna T. K. (2006). Zinc status in commonly consumed grains and vegetables: A molecular and fortified food processing techniques to enhance the content and bioavailability. Paper presented

Pp: 21 International Conference on BIOTECHNOLOGY APPROACHES FOR ALLEVIATING MALNUTRITION AND HUMAN HEALTH. Bangalore, India

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Yamuna Rani, B. R. and Shankar, A. G. (2006). Probing the differential Zinc uptake and translocation among contrasting finger millet genotypes- Zn65 studies. Pp:23 National seminar on plant physiology, Indian Society for Plant Physiology, New Delhi, India

Agarwal M., Vijayakumari J., Shankar A. G. and Rajeshwari Y. B. (2005). Bioavailability studies on Zinc Using Various Post-harvest processing Techniques. Paper presented in ICFOST 2005, NIMHANS Convention Centre, BANGALORE-Pp:19 ICFOST 2005.

Khin Myat Lwin, P.H.Ramanjini Gowda, S.Chandrashekar, T.K.S. Gowda, M.Vasundhara, Nagesha, N., U.P.Virupakshagouda, S.Sreenevasa, and T.Manjunatha. (2006), In vitro regeneration and transformation of Coleus forskohlii with glucanase-chitinase gene. Journal of Non-Timber Forest products, 13(3): 193-197.

Khin Myat Lwin, Ramanjini Gowda,P.H.,T.K.S.Gowda, Nagesha, N., Sreenivasa,S., Manunatha,T., Virupaksha U Patil, Vasundhara,M. and Chnadrashaker,S.C.2004. “In Vitro regenration and transformation of Coleus forskohlii with glucanase-chitinase gene. Proceedings of ICAR National Symposium on Biotechnological Interventions for improvement of Horticultural Crops: Issues and Strategies, Thrissur, Kerala(India). Page No. 340-342.

Krishna Murthy, K., Ramanjini Gowda, P.H., Gowda, T.K.S., and Prakash M.K., 2003. Transformation of groundnut (Arachis hypogaea L.) using BAR with GUS gene through Agrobacterium tumefaciens. Mysore J. Agric. Sci., 37(4) 337-343.

Suvarna, C.V., 2005. Lactic acid bacteria in fermented foods –idly and pickles: their antimicrobial properties on growth and activities of faecal indicator and food poisoning bacteria. Ph. D thesis Submitted to Univ. of Agril. Sciences, GKVK, Bangalore Reddy, B.V.S., Ramesh, S. and Longvah, T. 2005. Prospects of Breeding for Micronutrients and ί-Carotene-Dense Sorghums. International Sorghum and Millets Newsletter, 46: 10-14.

Underwood, R. A. 2000. Overcoming micronutrient deficiencies in developing countries: Is there a role for agriculture? Food and Nutrition Bulletin, 21 (4): 356-360.

Velu, G., Kulkarni, V.N., Muralidharan, V., Rai, K.N., Longvah, T., Sahrawat, K.L. and Raveendran, T.S. 2006. A Rapid Screening Method for Grain Iron Content in Pearl Millet. International Sorghum and Millets Newsletter. 46: 10-14.

Welch, R.M. and Graham, R.D. 2002. Breeding crops for enhanced micronutrient contents. Plant and Soil, 245: 205-214.

Thematic area: Exploiting the potential of Bio-farming in the production of useful pharmaceuticals

Infectious diseases have been a major cause of mortality all over the world, but more so in the developing countries. About 16.5 million people died globally owing to infectious and parasitic diseases, of which million belonged to the developing countries and most of these diseases are vaccine preventable diseases. The global vaccine market has grown at an average of 10 percent every year since 1990 from $2.9 billion to $ 6 billion in 2002.

Vaccines are primary tools in programmes of health intervention for both humans and animals. Fermentation based vaccines are very costly accompanied by low production yields. The purification steps require sterile conditions. Hence development of safe, efficient and inexpensive oral vaccines thus assumes strategic importance.

The recent spectacular advances in molecular biology and genetic engineering have thrown up one of the best alternatives: that is the production of recombinant protein in plants. The recent progress in the area of transgenic plants has however, once again attracted the attention of scientists and plants are being looked upon as potential bioreactors for the production of immunotherapeutic molecules.

The use of plants for recombinant protein production offers reduced capital costs relative to fermentation methods. The production can also be scaled up. Multemeric proteins, such as antibodies can be produced in the correct assembly in plants. The major advantage being, the plants do not serve as hosts for human pathogens.

The yeast is better system for expression of heterologus genes because its advantages over other systems like post translational modifications, glycosylation will be better in yeast expressed recombinant proteins. Also yeast system can be used to express relatively larger fragment, protein folding will be proper which is required for protein to be biologically active. Upon induction higher yields of recombinant protein can be obtained and purification of recombinant protein will be simple as the heterologus gene product will be excreted in the media which is helpful in easy purification of recombinant protein there by the cost of production and purification of protein will be low than other systems.

Rabies is one of the most fearsome diseases known to animals and mankind. Active immunization campaigns in North America and Europe have controlled rabies in dogs. However in India rabies is highly prevalent in dogs and is transmitted to humans through bite.

Rabies is acute fatal encephalomyelitis, which can be transmitted to man by contact with infected animals. It was one of the first human diseases recognized through out the world (Dreesen, 1997). World wide the number of human rabies deaths is estimated to be between 35,000 to 50.000 annually. The highest incidence is in Asia and India alone accounts for 30,000 deaths. Around 10 million people receive post bite treatment globally every year. The World Health Organization estimates that about 4 million people per year are given at least partial post exposure treatment globally. The rabies vaccine produced in cell culture methods have disadvantage like contamination and high cost. A cheap, easily available and effective rabies vaccine is the need of the hour for developing nations. Recombinant protein production in plants and yeast would yield a cheap vaccine for the need.

Another important disease next to HIV is the Hepatitis B, which is one of the major diseases of mankind and is a serious global public health problem. Of the 2 billion people who have been infected with the hepatitis B virus (HBV), more than 350 million have chronic (lifelong) infections which contribute about 5% of the total world population. About 6.0 lakh peopledie each year from HBV-related liver disease or hepatocellular carcinoma. HBV may be the cause of up to 80% of all cases of hepatocellular carcinoma worldwide, second only to tobacco among known human carcinogens. India has been placed into the intermediate zone of prevalence of hepatitis B (2-7% prevalence rates) by the WHO. In India, there are 43 million people who are estimated to be HBsAg carriers with a carrier rate of 3%, which contribute nearly 10% of the HBV carriers in the world.

Immunization with hepatitis B vaccine is the most effective means of preventing HBV infection and its consequences. As the available recombinant vaccine is expensive, there is still need for a less expensive vaccine source in most developing countries.

Diabetes is becoming something of a pandemic and despite the recent surge in new drugs to treat and prevent the condition; its prevalence continues to soar. Perhaps the most worrying aspect of all is that the rise is even reflected in children. There is an estimated 143 million people worldwide suffering from diabetes, almost five times more than the estimates 10 years ago. There are 2 main categories of the disease. Type I Diabetes mellitus also called insulin dependent Diabetes mellitus (IDDM) accounts for 5 – 10% of all diabetes cases but the incidence is rapidly increasing. Type II Diabetes mellitus is the non insulin dependent Diabetes mellitus.

Type I Diabetes mellitus / IDDM is an autoimmune disease in humans. Oral administration of antigens is a long recognised method for inducing systemic immunological tolerance and has been proposed as an approach for treatment or prevention of allergic and autoimmune diseases.

The Transmembrane glycoprotein is known to produce antibodies which can give immunization against rabies infection. Transgenic plants and yeast would be cheap source of such antibodies.

Plants are a potential source of HBsAg that is not dependent upon process technology to ensure protein folding and particle assembly. In addition, a plant based HBsAg expression system makes possible the testing of an oral immunization strategy by simply feeding the plant samples.

The development of transgenic plants expressing GAD65 will help in production of the autoantigen in large scale at less cost. This would help in the treatment of insulin dependent Diabetes mellitus which is an autoimmune disorder since one of the treatments in case of autoimmune disorder calls for the oral administration of the antigen so as to induce systemic immune tolerance to the autoantigen.

.Transformation studies have been carried with ERA strain of rabies glycoprotein gene in tobacco, muskmelon and coleus. Protein was isolated and purified from the transgenic plants, immunization studies were carried with mice.

Cloning of CVS strain of the rabies glycoprotein gene into Pichia pastoris is under progress in collaboration with NIMHANS ,Bangalore.

Standardization of transformation and regeneration of Coleus forskohlii and muskmelon with different genes by both Agrobacterium mediated and biolistic methods have been carried out. The leaf explants of Coleus forskohlii were transformed with hepatitis B surface antigen gene along with npt-II as an antibiotic selection marker gene. The presence of HBsAg gene in putative transformants was confirmed by PCR and southern blot analysis.

Generation of transgenic potato plants that synthesise human insulin, a major insulin dependent Diabetes mellitus autoantigen, at levels upto 0.05% of total soluble protein has been reported by Takeshi Arakawa et al.,(1998). To direct the delivery of plant synthesised insulin to gut associated lymphoid tissues insulin was linked to the C- terminus of the cholera toxin B subunit (CTB). Non obese diabetic mice fed with transformed potato tuber tissues

containing microgram amounts of the CTB-insulin fusion protein showed a substantial reduction in pancreatic islet inflammation (insulitis) and a delay in the progression of clinical diabetes.

Human GAD65, the smaller isoform of the enzyme glutamic acid decarboxylase is a major autoantigen in human insulin dependent Diabetes mellitus. Andrea Porceddu et al.,(1999) have reported the generation of transgenic tobacco and carrot with GAD65 where the expression levels were between 0.01% and 0.04% of total soluble proteins.

At our lab standardization of transformation of Coleus forskohlii and muskmelon with different genes by both Agrobacterium mediated and biolistic methods have been carried out.

Sl. No	Position and no	Consolidated Emolument	Year 1	Year 2	Year 3	Total
1	Research assistant-6	16, 000 per month + 13 % HRA	1301760	1301760	1301760	3905280
2	Senior Research Fellow-6	12, 000 per month + 13 % HRA	976320	976320	976320	2928960

2.2 Consumables

PATENTS:

36/MAS/2003. Dr.P.H.Ramanjini Gowda,Department of Biotechnology, Univesrity of Agricultural Sciences, Bangalore, Improved Rabies vaccine production in plants. The Gazette of India,September,6,2003 (Part III-Sec2)".

Nagesha, N and Ramanjini Gowda, P.H. (2006), a comparison of ERA rabies strain Nucleotide and Deduced amino acid sequences with the different strains of rabies virus for sequence homology. Research Journal of Biotechnology, 1(2): 26-30.

Nagesha, N., P. H. Ramanjini Gowda, S. N. Madhusudana, J. Lokesh,N. J. Vinay, K. Michelle, B.N. Devaiah, R. Madhuvanthi,vanikulkarni,S. Saraswathi, A. N. Dinesh, T. K. S. Gowdaand K. Mehamooda.Genetic transformation of cantaloupe melon (Cucumis melo L.) with the rabies virus glycoprotein gene (PRGSpRgp) and immunization studies in mice Journal of Horticultural Science & Biotechnology (2007) 82 (3) 383–386

Khin Myat Lwin, P.H.Ramanjini Gowda, S.Chandrashekar, T.K.S. Gowda, M.Vasundhara, Nagesha, N., U.P.Virupakshagouda, S.Sreenevasa, and T.Manjunatha. (2006), In vitro regeneration and transformation of Coleus forskohlii with glucanase-chitinase gene. Journal of Non-Timber Forest products, 13(3): 193-197.

