DEVELOPMENT AND TECHNOLOGY TRANSFER
TISSUE CULTURE PILOT PLANT/MICROPROPAGATION TECHNOLOGY PARK
The Two Tissue Culture Pilot Plants achieved their targetted production and field evaluation continued. In order to effectively transfer the proven technology to entrepreneurs, utilizing the existing infrastructure and expertise of the Pilot Plants, two Micropropagation Technology Park (MTP) – Pilot Plants were supported. The MTPs are serving as a platform for transfer of proven technologies, which would include providing the required infrastructure, know-how training etc. Field data for the demonstration of tissue culture raised forest trees is being collected and analysed. Studies have also been taken up for development/refinement of tissue culture protocols of economically important species – especially horticultural crops.
Large scale production of the important tree species continued and over 40 lakh plantlets planted in an area of approx. 3500 ha. for field demonstration. The field data recorded indicated better performance of the tissue culture raised plants vis-a-vis the control. A preliminary cost benefit analysis indicated about 5 times higher return in the case of tissue culture raised plants.
Micropropagation Technology Park – NCL, Pune
Protocols were developed and upscaled for large scale micropropagation of different varieties of banana, sugarcane, turmeric and ginger. A single step process was standardized for micropropagation of sugarcane, turmeric and ginger. A single step process was standardized for micropropagation of sugarcane, replacing the normal two step process. Two thousand tissue culture raised plants of economically important varieties of Banana viz. Basarai and Srimati were field planted in a farmer’s field near Pune to get first hand information on field performance with respect to growth, yield, time of first harvesting etc. Micropropagation technology was developed and upscaled for three economically important varieties of Phragmites viz. P. karka, P. communis and P. australis. Phragmites is a common seed belonging to the family Graminae. P. australis is used for the treatment of many industrial wastes by the method called “The Root Zone” treatment. Five thousand tissue culture raised plants of P. australis were supplied to a private company situated in Pune. The technology on micropropagation of teak was transferred to International Plant Laboratories Ltd. (IPL), Baltonsborough, UK. Hands on training on micropropagation of teak protocol was imparted to a representative from IPL based on the agreement made between NCL and IPL. The technology has been well adopted at IPL and has successfully completed its agreement period. Technologies for three forest tree species and economically important horticultural and plantation crops are also being transferred to the Industry. In addition to transfer of technology for commercialization, the MTP is also providing a complete technology package on a turnkey basis to State Government Research Institutes/Universities. This includes setting up of the laboratory, species protocols and training. A major component of the MTP activities is training, being provided at all levels – scientists, technicians and students.
Micropropagation Technology Park – TERI, New Delhi
While continuing with the mass propagation of tree species, a large number of tissue cultured plants of various forest species were produced at the MTP. These plants were distributed to different forest departments and private growers for field trials