Ramanjini Gowda, P.H. Madhusudana, S.N., Dinesh, A.N.,Devaiah, B.N., Nagesh.N,Madhuvanthi. R, Saraswathi.S, Prakash, C.S., and Gowda, T.K.S. Expression and immunogenicity of Rabies glycoprotein produced in tobacco and muskmelon plants by Agrobacterium mediated transformation. Proceedings of the 10^th IAPTC&B congress, Plant biotechnology and beyond. June 23-28, 2002, Orlando, Florida, USA. Abstract No. 1153.

Ramanjini Gowda, P.H., Madhusudhana, S.N., Shilpa R., Devaiah, B.N., Nagesh, N., Dinesh, A.N., Saraswathi and Gowda, T.K.S 2002 March 1-2. Studies on biochemistry and amino acid profiles of rabies glycoprotein produced in transgenic crop plants. Book of

abstracts of the 4^th National symposium on Biochemical Engineering and Biotechnology, IIT, New Delhi, India

Madhuvanthi,R., Ramanjini Gowda, P.H, Madhusudan,S.N.,Saraswathi., Nagesh,N., Devaiah, B.N., Krishnamurthy,G.V., and T.K.S. Gowda. Comparison of rabies glycoprotein produced in transgenic plants with the rabies virus glycoprotein. Proceedings of the international symposium on Molecular approaches for improved crop productivity and quality, Coimbatore, India, May22-24. 2002.

Khin Myat Lwin, Ramanjini Gowda,P.H.,T.K.S.Gowda, Nagesha, N., Sreenivasa,S., Manunatha,T., Virupaksha U Patil, Vasundhara,M. and Chnadrashaker,S.C.2004. “ In Vitro regenration and transformation of Coleus forskohlii with glucanase-chitinase gene. Proceedings of ICAR National Symposium on Biotechnological Interventions for improvement of Horticultural Crops: Issues and Strategies, Thrissur, Kerala(India). Page No. 340-342.

Nagesha, N., Devaiah, B.N., Ramesh,B.N. and Ramanjini Gowda,P.H.2004. In vitro Micro propagation and transformation of Cantaloupes (Cucumis melo L) with GUS gene through Agrobacterium mediated transformation. Proceedings of ICAR National Symposium on Biotechnological Interventions for improvement of Horticultural Crops: Issues and Strategies, Thrissur, Kerala(India). Page No. 343-345.

RamanjiniGowda.P.H .Madusudana.S.N., Dinesh.A.N and Gowda.T.K.S. 2000, Production of Rabies vaccine in Tobacco plants by Agrobactrium mediated transformation, Journal of APCRI, vol. 1,Issue 2,2000

Nagesha, N and Ramanjini Gowda, P.H. (2006), Comparison of homology of sequences between different strains of rabies Glycoprotein gene. Abstract published in the International Conference on Biotechnology approaches for alleviating malnutrition and human health, 9^th -11^th June, PP 94.

Nagesha, N., Virupakshagauda U Patil., Sumita kumari, P., Ramya,B., Kumaraswamy, G.K., Ramachandra, R and Ramanjini Gowda, P.H.(2006), Production of recombinant

proteins in plants. Abstract published in National Seminar on Gene constructs, May 17-18, 2006, IIHR (Indian Institute of Horticulture Iresearch, Bangalore, PP-58.)

No of students worked on vaccine production under the guidance of Dr.Ramanjini Gowda; M.Sc. – 15, Ph.D- 1

Thematic area: Microbial biotechnology to prospect the rich microbial biota of the State for green energy and novel trait specific genes

Microbial biotechnology is the use of living organisms or their products to modify soil ecosystem function to provide higher and the human environment Microbial biotechnology is growing rapidly through the invention of new technologies such as genomics, transcriptomics, and proteomics .These genomic data are now exploited in thousands of applications, ranging from those in medicine, agriculture, organic chemistry, public health, biomass conversion, to biomining. Microbial Biotechnology focuses on important issues related to agriculture to have an in-depth analysis of the critical input applications leading to sustainability and adoptability in the fields of energy, food safety, nutrient availability, soil and plant health, wastewater treatment and recycling. The marked increase in understanding of these organisms and their cell products gains us the ability to control the many functions of various cells and organisms. Using the techniques of gene splicing and recombinant DNA technology, we can now actually combine the genetic elements of two or more living cells. Functioning lengths of DNA can be taken from one organism and placed into the cells of another organism. As a result, for example, we can cause bacterial cells to produce human molecules. Cows can produce more milk for the same amount of feed. And we can synthesize therapeutic molecules that have never before existed. An understanding of the scientific principles behind fermentation and crop improvement practices has come only in the last few decades guiding natural processes to improve agricultural productivity and the farmers economic well-being. Harnessing Microbial processes for crop productivity and soil health is very important technology ,genes extracted from microorganisms for effective in converting biomolecules into beneficial chemical molecules , had proved possible to generate certain key industrial chemicals (glycerol, acetone, and butanol) using bacteria., of large-scale sewage purification systems based on microbial activity..

In the University of Agricultural Sciences, Bangalore research in these areas are being carried out. Under green energy programme the biomass conversion to ethanol particularly with crop residues and food processing wastes have been initiated. Improvement of biofuel crops like sweet sorghum is taken up for high sugar content required for alcohol production.

The microbial systems are exploited for potential genome for application to improve nutrient mobilization, solubilization through molecular techniques, such as introduction of P solubilising gene into vectors like E.coli and Rhizobium. Significant work is being carried out in Bt genes and in development of of Transgenic plants.

Biodiversity of below ground ecosystem is studied particularly with reference to the beneficial microorganisms and its appropriate application in crop production.

The three main areas under microbial biotechnology are: (A) Green energy (B) Microbial systems as source for genes for nutrient acquisition and insecticidal toxin proteins (C) Below ground biodiversity

Green energy is a term describing the environmentally friendly sources of power and energy. Basically this refers to renewable and non-polluting energy sources. Green energy includes natural energetic processes that can be harnessed with little pollution. Under this category the important are cellulose digestion, anaerobic digestion, power generation by microbial cells, including solar power, biomass power, tidal power and other sources of energy.

Energy crops will allow farmers to use more of their under utilized land productively.

The reason for the renewed interest in ethanol is its pollution free environment when used in both transport and non-transport sectors. Since ethanol contains oxygen, using it as a fuel blend reduces carbon monoxide emissions. This ethanol blend called Gasoline, E-2 to E-10 depending on the percentage of ethanol in the blend, containing up to ten percent ethanol is widely used, results in up to 25 percent fewer carbon monoxide emissions than conventional gasoline. Using ethanol can also reduce total carbon dioxide emissions. Ethanol is made from crops that absorb carbon dioxide and give off oxygen. This carbon cycle maintains the balance of carbon dioxide in the atmosphere when using ethanol as a fuel. As new technologies for producing ethanol from all parts of plants and trees become available and economical, the production and use of ethanol should increase in a large scale.

B. Microbial systems as source for genes for nutrient acquisition and insecticidal toxin proteins:

All most all the soils in our country are reported to be deficient in nutrient availability to crops. Hence, supply of nutrient source to soil is inevitable. Most of the nutrient sources used for crop production is imported since we do not have fertilizer grade nutrient deposits in the country. Several soil microorganisms are known to minaralise and mobilize a variety of nutrient complexes in the soil through elaboration of a variety of enzymes. Such microorganisms are presently being used as Biofertilizers to improve nutrients such N & P availability to crops from the soil. Genes responsible for production of Citric acid (Citrate synthase), Gluconic acid (pqq synthase) have been isolated from P solubilizing bacteria and are cloned and expressed in non P solubilizing bacteria, and also in plants through transgenic approach. These transgenic bacteria and plants are reported to perform better in P deficient soils utilizing insoluble P sources present in the soil (viz. Al.-P).They were also found to help the plant to withstand Aluminium and other heavy metal toxicity. Microbial like AM fungi are involved in Phosphorus nutritents along with plant growth promotion and Biocontrol activities. They have the potential to increase the nutrient mobilization through Phoshate trans porter genes.

The microorganisms produces various toxins commonly termed as Toxin complex (Tc) which then kills the insect which facilitate the multiplication of bacteria and nematode. This project aims to study the extent variability with respect to the pathogenicity to the insect as well as the at the molecular level by the subjecting the bacterium to AFLP analysis . These bacterial cultures will also be subjected to intensive screening for various antibacterial

and anti fungal characteristics especially with respect to the plant pathogens. Attempts will be made to isolate the Tc genes of the bacterium and compare it with the existing sequences available in database to determine the variability of these genes. In order to be one step ahead in our fight with insects it is necessary to explore novel genes that has not yet been exploited. As it is present in the gut of nematodes that infect insects, it highly successful in destroying the insect in to which it enters.

Soil is the habitat of a diverse array of soil organisms, bacteria, fungi, protozoa and invertebrate animals– the activities of which contribute to the maintenance and productivity of agro-ecosystems by their influence on soil fertility. Due to intensification in agriculture, the biological regulation of soil processes is altered with loss in soil biodiversity. The assumption is often made that the consequent reduction in the diversity of the soil community and eventual extinction of species may cause a catastrophic loss in function and reduces the ability of agricultural systems to withstand unexpected periods of stress.

The soil organisms contribute to the soil fertility and hence to the ecosystem productivity and sustainability. Because of the great diversity at the species level, for purposes of studying the significance of biodiversity in ecosystem functions they have been grouped as N₂-fixing organisms and AM fungi, associated with nutrient acquisition; Earthworms, Termites and Nematodes, the groups of soil fauna which most strongly influence comminition, decomposition of organic matter and nutrient redistribution and bioturbation; and taxonomically – broad groups of the other fungi and bacteria have primary roles in decomposition and recycling. There is a widely accepted working hypothesis that there is high degree of functional redundancy within many functional groups and that this varies relative to the degree of specialization exhibited.

Biomass is the major resource available that needs to be biologically treated for production of bioenergy particularly ethanol to application in transport and non-transport sectors. As microbial trains have more diversity they can be isolated from diverse natural habitats like fruit surface, nectar rich flowers, soil and Insects. But microbial strains isolated from sugar cane juice are known to tolerate to high ethanol concentrations. This property is very useful for microbial strains to be utilization in the production of bioethanol. Therefore microbial strains from different environments are efficient for production of bioethanol.

Biomass is the form tuberous vegetables such as, potato, tapioca and sweet potato; of cellulosic and pectin material from urban solid wastes with high proportion of fruit and vegetable matter can be efficiently converted into a fermentable liquid by developed bacterial and fungal cultures into final product, the ethanol. This ethanol can be used wholly or as a blend and reduce the environmental pollution and depending on fossil fuels. Naturally cellulose producing microbes can easily hydrolyze cellulose present in crop residues. In combination of hydrolyzing cellulose and production of ethanol.

PROJECT -I

Biomass in the form tuberous vegetables such as, potato, tapioca and sweet potato; of cellulosic and pectin material from urban solid wastes with high proportion of fruit and vegetable matter can be efficiently converted into a fermentable liquid by developed bacterial and fungal cultures into final product, the ethanol. Nearly 25 percent of the produce are obtained as post harvest wastes that can be utilized economically. This ethanol can be used wholly or as a blend and reduce the environmental pollution and depending on fossil fuels. The current production of ethanol is in the range of 8% - 10a%. The microbial improvement is needed for increase in the concentration of fermentable liquor and finally to ethanol production in the range of 15% - 20%.

CO.PI: Dr. RAMANJINI GOWDA, Professor and Head, Department of Biotechnology.

It is important that the yeast and other microbial strains used be able to survive the highest ethanol concentration production. For beer these concentrations range in 3-9%, for grape wine 11-15% and for honey wine up to 17% . Alcohol tolerance is particularly important for alcoholic fermentation, while production for fuel. We need to have microbes with a tolerance more than 25%.

FUNCTIONAL HETEROGENEITY AND GENETIC DIVERSITY OF SACCHAROMYCES CEREVISIAE, ISOLATED FROM THE NATURALLY FERMENTING FRUITS OF WESTERN-GHATS OF KARNATAKA

Production of Saccharomyces cerevisiae biomass is an economically important process. Industrial strains of S. cerevisiae are used by many food companies as starters for fermentative processes in the making of bread, wine, beer, and other alcoholic beverages. The technological characteristics of commercially produced yeast determine, to a high extent, the quality and fermentation performance of those processes. Several studies have evaluated the energetic, kinetic, and yield parameters of the yeast biomass production. Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, the cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation.

MICROBIAL SYSTEMS AS SOURCE FOR GENES FOR NUTRIENT ACQUISITION AND INSECTICIDAL TOXIN PROTEINS:

Arbuscular mycorrhizal fungi is commonly used for plant growth and biocontrol activities. The plant growth promotion activities is due to the increased root surfaces in the inoculated plants. As a result of this increased biomass and surfaces area it can absorb the nutrients from larger area in the soil. Among many of the nutrients absorbed Phosphorus is one major nutrient that mycorrhiza transport to the plant. In addition the AM fungi also retains some amount of moisture which helps in drought tolerance. The AM fungi cannot be grown with out the host plant.

B.HYPOTHESIS: A M fungi is known to improve the plant growth in general however there are reports that strains of AMF vary greatly in their ability to improve the plant growth. Under this project it is envisaged to collect different dominant strains of these fungi and test it on the

APPLICATION OF AM FUNGI OF DIFFERENT AGROCLIMATIC ZONES OF KARNATAKA ON PLANT GROWTH AND MOLECULAR CHARACTERIZATION OF THEIR PHOSPHATE TRANSPORTER GENES

The current status of the AMF research is that few randomly isolated strains of the AM fungi are tested on plants for their response. It is there fore necessary to carry out a systematic and specific work in order to select a suitable strain for the crop in Karnataka. There are also some studies on the P transporter genes in AMF and is comparison with other P transporter genes. It would also be very important work if the variations in the P transporter genes are determined and efficiency can be correlated with the differences in genes.

CLONING AND CHARACTERIZATION OF PHOSPHATE SOLUBILIZING GENE/S TO IMPROVE PHOSPHORUS UPTAKE OF CROPS THROUGH TRANSGENIC APPROACH.

Several P solubilizing bacteria are isolated from soil and rhizosphere samples which are capable of solubilizing different insoluble P sources (i.e. Di-Calcium, Tri-Calcium and different low grade Rock phosphates available in India.) Recently we have been able to isolate a gene namely Glutamine synthase from a P solubilizing bacteria, (Serratia marscesans) and the DNA is sequenced. Through complementation studies using genomic DNA library of the wild, the mutant lacking P solubilizing ability due to lack of production of Gluconic acid, was possible to restore the P solubilizing ability in the mutant. The above was confirmed by HPLC analysis for Gluconic acid production in the mutant complimented with genomic DNA from the wild strain.

ISOLATION AND BIOCONTROL APPLICATION OF NOVEL TOXIN COMPLEX GENES FROM PHOTORHABDUS LUMINESCENS

In order to be one step ahead in our fight with insects it is necessary to explore novel genes that has not yet been exploited. As it is present in the gut of nematodes that infect insects, it highly successful in destroying the insect in to which it enters.Insects are killed by the toxins produced by the bacteria Photorhabdus and Xenorhabdus spp . It is similatr to the Bt toxin genes only that it has not been explored. The Toxin complex genes in the bacterial genome are responsible for the production of the toxins. These genes will be isolated and cloned into other bacterial system for its expression.

The Bt genes have become the mainstay of biotechnology. Various crops are being developed with Bt genes. It is necessary to isolate newer and different Bt. Genes and incorporate it into plants to reduce the use of pesticides.The soils were collected from different parts of Karnataka including western Ghats. The Bt strains were grown on the medium amd isolated the cry proteins and tested on different types of insects for their specificity. The cry genes obtained from IARI were used to transform Dolichos and other crop plants for resistance against lepidoptern pests.

Rationale: The Bt cry proteins known to adhere to the receptors of the insect gut and the protoxin is cleaved and the protein is dissolved in the alakaline medium of the insect gut and septicemia occurs.

Hypothesis: The cloning and transformation of Bt genes resulted in plants reistant to pests. Bt cotton grown in India are resistant to cotton bollworm.

THEMATIC AREA –III: BELOW GROUND BIODIVERSITY

BELOWGROUND BIODIVERSITY IN SUSTAINABLE AGRICULTURAL SYSTEMS

PI: Dr. A.N. BALAKRISHNA

CO-PI: Dr. BALAKRISHNA GOWDA

Rationale: Soil is the habitat of a diverse array of soil organisms, bacteria, fungi, protozoa and invertebrate animals– the activities of which contribute to the maintenance and productivity of agro-ecosystems by their influence on soil fertility. Due to intensification in agriculture, the biological regulation of soil processes is altered with loss in soil biodiversity. The assumption is often made that the consequent reduction in the diversity of the soil community and eventual extinction of species may cause a catastrophic loss in function and reduces the ability of agricultural systems to withstand unexpected periods of stress.

Hypothesis: The maintenance and enhancement of soil biodiversity may be particularly critical for sustainable agricultural systems.

Project X:

Isolation of Beneficial Microflora from Weed Rhizosphere to improve nutrient acquisition in crop plants.

RATIONALE:

Weeds are wild plants growing as competitive among the crops and many of them possess the capacity to grow even under adverse environmental conditions. Most of the weeds possess special characters like drought resistance, prolific multiplication in different agro-eco systems, capacity to regenerate from dead or dormant parts there by it encourages the growth and development of many micro organisms in their rhizosphere. The development of rhizosphere microflora varies with the root secretions of the crop and type of soil (Schippers et al., 1987; Weller,1988). In this context, analysing the rhizosphere microflora of such weeds and isolation and characterization of beneficial flora will be of immense use for their exploitation in crop production especially under adverse situations like drought and saline conditions (Subba Rao, 1997).

Weeds help in keeping the soil structure and texture besides, providing congenial environment for microbial activity. Bowen and Rovire (1976) reported that microbial colonization was more near the plant roots due to rhizosphere effect. Several factors such as the soil type, moisture, pH, availability of nutrients, temperature, age and condition of the plants are known to influence the microbial growth in the rhizosphere. Bhuvaneshwari and Subba Rao (1957) and Bangar (1990) reported that root exudates like amino acids, sugars and various growth promoting substances secreted by the plant will have a profound influence on building up of various types micro-organisms in the rhizosphere.

The availability of Agriculturally beneficial microorganisms tolerant to drought, acid and saline conditions is very meager and needs in depth study to bring out efficient strains under such adverse situations.

This project will be of immense use in exploring highly competitive and efficient nutrient mobilizing micro organisms which can be efficiently utilized in crops of Agriculture, Horticulture, Sericulture and in Silivicultural plants. The isolated strains may be of better adaptation to adverse situations like drought , salinity etc. and may be of much use in Agricultural crops.

1. A.T. Pereira, 1990, Endorhizosphere bacteria of wetland rice, their H2-dependent Chemolithotrophy, N2-fixation, P Solubilization and interaction with Rice genotypes.

2. H.S. Gopala Gowda, 1989, Studies on the occurrence and distribution of H2-dependent Chemolithotrophic Rhizobia of Glycine (Neonotonia wightii, Lackey) in different agro climatic zones of Karnataka and their symbiotic efficiency.

3. N.G Lakshminarayan, 1991, Studies on Phosphate solubilizing fungi associated with Vascular-Arbuscular Mycorrhizal Inocula.

4. H. Gireesh Kamath, 1993, Studies on Phosphate solubilizing bacteria associated with wet land rice.

5. S. Suresh, 1995, Studies on phosphate solubilizing bacteria and their plasmid profile.

6. Suhas B. Nimbalkar.1999, Characterization of Tn-5 mutants for P Solubilization and requirement of pqq.

7. S.H. Prakash, 2000, Isolation and molecular characterization of P solubilizing bacteria.

8. Jayalakshmi, 2001, Development of transgenic tomato using citrate synthase gene for improving phosphorous use efficiency.

9. S.D. Yogesha, 2001, Studies on transformation of citrate synthase gene to Indica rice cultivars and tobacco employing Agrobacterium mediated transformation technique.

10. Amit Kumar Singh,2002, Development of transgenic groundnut with citrate synthase gene through Agrobacterium mediated transformation for improving phosphorous use efficiency,

11. 11. S.B. Rachappa, 2004, Development of transgenic rice with citrate synthase gene and studies on citrate synthase gene in R1 transgenic tobacco plants.

12. A.S. Brijesh, 2004, Isolation and molecular characterization of Glutamine synthase gene of Serratia marcesans involved in the Solubilization of inorganic phosphates.

13. A. Radhakrishna, 2004, Studies on molecular biology of phosphate Solubilization in bacteria.

14. Earanna. N., Suresh. C.K., and Bagyaraj D.J.1999. VA mycorrhizal selection for Coleus barbatus. – Journal of Soil Biology and Ecology. 19: 20-24

15. .Jayanthi S, Mamatha G, Sudarshan L and Suresh C.K. (2001) Microbial profile of sandalwood plants in lateritic soil of Bangalore.- Journal of Medicinal and Aromatic Plant Sciences.23: 605-608.

16. Mamatha G, Jayanthi S, Bagyaraj D.J and Suresh C.K. (2001). Microbial and enzymatic analysis from sandal root zone soil growing in red sandy loam. - Indian Journal of Microbiology. 41: 219- 221.

17. Jayanthi S, Mamatha G, and Suresh C.K. (2001). Analysis for microbial and biochemical parameters from sandalwood rhizosphere of Mysore. - Indian Phytopathology ,54: 171 –174.

18. Earanna. N., Tanuja. B.P., Bagyaraj, D.J.and Suresh, C.K. 2001. Vesicular arbuscular mycorrhizal selection for increasing the growth of Rauwolfia tetraphylla. Journal of Medicinal and Aromatic Plant Sciences. 24: 695-697

19. Earanna. N., Suresh. C.K., Mallikarjunaiah, R.R. and Bagyaraj D.J. (2001). Response of Coleus aromaticus to VA mycorrhiza and PGPR– Journal of Spices and Aaromatic crops. 10:141-143.

20. Mamatha, G.Shivayogi, M.S.Bagyaraj, D.J. Suresh, C.K. (2001). Variations in microbial load and their activity in Sandalwood growing in Hilly zone of Karnataka. In “Bioinoculants in sustainable agriculture and forestry ”. Ed. S.M. Reddy Scientific Publishers . Jodhpur 119-123

21. Jayanthi S, Mamatha G, and Suresh C.K. (2001). Analysis for microbial and biochemical parameters from sandalwood rhizosphere of Mysore .- Indian Phytopathology ,54:171 –174.

22. Bagyaraj, D.J.,Mehrotra, V.S.and Suresh, C.K. (2002). Vesicular arbuscular mycorrhizal biofertilzer for tropical forest plants. In: Biotechnology of Biofertilizers Ed. S.Kannian Narosa Pub. House N.Delhi. 299-311

23. Suresh, C.K. and Bagyaraj,D.J. (2002). VA mycorrhiza -microbe interactioneffect on rhizosphere In: VA mycorrhiza. Interactions in plants, rhizosphere and soils. Ed B.N.Johri. Pantnagar,Uttar pradesh.

24. Earanna. N., Faooqi.A.A., Bagyaraj, D.J.and Suresh, C.K. (2002), Influence of Glomus faciculatum and Plant growth promoting Rhizomicroorganisms on Growth and biomass of Periwinkle, J soil Boil Ecol, 22(1&2):22-26

25. Shivakumar, B.S., Earanna. N., Faooqi.A.A., Bagyaraj, D.J.and Suresh, C.K. (2002), Effect of AM fungus and Plant growth promoting Rhizomicroorganisms (PGPR’s) on Growth and biomass of Geranium (Pelargonium graveolens), J soil Boil Ecol, 22(1&2):27-30

26. Mamatha,G., Jayanthi,S., Bagyaraj, D.J., Theerhaprasad,D. and Suresh,C.K. (2002). Enzymatic activities and biodiversity in the rhizosphere of Sandalwood of central dryzone.In recent trends in Biotechnology. Edt by V.S.Harikumar ,Agrobios india,53-58

27. .Shivakumar. B. S., Radhakrishna. D. and Suresh. C.K.., 2003.Effect of enriched compost with different inoculum levels of Glomus mosseae on Coleus forskholi plants.J Soil Boil Ecol.23,106-110

28. Mamatha.G, Shivayogi.M.S,Bagyaraj.D.J and Suresh.C.K., 2003. Influence of different VA Mycorrhizal isolates on growth and nutrition of sandal wood plants. My Forest, 39:137-145.

29. Shivashankar,C, Govindsamy,y., Earanna,N., Farooqi,A.A., And Suresh.C.K, (2004), Inoculation effect of Am fingus and plant growth promoting rhizosphe microorganisms on growth and biomass of Solanum virum Dunal, Karnataka J Agric Scinence,17:808-810

30. G. Mamatha, S. Jayanthi, D.J. Bagyaraj and C.K. Suresh. (2004) VA mycorrhiza and PGPR isolated from sandalwood rhizosphere growing in different agro climatic zones. Journal of Tropical Forest Science. Malaysia. 16: 283-293

31. Govindasamy , V and Suresh,C.K. (2004). Effect of ecto and Endo mycorrhizal symbiosis on nutrient Uptake and suga contetent of Eucalyptus tereticornis. J.Soil Biol., 1&2:127-133.

(2006). Effect of Glomus fasciculatum and plant growth promoting Rhizobacteria

on growth and Yield of Oscimum basilicum. Karnataka J. Agril. Sci. 19 (1): 17-19.

34., V.U., and Balakrishna, A.N., 2005.Detection of increased root colonization of mycorrhizal fungus in presence of soil yeasts by PCR based method. Biosci. Biotech. Res. Asia, 3(1):161-162.

35. Lmipathy, R., Balakrishna, A.N. and Bagyaraj, D.J., 2005. AM colonization pattern

different land use types of Western Ghats of Karnataka. . J. Soil Biol. Ecol., V. 24: 78- 84.

36. Vinutha, M. C. and Balakrishna, A.N., 2005. Population and diversity of Azotobacter

in different land use types in Western Ghats of Karnataka. Journal of Soil Biology and Ecology, V:24: 54-62.

37. Vinutha, M. C. and Balakrishna, A.N. Population and Diversity of PO₄-Solubilizing

38. Lakshmipathy, R. and Balakrishna, A.N., 2005. Abundance and diversity of AM fungi across a gradient of land use types in Western Ghats. In: the National Workshop on Conservation and Sustainable Management of Belowground Biodiversity held at Kerala Forest Research Institute, Peechi, Kerala from 21-23^rd June 2005. pp. 353-370. 38.Balakrishna, A.N. Maikhuri, R.K. and Sankaran, K.V., 2005. Diversity of AM fungi across a gradient of land uses in Western Ghats and Nanda Devi biosphere. In: theAnnual meeting of the TSBF-CIAT project on Conservation and Sustainable Management of Below-Ground Biodiversity held at Manaus, Brazil in April 2005.

39. Balakrishna, A.N., Balasundaran, M., Singh, R.K., Maikhuri, R.K., Shankar, S., Devyani Sen, Binisha, S. and Chandra, A., 2005. Assessment of diversity of legume nodulating bacteria (LNB) in Nilgiri and Nandadevi Biospheres of India. In: the Annual meeting of the TSBF-CIAT project on Conservation and Sustainable Management of Below-Ground Biodiversity held at Manaus, Brazil in April 2005

40. Radhakrishna.D, Shivram.S and Shivappa Shetty,K.,1991.Carbon utilization by stem nodulating Rhizobium spp. Zentrabl.Mikrobiol.,146:221-225.

41. Radhakrishna.D and Bagyaraj, D.J., 1998. Micrbial degradation of solid wastes as a source of plant nutrients. New trends in Microbial ecology. Eds: Bhart rai and Dkhar, ISCNR, Shillong pp108-117.

42. Radhakrishna.D and Bagyaraj D.J., 2000. Recent advances in composting. Vistas in Mycology and Plant pathology. Eds.Gangawane, Common Wealth pub. 88-100.

43. Radhakrishna K.R. 2007 Isolation, screening and molecular characterization of ethanol tolerating yeast (saccharomyces) strains. Thesis submitted to UAS Bangalore under the guidance of Dr K.M. Harini Kumar

44. Ranjitha patil.S.P 2007 biochemical and molecular studies on sweet sorghum ( sorghum biocolor L. Moench) Thesis submitted to UAS Bangalore under the guidance of Dr D.Dayal Doss.

Thematic area: Development and exploitation of tissue culture technology

P.I: Dr. B.N. Sathyanarayana, Professor, Department of Horticulture, UAS, Bangalore-65

Co-PI: Dr. T. H. Ashok, Professor, Department of Biotechnology,UAS,Bangalore-65

Plant tissue culture has developed into a technique of great reckoning for its plethora of applications in agriculture. Though initially started as an aspect of academic curiosity, of late progressed itself into a technique for commercial exploitation in the production of plants as well as in the improvement of crop plants. Though tissue culture is an integral basic of the present day biotechnology, but has its own standing as a technology of commercial importance. Tissue culture technology is now being employed for commercial production of economically important plants of agriculture, horticulture and forestry importance. In horticulture, the use of this technology for production of clonal plants varying from fruits to flower crops has assumed greater proportions. Production of plants through tissue culture is itself an industry. Over 800 companies are involved in this business world over. Of that nearly 50 companies are located in Bangalore itself. However, as for as the crop improvement is concerned, Tissue culture technology is yet to be used to its potential. Technique such as in vitro mutagenesis or induction of somaclonal variants could be an easy and amenable technique to obtain new variants of desirable characters of greater advantage, say for example increasing the drug yield from medicinal plants, increasing essential oils from the aromatic plants, over all, improving the quality and quantitative traits in the plants of horticultural and forestry importance.

Horticulture encompasses growing fruits, flowers, vegetables, plantation crops including medicinal and aromatic crops and of course and processing of horticultural crops for value addition. Value addition has attained a great momentum in the world at present. Production of horticultural crops is on an unprecedented upswing at present owing to globalization and free world market. It is also a fact that the consumption of horticultural produce has increased considerably owing to richer life standards of people across the world. It is also true that people are becoming more conscious of the benefits of horticultural produce to the human health and the absolute need for it. Junk consumerism is on the wane and neutraceuticals are steadily encompassing the living styles of people. People are now becoming very aware of the need to be with the nature. Talking about the nature, people are extremely becoming alert in preserving forests and growing and nurturing forest plant species. The products of forest plants specifically of those with immense health benefits are going to be sought after in an unprecedented scale. There is not only a need to cater to the global demands for the forest plant products but also reduce the exploitation on natural forest resources. Further, Forest species are now an established source as alternative energy sources such as fuel. The concept of Bio-fuel is now being implemented with lot of hopes world over. This could only lead to lead to a situation of severe exploitation. Considering the importance of Horticulture and Forestry on a global scenario The science and practice of plant tissue culture has a tremendous role to play to augur to the crisis which looks imminent under the present circumstances. Plant Tissue Culture is the technology which could serve the purpose of

increasing the availability of plant material be it horticultural or forest species. Increase in availability could be achieved on a very high scale at a rapid pace. The horticultural and forest species could also be improved genetically to yield more. Yield described here is not only quantitative terms but also on qualitatative terms. In Medicinal and Aromatic plants, the active principles constituting the drugs could be enhanced by altering the plants at a genetic level. There are so many species of forest plants including the invaluable medicinal plants are on the verge of extinction. Tissue Culture is the only method to save them from extinction. Hence, Plant Tissue Culture Technology isa high ly relevant and augmentative technology to enhance the availability and increase the production, not withstanding the technology’s great impact conservation of endangered species of plants.

Tissue culture technology is a reliable means of large scale clonal propagation, and as a technique of crop improvement for qualitative and quantitative traits of horticultural and silvicultural crops. Through Plant Tissue Cultural Technique a great number of forest species which under the threat of extinction could also be conserved thus saving biodiversity for the future. The world could never suffer from inadequacies particularly in relation to human needs as long as the technologies such as Plant Tissue Culture could be used to foster our resources to use them to the optimum and as well as to harness higher yields.

Though India is a leader in all the horticultural endevours such as production of fruits and vegetable and many plantation crops, next only to China, our contribution to the world market is a meager less than 10 per cent India has not lagged behind in flower production as well. India is fast competing with the advanced and traditional flower growing countries such as Holland, many European countries and newly added developing African Countries. India has become global player now and which can be measured by the fact that India is one of the leaders in the export of flowers mainly Hybird T Roses, and ornamental foliage and flowering plants. Bangalore and Mysore are becoming big reckoning centres in this scenario. Orchid production is a grey concern. Though we have the rich source, but are fast depleting due to unscrupulous harvest from the wild. Organised cultivation of Orchids on a large scale could augur well for the country and Karnataka as well. Though Karnataka is not a name to reckon with, but our Plant Tissue Culture Facilities strengthened could do well to raise the status of orchid production. In general, increased help from the Karnataka Government could greatly boost the horticulture Industry. Western ghats of Karnataka is a home for a rich wealth of medicinal and aromatic herbs. When the world is going gaga over the use of herbal products be, it for medicine or cosmetic or be it for the use as a neutrceutical, I see a huge potential for the Karnataka State in Particular and India in general. As regards to Forestry, India is next to none in its wealth. Karnataka has a high forest cover. Conservation of plant resoureces, increased production of forest plant products, will alter the Karnataka,s silvicultural scenarion.. What is being done now in horticulture and forestry sector, though not depressing, what needs to be done is calling. Under this scenario, Plant Tissue Culture as a technique for large scale clonal propagation, crop improvement for enhancing qualitative and quantitative traits and saving the red listed

forest species is definitely beckoning serious attention from the Government of Karnataka. Hence, this project.

Objective 1: Production of plants of high value crops such as Banana Cvs Nanjangud Rasabale and Elakki bale, elite clones of Jackfruit, Guava, v.Allahabad safeda. Among forest plants of biofuel importance, elite clones of Pongamia and Neem.

Objective 2:Crop improvement via in vitro mutagenesis. Banana Cv, NanjungudRasbale for somaclone variants resistant to fusarium wilt malady.

Objective 3: Conservation of endangered Forest species more particularly of medicinal and aromatic species which are listed in the Red Book, Particularly species relevant to Karnataka. Such as Orozylum indica, Saraca indica and Kinzeodendron Pinnata

1. Satyanarayana, B.N. Deepu Mathew and Sumaya Nikhat, 2001, First report on Effect of Thidiazuron (TDZ) on in vitro shoot proliferation in Gerbera (Gerbera jamesonii) Science and Culture, 67(1-2): 61-62

2. Jagannath, J., Ashok, T.H. and Sathyanaryana, B.N, 2001, In Vitro propagation in Carnation cultivars (Dianthus caryophyllus L), Journal of Plant Biology, 28 (1): 99 –103.

3. Jagannath, J., Ashok, T.H. and Sathyanaryana, B.N., 2002, In Vitro Micro-propagation studies in carnation, Karnataka Journal of Agricultural Sciences, 15(1): 109-114.

4. Endale Gebre and Sathyanaryana, B.N., 2001, TAPIOCA-A new and cheaper alternative to agar for direct in vitro shoot regeneration and microtuber production from nodal cultures of potato. African crop Science Journal, 9(1): 1-8.

5. Dinakara Adiga, J., Khan, M.M. and Sathyanaryana, B.N., 1998, Effect of carbon Source, culture vessels, gelling Agents and GA₃ on in vitro shoot proliferation of Singapore Jack (Artocarpus heterophyllus Lam.), Karnataka Journal of Agricultural Sciences, 11(3): 632-636.

6. Kalpana Sachdev, Prakash, K.S., Prasad, T.G. and Sathyanarayana, B.N., 2002, Somatic embryogenesis and plantlet regeneration from node and leaf callus cultures of Gloriosa superba L. Journal of Plant Biology 29 (2).

7. Kalpana Sachdev and B.N.Sathyanarayana, 2002, Effect of coconut water and banana pulp on in vitro culture of Dendrobium Cv. Jo Mutant. Journal of Plant Biology 29 (2).

8. Prashanth, K.G., Sathyanarayana, B.N., Suresh, N. Sondur and Deepu Mathew, 2002, In vitro shoot proliferation and callus induction in carambola (Averrhoa carambola). Tropical Science

9 B.N. Sathyanarayana, G.B.S. Suma, Deepu Mathew K.G. Prashantha and Suresh N. Sondur, 2003, Somatic embryogenesis and plant regeneration from stem, leaf, root and petal explants of sweet type carambola (Averhhoa carambola). Journal of Plant Biology (In print)

10. K.G. Prashantha, B.N. Sathyanarayana and Deepu Mathew, 2003, In vitro callus induction and plantlet regeneration in rose apple. Journal of Plant Biology, 30(1): 1-4

11. Sathyanarayana, B.N. and Jennet Blake, 1994, The effect of nitrogen sources and initial pH of the media with or without buffer on in vitro rooting of jackfruit (Artocarpus heterophyllus Lam.). Physiology, Growth and Development of Cells in Culture. Kluwer Academic Publishers, Netherlands.

12. Dalia Mathews, B.N. Sathyanarayana and A.V. Meera Manjusha, 2003, Modulation of flower colour by metabolic engineering: Providing ‘New’ functions for an ‘Old’ pathway. One Hundred Research Papers in Floriculture (Ed. Rajeevan,

P.K., P.K. Valsala Kumari and R. L. Misra ). Indian Society of ornamental Horticulture, New Delhi, pp.190-195.

13. Srinath, J., Bagyaraj, D.J. and Sathyanarayana, B. N., 2003, Enhanced growth and nutrition of micropropagated Ficus benjamina to Glomus mossae coinoculated with Trichoderma harziannum and Bacillus coagulens. World journal of microbiology and Biotechnology, 19: 69- 72.

14. Prashantha,K.G., B.N. Sathyanarayana and Deepu Mathew, In vitro shoot proliferation and plant regeneration in sweet carambola (Averrhoa carambola). Tropical Science, 44: 108-110.

15. Shreevatsa, K.S., K.V. Jayaprasad, P. Narayanaswamy and B. N. Sathyanarayana, 2004, Establishing in vitro cultures of Strelitzia reginae using shoot tip explants. Journal of Ornamental Horticulture, 7(3 and 4): 341- 344.

16. B.N. Maruthi Prasad, B.N.Sathyanarayana, Gowda Balakrishna, R.Sharath 2007, In vitro regeneration of Drug Yielding tuber crop, Chlororphytum borivilianum Medicinal and Aromatic Plant Science and Biotechnology, Global Science Books.

17. B.N. Maruthi Prasad, B.N.Sathyanarayana, Jaime A, Teixeira da Silva, Gowda Balakrishna, R.Sharath 2007, Regeneration in Chlororphytum borivilianum through somatic embryogenesis. Medicinal and Aromatic Plant Science and Biotechnology, Global Science Books.

18. R.Sharath, V. Krishna, B.N.Sathyanarayana, B.N. Maruthi Prasad, B.
G Harish, 2007, High Frequency Regeneration throough somatic embryogenesis in Bacopa monnieri (L) Wettest, an important medicinal plant. Medicinal and Aromatic Plant Science and Biotechnology, Global Science Books.

19. Dalia B. Varghese and B.N.Sathyanarayana, 2007, Induction of variation in two cultivars of Bacopa monnieri by Gamma Irridiation of in vitro culture. Medicinal and Aromatic Plant Science and Biotechnology, Global Science Books.

The transfer of desirable genetic traits across species barriers by rDNA technology has shown promise for solving various constraints that affects to realize the potential yield of our crops. Several transgenic crops developed have shown the possibilities of improving pest, disease and herbicide resistance, abiotic stress tolerance and nutritional quality improvement. Besides India, 20 other countries grow biotech crops. Though the major contributions come from 3 crops - cotton, soybean and maize, a study in these areas testifies the potentiality, slow but steady acceptance, and the economic benefits. BT cotton transgenics in India also showed a similar trend, now occupying approximately 5.6 million hectares and it has significantly contributed and in reducing pesticide consumption increasing the yield from 300 to 475 Kg lint per hectare.

Realizing the potential of transgenics, several research programmes have been initiated in public and private R&D centers.

Over 300 IBSCs have been constituted in India indicating that rDNA programme have been initiated in these institutes and more than 80% are directly or indirectly associated with developing transgenics of different crops. However, no event has been released for commercialization (Fig.1).

Even the collaborative ventures from other countries with efficient genes and gene constructs did not yield any commercialized technology so far. Further, in spite of initiating several backcross programmes, the commercial release of such products is yet to takes place.

a. It has been increasingly realized that the slow progress for generating commercially viable transgenics as stipulated by biosafety guidelines, is the lack of interface from the development of primary transformants to the commercially viable transgenic product.

b. The research/academic institutions have the intellectual capacity to ‘create’, but lack resources, skills, infrastructure and capabilities to advance their innovative creations to a commercial level of applicability.

c. The time taken from innovation to commercialization is quite lengthy (Fig) and hence became one of the major constraints to exploit transgenic research. There is a need to reduce the time gap between the events.

comprehensive characterization before commercialization. It includes, besides the agronomic evaluation (productivity/production to target pest and diseases) of several biosafety issues like safety to environment, food and feed safety, toxicity issues and soil, water and air pollution.

In view of this, several biosafety tests as stipulated by regulatory authorities (IBSC, RCGM and GEAC) have to be carried out. In addition, bio-efficacy of the transgenics developed needs a thorough investigation. Further, there is a need to provide comprehensive information on molecular characterization of the transformants. All these information has to be generated by raising the transgenic crops under DBT approved containment facilities, contained strip trials and multi-location research trials.

To identify the transgenic event with improved target trait, several primary transformants are to be generated and rigorously examined. Such analysis alone will lead to identify superior events with commercial significance. One of the major constraint is a holistic facility for evaluation of the initial events and development of required biosafety measures for advanced material and finally to interface with private sector for its commercialization.

Translation Research Centre (TRC) will facilitate the translation of technologies for the development of commercially viable transgenics/products. It forms the interface between innovation and commercially viable product development.

The Translational Research Centre proposed to be established in this project will substantially hasten the process of generating most viable transgenics. The TRC has well defined functions to address the diverse aspects for commercialization of the transgenics.

The focus is to develop tolerant cultivars for pest and diseases and to the major abiotic stress – drought in important dry land crops. There would substantial economic benefit in transgenics tolerant to these stresses are developed.

The university has made considerable efforts in this direction to develop transgenics to manage the important pest and diseases besides tolerance to abiotic stresses, especially to drought.

One the major constraints in developing transgenics in dry land crop species like redgram, groundnut, field bean etc., been the lack of efficient transformation protocols. Recently, our group has made a significant break through in developing transgenic protocols and transgenics. Based on this progress, the group at UAS Bangalore launched a massive program to develop transgenics expressing transgenics in several dry land crop species.

Many of the transgenics developed by the group are quite promising. The progress made so far has demonstrated the ability of the team at UAS Bangalore to develop transformation protocols but also to validate the tolerance levels of the transgenics against pest and diseases. Furthermore, the team also has expertise in determining the responses of these transgenics to drought and other abiotic stresses.

The major goal is to thoroughly characterize and evaluates the large number of transgenics developed in several crop species, ultimately to identify promising ones with high degree of field level tolerance. Being genetically modified organisms, it is also essential to develop the required information for bio-safety clearance. The specific objectives therefore are

1. To evaluate the transgenics developed in the following crops to identify promising ones with high degree of field level tolerance.

3. To generate information to clear bio-safety clearance and consumer acceptance.

To begin with the Translational Research centre has the onus of taking these events further to the level of products for public consumption. A list of the crops developed and currently available at UAS, Bangalore along with the proposed work for each event under the TRC are appended. A summary of these works in progress are indicated below.

Control NPR1/defensin Transgenic Control NPR1/defensin+ Glucanase Transgenic

1) Dehydration tolerance of transgenics expressing P5CS (pyrroline 5-carboxylase

2) Salinity tolerance of transgenics expressing NHX (Na⁺ - H⁺ antiporter)

Several transgenics crops expressing genes to improve specific agronomic traits are being developed both in the private and public sector. One of the approaches is to take advantage of the efficient and validated transgenic events by using them in back cross programme in to the other superior germplasm.

The TRC provides the required platform for such a programme. This substantially adds value to our germplasm and hasten the process of developing desired transgenic with adaptation to the local conditions.

Several multi location research trails from the different organizations (private and public institutions will be conducted to generate data information on Agronomic superiority (traits specific) and molecular characterization.

Several organizations are proposing to develop contractual research for the initial evaluation of the transgenics.

Nuziveedu seeds, Ankur seeds, Beejo sheethal, Monsanto, Central University, South campus, Delhi university etc..

5. To take up long term experiments on risk assessment of transgenics developed

One of the major requirements is risk assessments is terms of long term effects of transgenics on soil micro organisms, soil insects and other beneficial insects.

TRC will be a self-contained structure and facility for dealing with all aspects of transgenic crop evaluation and subsequent commercialization. Infrastructure will include the following:

This interface facility substantially hastens product development from innovations. This forms a platform product beneficial not only to the scientific community of U.A.S., Bangalore, but also for many institutions and universities of the country.

Several R&D centers are established in the private sector in developing transgenics and also have initiated several molecular breeding programs. Apart from facilitating to generate information needed for regulatory authority, the centre also provides authenticity of the material developed by the private sector.

The mandate of the centre is to provide a comprehensive analysis of transgenics under the guidance of the diverse experts of various disciplines, the acceptability of the products developed will be greater and it hastens the dissemination of this technology.

1. K. Sankara Rao, Rohini Sreevathsa, Pinakee D. Sharma, Keshamma E and Udaya Kumar M. In Planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro. (2007). Submitted to Physiol. Mol. Biol. Plants.

3. Rohini V. K. and Sanakara Rao K. (2006). Recent advances in legume transformation. A Book Chapter on Legume transformation (to be published in 2007).

4. Rohini V. K., N. H. Manjunath and K. Sankara Rao (2005). Studies to develop systems to mobilize foreign genes into parasitic flowering plants. Physiol. Mol. Biol. Plants. 11(1): 111-120.

5. Sankara Rao, K and Rohini, V. K. (2003). Genetic transformation and regeneration of sandalwood (Santalum album L.) – In Plant genetic engineering Vol. 3; Improvement of commercial plants – I. Eds. Pawan K. Jaiwal and Rana P. Singh.

6. Sankara Rao, K and Rohini, V. K. (2003). Biotechnology of safflower (Carthamus tinctorius L.): Strategies for gene transfer – In ‘Plant genetic engineering Vol. 4; Improvement of commercial plants – II. Eds. Pawan K. Jaiwal and Rana P. Singh.

7. Rohini, V. K. and Sankara Rao, K. (2002). In planta strategy for gene transfer into plants: Embryo transformation (a Review). Physiol. Mol. Biol. Plants 8(2): 161-169.

8. Rohini, V. K. and Sankara Rao, K 2001. Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease. Plant Sci. 160 (5): 883-892.

9. Rohini V. K and K. Sankara Rao. (2000). Embryo transformation, a practical approach for realizing transgenic plants of safflower (Carthamus tinctorius L.). Ann. Bot. 86: 1043-1049.

10. Rohini, V. K. and Sankara Rao, K. (2000). Transformation of peanut (Arachis hypogaea L.): A non-tissue culture based approach for generating transgenic plants. Plant Sci. 150(1): 41-49.

11. Sankara Rao, K. and Rohini,V. K. (1999). Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): A simple protocol. Ann. Bot. 83: 347-354.

12. Sankara Rao, K. and Rohini, V.K. (1999). Gene transfer into Indian cultivars of safflower (Carthamus tinctorius L.) using Agrobacterium tumefaciens. Plant Biotechnology. 16 (3): 201-206.

Human Resource Development in Agricultural Biotechnology

To sustain the ongoing R and D programmes in Agricultural Biotechnology, and also to develop Research Leadership in this potential area, a strong Human Resource Development programme is essential. In view of this, one of the components of the Agricultural Biotech centre is to strengthen the on going programmes in Agri biotechnology and to start new initiatives. The approaches and strategies are

· Develop and run Undergraduate and Post graduate level courses on Crop Physiology, Molecular Biology and Biotechnology.

Human Resource Development in Agricultural Biotechnology is an important component to cater the needs of Agricultural universities and Agri Industries. The Agricultural University can alone develop human resource in Agricultural Biotechnology, since the scientific development in Agricultural Biotechnology needs a thorough knowledge of crops, their cultivation, breeding, pests and disease resistance etc.

In view of this, B.Sc (Agri. Biotechnology) programme has been started in the university to train the students in the emerging areas of Biotechnology with a strong background in basic sciences and the allied Agricultural subjects. University has also started degree programmes in M.Sc (Agri.) Plant Biotechnology and Ph.D in Plant Biotechnology.

The University is planning to start the M.Sc (Agri.) and Ph.D in Plant Physiology and Molecular Biology to study the basic aspects of Genetic Engineering.

The university is also planning to start a Post Graduate Diploma in ‘Biosafety in Plant Biotechnology’ in collaboration with U N University of Japan.

Post Graduate Diploma in” Biosafety in Plant Biotechnology”

The new and emerging tools of biotechnology offer significant opportunities to enhance agricultural productivity, food and nutritional security, and environmental quality worldwide. Some countries have already developed and commercialized genetically engineered transgenic crops. Many developing countries have initiated biotechnology research and development programs to benefit from the new tools of biotechnology.

The commercialization of transgenic crops has sparked off intensive debates world wide regarding biosafety and the impact of this powerful technology on agriculture, human health and environment. The biosafety concerns of the transgenics owe their origin to the fact that tools of recombinant DNA technology have clubbed all biological systems into a “single gene pool” providing access to even genes from completely unrelated or sexually incompatible organisms. Therefore, the transgenic technology, while providing unlimited scope for crop improvement, also imposes tremendous responsibility on the scientific community and the regulatory authorities towards ensuring biosafety.

At the end of the programme, successful trainees will be able to conduct risk assessments and apply risk management options. They will also have acquired skills to deal with public policy issues at the interface of science, government, industry and civil society. The University of Agricultural Sciences is a pioneer in offering Postgraduate course on Biosafety in conjunction with Intellectual Property Rights since 1999-2000. Further, the curriculum of B.Sc. (Ag. Biotech) to be offered from the current year by UASB has a separate course on this aspect Therefore UN university of Japan chose UAS, Bangalore for shaping a Biosafety curriculum by holding a Brain storming session on 19^th & 20^th of July 2007.

Currently there are no institutions in India offering academically accredited Programmes in biosafety even at diploma level. The Diploma is targeted at individuals interested in or being engaged as biosafety professionals in government agencies or industry. It is also tailored for individuals with an interest in public policy, legal and ethical aspects of biotechnology. This is urgently warranted to augment the HRD component to enhance the pace of Agricultural Biotechnology in the country

Keeping in view the need, University in association with UN University Japan and DBT, New Delhi, structured a programme on “Regulatory affairs in Biotechnology” to be offered as a Post Graduate Diploma. The proposed modules are,

Module 8: Hands-on activity to deal with applications on risk assessment and management

Module 10: Relationships and opportunities to work with industry, media, NGOs, communities

Indian agriculture faces the formidable challenge of having to produce more farm commodities for our growing human and livestock population from diminishing per capita arable land and water resources. Biotechnology has the potential to overcome this challenge to ensure the livelihood security of 110 million farming families in our country.

Biotechnology Parks as envisaged both by DBT and the Karnataka Government, can provide a viable mechanism for licensing new technologies to upcoming biotech companies to start new ventures and to achieve early stage value enhancement of the technology with minimum financial inputs. These biotech parks facilitate the lab to land transfer of the technologies by serving as an impetus for entrepreneurship through partnership among innovators from universities, R&D institutions and industry.

In view of the above imperative, emphasis will have to be given on training of high quality technicians and technologists in skills required by the industry by establishing Regional training centers.

The trainees will learn recombinant DNA techniques in a context that relates to the job functions of a bioscience technician: performing tests, making products, obtaining and processing materials, controlling inventory, using and maintaining equipment and facilities, performing and documenting safe practices, complying with regulations, maintaining quality assurance, evaluating and documenting results, and communicating information. Students must be able to effectively and consistently practice aseptic technique, observe all safety rules, keep a scientific notebook, calculate and make up solutions/dilutions, practice scientific honesty, and follow procedures as they perform the recombinant DNA experiments.

Keeping the importance of human resource development in this area in mind, it is recommended to develop the following facilities/improvements for human resource development under the proposed programme

An estimated 112 lakh ha area covering nearly 74 per cent of cultivable land in the State is under semi-arid tracts. The entire area is prone to repeated drought besides facing innumerable pest and disease problems. The losses in crop productivity due to drought is as high as 60 per cent and there is an imperative need to address this issue from all directions. Biotic stresses such as pests and diseases are known to substantially reduce yields to the tune of nearly 30 to 35% in most crops and consequently accounting for nearly Rs. 1136 crores in loss of revenue annually. Needles to say that the impact of drought is much higher.

Options to improve the crops by biotechnological solutions should greatly reduce the impact of some of these problems and are in the realm of possibility for many crops. The programmes envisaged to mitigate Biotic and Abiotic stresses through molecular breeding and transgenic approaches are likely to provide suitable technologies to improve the productivity of important dry land oil seed and pulse crops. Even an adoption rate of just around 20 per cent is estimated to provide substantial annual benefit.

The programme on aerobic rice is expected to develop high yielding rice cultivars that require less water will substantially save the irrigation water, which is the major limiting input in Agriculture.

Productivity of over 4.0 lakh ha of problematic soils of Karnataka is expected to increase with the development of salt tolerant transgenic crop varieties. Similarly, cultivation costs through fertilizer input is expected to greatly reduce for the dry land farmers through programmes that aim at development of varieties with efficient utilization of soil nutrients such as phosphorous and zinc.

Besides the productivity of land the programme also envisages to foster the other economic and health benefits of the farming community. Enhanced quality traits such as improved nutritive value by enhancing the Fe, Zn, oil type or protein content in crop plants as envisaged through biotechnological tools is expected to provide enhanced nutrition to the rural masses. Improvement in crop quality such as longer shelf life would reduce the post harvest losses of many vegetable crops.

The programme also envisages producing many pharmaceuticals in crop plants that would contribute to value addition of crop plants leading to higher price realization by the farmers for their produce.

In addition, the work on functional genomics envisages developing many potential genes for use in mitigating the problems of drought, salt tolerance, water use efficiency, insect pests, diseases, quality traits, etc. The rich biodiversity existing in the state of Karnataka will be explored for genes of various traits such as high alcohol production, P solubilisation, N fixation, salinity tolerance, and toxins for pest and disease management.

Crop productivity can suffer due to lack of availability of elite plant material in many perennial cropping systems. Therefore the proposed programme envisages developing robust, high through put methodologies of mass producing a number of perennial horticultural plants such as jack, cardamom, banana, guava etc., for distribution to the farming community. Further somaclonal variants tolerant to diseases in locally valuable ecotypes in Banana would be exploited for the economic benefits of the farmers and to conserve the rare resource.

The training and education would help develop human resources to meet the manpower needs of the public and private Agri-biotech sector in the near future and help develop long term technological capabilities of the State.

Establishment of the Centre for Agricultural Biotechnology at the University of Agricultural Sciences, Bangalore is expected to help realize the needs of food security of the country in general and enhance the nutritional and economic security of the farming community of the State by facilitating the growth of agri-sector to reach 4 per cent as envisaged by the National Development Council.

Crop	Area (ha)	Production (ton)	Expected loss due to insects (Kg/ha)	Price per quintal (Rs)	Overall loss experienced by the state (crores of Rs.)
Groundnut	850000	864450	208	1200	212.16
Pigeon pea	580000	276080	411	1400	333.732
Castor	20000	17800	180	800	2.88
Field bean	80000	23520	170	850	11.56
Bengal gram	369000	251658	180	1000	80.442
Tomato	46788	1777944	10000	200	93.576
Cabbage	11000	209000	50000	200	11
Cotton	652000	521600	300	2000	391.2
Total					1136.55

*Approach*	*Deliverables*
*Identification of highly expression stress genes* Subtracted stress library developed from crops such as ground nut, finger millet, will be used for gene expression studies to identify highly stress responsive genes. The gene expression studies will be done at different stressful environments and developmental stages.	Identification of highly stress responsive genes
*Cloning of stress inducible promoters* The promoters of the highly expressed genes will be cloned from species of choice	Stress specific promoters
*Promoter validation* The clones promoters will be used to drive reporter genes in model systems under various stressful environments	Validated stress specific promoter

Approach	Deliverable
Genome wide screening using SSR and DArT markers.	1. Assessing the genetic diversity and construction of the population structure. 2. Assessment of Linkage disequilibrium on the groundnut genome.
Stable isotope ratios of carbon and oxygen will be measured using the IRMS. Plants will be raised in Root structures to assess the root traits in two locations or two seasons. TIR technique will be adopted to assess genetic variability in intrinsic tolerance	1. Phenotypic characterization of germplasm for WUE, root traits and other related drought tolerance traits. 2. Identification of promising “Trait donor” parents that can be used in conventional breeding programs or to develop trait specific mapping populations.
The genotyping and phenotypic data will be used for Marker-Trait association.	1. Association of markers to relevant physiological traits.

Approach	Deliverables
Raising of inbred lines in root structures to assess the variability in root traits and WUE	Contrasting sunflower inbreds differing in WUE and root traits with reasonably high intrinsic tolerance
Contrasting lines differing in root traits and WUE with reasonably high intrinsic tolerance will be crossed.	Trait specific mapping populations segregating for WUE and root traits will be developed.
A large number of SSR and other marker systems such as AFLP and RAPD will be used to characterize the mapping populations	Sunflower linkage map will be developed.
Root structure experiments coupled with stable isotope ratios and TIR	Identification of QTL’s conditioning WUE and root traits.

Objectives	Deliverables
Identification of molecular markers linked to root traits in sesame	Molecular markers inked to root traits identified
To identify distinct genotypes with the desirable root morphological traits in sesame	The genotypes with improved ability to put forth roots deep in the soil under moisture stress conditions isolated
To develop drought tolerant sesame genotypes under elite agronomic background	Superior drought tolerant and high yielding sesame genotypes identified and evaluated under field conditions

Crop	Area under rain-fed (mha)	Potential productivity (kg. ha^-1)	Realized under rainfed (kg. ha^-1)	% reduction	Loss in revenue (Crore Rs)	Gain in revenue due to technological intervention
Groundnut	0.86	2000	700	65	1000	200
Sunflower	0.36	1338	520	62	800	170
Redgram	0.58	900	470	52	1000	250

Approach	Deliverables
Identified transcription factor genes will be introduced into groundnut.	Development of groundnut transgenics with better drought tolerance capacity.

Approach	Deliverable
A expression cassette will be constructed with the a few most relevant C₄ genes. Rice cells will be transformed either by conventional methods and/or by inplanta transformation techniques.	* An expression cassette with all the relevant C₄ genes will be constructed. * Transformed Rice plants over expressing C₄ genes will be generated.
Carbon isotope signatures will be adopted as the approach to identify the putative transormants.	Several positive transformants of rice expressing the C₄ genes will be identified based on carbon isotope signatures.
Stable transformants will be used for assessing photosyntheis, water use efficiency, photorespiration, abiotic stress tolerance using several physiological screens	Comprehensive physiological characterization of the stable transformants will be assessed.

Approach	Deliverables
Over expression studies using either Agbac. or Inplanta technique	Transgenics for salt tolerance.
A meticulous screening technique will be adopted to determine the extra or enhanced tolerance accrued due to a double gene transfer for two functions of ion homeostasis.	Transgenics with tested tolerance at greenhouse /field level.

Approach	Deliverable
Development of transgenics	Transgenics for higher P acquisition capacity in Pigeon pea

	Pigeon pea	Chickpea	Cowpea	Mungbean	Horsegram	Field bean
Bacterial leaf blight		√
Bacterial blight			√	√
Anthracnose					√
Brown blotch			√
Powdery mildew	√			√	√
Wilt	√
Leaf spot	√		√	√	√
Seedling blight	√
Rust			√	√
Root rot		√
Ascochyta blight		√
Foot rot		√
Collar rot		√
Leaf blight		√
Yellow Mosaic		√		√	√	√
Leaf crinkle		√
Sterility Mosaic	√
Stunt disease	√

No	Title	PI
1	Development of SSR markers for Fusarium wilt and sterility mosaic resistance and pyramiding of genes for multiple resistance in pigeonpea.	M. Byre Gowda
2	Use of molecular markers and marker assisted breeding for the development of yellow mosaic virus resistant horsegram varieties	Dr. K. P Viswanatha
3	Development of SSR markers and marker assisted Selection for Mungbean yellow mosaic virus and Powdery mildew in green gram and blackgram	Dr. D. L. Savitramma
4	Development and Utilization of DNA based molecular markers for biotic and abiotic stress tolerance in chickpea (Cicer arietinum L.)	Ms. M. K. Jayashree
5	Marker-assisted selection for rust resistance in Cowpea	Dr. M. S. Uma
6	Marker Assisted back cross breeding for Fusarium wilt (Fusninm species) resistance and tagging of markers linked to other economically important characters in Bambara groundnut (Vigna subterranere L.)	Dr. R. Nandini
7	Development of multiple disease resistance to Tomato Leaf Curl Virus, Bacterial wilt and nematode in Tomato by using molecular marker assisted selection.	Dr. R. S. Kulkarni
8	DNA Marker Assisted Introgressions of sheath rot, bacterial leaf blight and blast resistance into elite Rice Varieties of Karnataka	Dr. Shailaja Hittalmani
9	Development of Multiple disease resistance in TyLCV bacterial wilt and nematodes in Tomato by using MAS	Dr. R. S. Kulkarni
10	Development of Genomic Resources (SSR Markers), and mapping and validation of QTLs conferring wilt resistance in Chili/hot pepper (Capsicum annuum L.)	Dr. A. MohanRao

Pigeonpea area in Karnataka	Current production	Current productivity	Potntial productivity if diseases are controlled	Gain in yield to the state	Current pigeon pea grain value	Gain in income to the farmers
5.2 lakh ha	2.90 lakh tonnes	500 kg/ha	3.9 lakh tones	1.00 lakh tones	Rs 20/kg	Rs 200 crores

Objectives	Approaches	Deliverables
To develop new SSR markers in cowpea	Development of partial genomic libraries enriched with microsatellites Sequencing of positive clones and primer designing and synthesis	Genomic library of cowpea SSR markers of cowpea (for use in construction of marker map, genome mapping, marker assisted selection, diversity analysis and characterization studies).
To develop mapping population	Crosses will be made between highly susceptible and highly resistant parent to generate mapping popualtions	Development of mapping populations(F2 and Backcross populations)
To identify polymorphic SSR markers for parents and F2 & Back cross populations	phenotyping of each plant in mapping populations by natural incidence	Phenotyping and genotyping of progenies in mapping populations
To identify polymorphic SSR markers for parents and F2 & Back cross populations	Identification & tagging of Markers associated with rust resistance	Productive rust resistant lines

	Approach	Deliverable
1	The germplasm collection will be screened in the green house with artificial inoculation and also in the sick plots.	Disease resistant lines can be selected
2	The resistant and susceptible lines will be subjected to Bulk Segregant Analysis using molecular markers	The markers putatively linked to respective disease resistance can be identified.
3.	Disease resistant lines are intercrossed and the resulting progeny will be subjected to marker assisted selection for multiple resistance	Multiple disease resistant lines can be identified

Sl. No.	Target Crop	Problem to be tackled	Deliverables
Insect resistant Transgenics
1.	Redgram – varieties BRG 2 and the BRG 4	Pod borer using insecticidal gene – cry1AabcF	Pod borer tolerant transgenic BRG-2 and BRG-4 redgram varieties
2.	Chickpea – Vishal and Annigeri 1	Pod borer using insecticidal gene – cry1AabcF	Pod borer resistant transgenic Vishal and Annigeri varieties of chickpea
3.	Sunflower –	Head borer using insecticidal gene – cry1AabcF	Head borer resistant transgenic sunflower hybrid
4.	Castor - Jyothi	Capsule borer using insecticidal gene – cry1AabcF	Capsule borer resistant transgenic castor

Disease resistant Transgenics
5.	Groundnut – TMV-2, K-134 and VRI-2	Groundnut budnecrosis with nucleocapsid gene	Groundnut bud necrosis resistant transgenic TMV-2, K-134 and VRI -2
6.	Sunflower –	Sunflower necrosis disease with TSV coat protein genes N and Nss	Necrosis resistant transgenic sunflower hybrids

Herbicide tolerant Transgenics
7.	Groundnut – TMV-2, K-134 and VRI-2	Glyphosate resistant –EPSPS & IgrA –multiple gene constructs	Glyphosate resistant transgenic K-134 groundnut variety

Approach	Gene	Deliverables
Agrobacterium tumefasciens mediated in planta transformation	Synthetic construct Cry1Ec to be procured from NBRI, Lucknow and VIP from ICGEB New Delhi	Insect resistant transgenic -Jyothi – castor lines

Approaches		Deliverables
Objective 1	Assessment of genetic variation in Zn and Fe content in diversified germplasm lines/varieties of rice	Selection of high and low contrasting Types
Objective 2	Assessment of Zn and Fe acquisition in contrasting lines with high and low types under different Zn and Fe available conditions.	F1 population developed and used for QTL this marker is used to screen large Germplasm lines to identified donor Parent
Objective 3	To study the variation in Zn uptake using radio labeled Zn (⁶⁵Zn) in contrast lines differing Zn content.	Up take and translocation in contrast Types
Objective 4	Evaluation of F2 population developed using high and low types and development of QTL	Development of QTL using phenotypic and genotypic data. Advancement of selected few line based on QTL and phenotypic Zn content
Objective 5	Assessment of variation in transporters and their expression in root and shoot.	Differential expression of transporters (ZNT1, ZIP and IRT) which has been cloned and characterized – Northern analysis will be done to examine whether Zn transporter will result in constitutively high expression of these genes.
Objective 6	Development of transgenic plants over expressing transporters.	Transformation of Zn transporter gene into rice by using Agrobacterium mediated gene transformation/gene gun method.

Sl. No	Approaches	Deliverables
1.	Procurement of vegetables from different parts of Karnataka and isolation of Karnataka and isolation L AB from these sources.	Lactic acid bacterial isolates
2	Lactic acid bacterial formulations using different carrier materials for application to vegetables.	Formulations of LAB for application to vegetables.
3.	Standardizing the protocols for development of different products using Lactic acid bacterial isolates	Fermented products
4	Molecular approaches for strain improvement of isolates for increasing the shelf life and nutrient conc	New strains of isolates with high efficiency for the production of antimicrobial compounds
5.	Nutritional analysis, economics and Shelf life experiments for the products	Technology for preservation of vegetables and fruits

Sunflower and associated problem	Modification required	Utility of the end product
High Linoleic acid	Reduced linoleic acid OR Higher levels of Oleic acid	Increased oxidative stability i.e. longer shelf life, as well as having an improved health profile or benefits.

Objective 1: Cloning and expression of Challengevirus standard strain (CVS) Rabies glycoprotein gene in Pichia pastoris and plants and immunization of mice with the recombinant rabies glycoprotein
Approaches	Deliverables
1. Cloning and expression of challenge virus standard strain rabies glycoprotein gene in Pichia pastoris and plant binary vector	Yeast vector and plant binary vectors will be constructed with rabies glycoprotein gene
2. To study the biochemical and molecular characteristics of the rabies glycoprotein produced in Pichia pastoris.	The expressed protein will be confirmed with SDS and western blot analysis.
3.To evaluate the immunogenecity and protective capacity of recombinant vaccine produced in Pichia pastoris and Commercialization of vaccine	The purified protein will be tested for its immunogenecity in mice and produced in large scale for commercialization.
Objective 2: Plant expression of Hepatitis-B antigen and targeting molecules for immunization in mice
Approach	Deliverables
1. Transformation of Muskmelon and Coleus forskohlii plants with HBsAg gene under the CaMV 35 S promoter using the gene construct pHB118 Agrobacterium mediated and biolistic methods	Binary vector with HBsAg gene will be transformed to Coleus forskohlii for obtaining transgenic plants.
2.Testing of the integration and expression of the gene by various molecular techniques	The integration of the gene will be confirmed by PCR and Southern blot analysis and expression of the HBsAg gene in plants will be confirmed by SDS-PAGE and western blot analysis.
3.To study the feasibility of inducing immune response in hepatitis B animals by oral feeding / by injecting HBsAg being produced in transgenic plants and commercialization.	The animals will be immunized with the plant produced HBsAg protein by oral feeding and intramuscular injection and and tested for the immune response along with available commercial vaccine. Large scale recombinant protein produced in plants will be commercialized.
Objective 3: Transformation of crop plants with a gene encoding GAD 65, a major auto antigen in Insulin Dependent Diabetes mellitus via Agrobacterium
Approaches	Deliverables
Cloning of hGAD65 gene into a plant binary vector	GAD-65 gene will be cloned to plant binary vector under CaMV 35 S promoter for expression in plants
Standardization of protocol for the transformation of crop plants with GAD65 gene.	Transformation of crop plants with GAD65 gene by Agrobacterium mediated and biolistic methods.
Molecular characterization of integrated gene and its expression.	Transformed plants with GAD65 will be confirmed by PCR, Southern blot analysis
Immunization studies in NOD mice to estimate the insulin production	Recombinant protein from the plants will be isolated, purified and immunization studies will be carried in animals. The technology will be patented and sold to industries for large scale production.

Sl. No.	Item	Quantity	Year 1	Year 2	Year 3	Total
1	Enzymes, chemicals and glassware	As per requirement	15 lakhs	15 lakhs	20 lakhs	50 lakhs

Other items	Consolidated Emolument	Year 1	Year 2	Year 3	Total
2.3 Travel	Nil	0.75	0.75	0.75	2.25
2.4 Contingency	Nil	0.75	0.75	0.75	2.25
2.5 Overhead @ 15 % (If applicable)	Nil	589212	589212	664212	1842636

Sl No.	Title	Funding agency
1	Development of transgenic plants producing edible vaccine against Rabies virus	Department of Biotechnology, New Delhi
2.	Transformation of Groundnut with Glucanase Chitinase encoding genes using Agrobacterium tumefaciens	Indian council of Agricultural Research
3.	Testing the immunogenicity of Rabies glycoprotein produced in transgenic Groundnut crop	National Agricultural Technology Project
4	Scheme on immunization of mice and dogs with the rabies Glycoprotein produced in transgenic crops	Dept. of Biotechnology
5	Studies on glycosylation of rabies protein expressed in transgenic plants	DST/NSF
6	Transformation of crop plants with a gene encoding GAD-65, a major autoantigen in insulin dependent diabetes mellitus using Agrobacterium	UAS, Bangalore
7	Maintenance & training staff &students in using genegun and transformation of rice using genegun with Glucanase Chitinase gene against blast resistance	Kirk house trust, UK

Approaches	Deliverables
Isolation of microorganisms from different habitats and developing mutants. Screening of microbes for maximum ethanol production. Standardization of fermentation conditions for maximized product yield in crop residues.	Pure cultures will be ready for screening. Best strains will be tested for Ethanol production efficiency. The protocol for production of ethanol from different crop residues.

Project 1: Belowground biodiversity in sustainable agricultural systems

Project 2: Isolation of Beneficial Microflora from Weed Rhizosphere to improve

nutrient acquisition in crop plants.

Approaches and Deliverables

Objectives

Approaches and deliverables

5. Development of trait introgression lines resistant by crossing wilt and SMD resistant donor parents with agronomically elite cultivars.

Methodology

Sequencing of positive clones and primer designing and synthesis

Methodology:

E. Objectives: -

Objective 1: Cloning and expression of Challengevirus standard strain (CVS) Rabies glycoprotein gene in Pichia pastoris and plants and immunization of mice with the recombinant rabies glycoprotein

Approaches

Deliverables

1. Cloning and expression of challenge virus standard strain rabies glycoprotein gene in Pichia pastoris and plant binary vector

Yeast vector and plant binary vectors will be constructed with rabies glycoprotein gene

The expressed protein will be confirmed with SDS and western blot analysis.

The purified protein will be tested for its immunogenecity in mice and produced in large scale for commercialization.

Approach

Deliverables

The animals will be immunized with the plant produced HBsAg protein by oral feeding and intramuscular injection and and tested for the immune response along with available commercial vaccine.

Objective 3: Transformation of crop plants with a gene encoding GAD 65, a major auto antigen in Insulin Dependent Diabetes mellitus via Agrobacterium

Approaches

Deliverables

2.2 Consumables

Other items

PATENTS:

PROJECT -I

THEMATIC AREA –III: BELOW GROUND BIODIVERSITY

BELOWGROUND BIODIVERSITY IN SUSTAINABLE AGRICULTURAL SYSTEMS

PI: Dr. A.N. BALAKRISHNA

CO-PI: Dr. BALAKRISHNA GOWDA

Project X:

Isolation of Beneficial Microflora from Weed Rhizosphere to improve nutrient acquisition in crop plants.

RATIONALE:

1. K. Sankara Rao, Rohini Sreevathsa, Pinakee D. Sharma, Keshamma E and Udaya Kumar M. In Planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro. (2007). Submitted to Physiol. Mol. Biol. Plants.

3. Rohini V. K. and Sanakara Rao K. (2006). Recent advances in legume transformation. A Book Chapter on Legume transformation (to be published in 2007).

4. Rohini V. K., N. H. Manjunath and K. Sankara Rao (2005). Studies to develop systems to mobilize foreign genes into parasitic flowering plants. Physiol. Mol. Biol. Plants. 11(1): 111-120.

5. Sankara Rao, K and Rohini, V. K. (2003). Genetic transformation and regeneration of sandalwood (Santalum album L.) – In Plant genetic engineering Vol. 3; Improvement of commercial plants – I. Eds. Pawan K. Jaiwal and Rana P. Singh.

6. Sankara Rao, K and Rohini, V. K. (2003). Biotechnology of safflower (Carthamus tinctorius L.): Strategies for gene transfer – In ‘Plant genetic engineering Vol. 4; Improvement of commercial plants – II. Eds. Pawan K. Jaiwal and Rana P. Singh.

7. Rohini, V. K. and Sankara Rao, K. (2002). In planta strategy for gene transfer into plants: Embryo transformation (a Review). Physiol. Mol. Biol. Plants 8(2): 161-169.

8. Rohini, V. K. and Sankara Rao, K 2001. Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease. Plant Sci. 160 (5): 883-892.

Human Resource Development in Agricultural Biotechnology

Post Graduate Diploma in” Biosafety in Plant Biotechnology”

Sl. No	APPROACHES	DELIVERABLES
I	Isolation of Arbuscular mycorrhizal fungi from different agroclimatic zones of Karnataka	The number of isolates of AMF is quantifiable out put
II	Testing of different isolates on the different crop plants for their growth response	Establishment of suitable strains for different crops.
III	Genetic variability studies by AFLP and isozyme pattern. P transporter gene isolation and comparison with known P trans porter genes	Related ness of the strains with respect to their growth response. Gene sequences of different isolates could be determined.

Sl No	APPROACHES	DELIVERABLES
I	Isolation of Bacillus thuringensisfrom different soils of Karnataka	The isolates of Bacillus thuringensis are characterized for their specific activity on different type of insects.
II	Isolation of Bt genes	Bt genes isolated will be used in production of pest resistant transgenics.
III	Transformation of crop plants using Bt. Genes	The crop plants resistant to pests are tested for their insect resiatance and released for commercial cultivation.

Sl.No	Approaches	Deliverables
1.	Species composition of different functional groups of organisms like n-fixers, P-solubilizers, P-mobilizers like AM fungi, soil meso-fauna, in different cropping systems viz., mono-cropping, crop rotation, mixed cropping will be determined by following standard protocols.	This will give as an indication as to how soil organisms are associated with different cropping systems and crops
2.	Different functional groups of organisms will be mass multiplied and inoculated to plants or manage indigenous population by organic amendments.	Will enhance population of soil organisms and thus result in enhanced nutrient cycling and better soil fertility.
3.	Develop data base will be developed on species composition and determine various soil biological properties and relate to crop performance.	Indicators will be developed for soil fertility.

Crop	Variety	Character	Gene	Generation	Status
Field bean	HA-4	Insect resistance (Helicoverpa armigera and Spodoptera litura)	1. Chimeric cry gene	T₅ in progress	Screening of plants from 42 T4 lines is in progress
			2. cry1Aa3	T₃ in progress	4 lines identified so far
			3. cry1 Ac	T₃ in progress	3 lines identified so far

Sl.No.	Particulars	Proposed Outlay (in crore Rs.)
1.	Biotech innovation centre, Coordinating unit, mini-conference hall, seminar hall, incubator labs, bioinformatics lab	7.0
2.	Translation research centre , Contained field faciltity, transgenic containment, green house, insect rearing facility, microbial culturing unit, molecular characterization unit	9.0
3.	Biotech training centre, Training labs, lecture halls with video conferencing, mini auditoriums, dormitories, visiting scientist lab	2.0

Sl. No.	Particulars	Proposed Outlay (in crore Rs.)
1.	Instrumentation for all associated projects and labs, computers, AV equipments and minimal office communication equipment	8.0

Sl. No.	Particulars	Proposed Outlay (in crore Rs.)
1.	Recurring expenditure and contingency For chemicals, lab ware, consumables, and labour	9.0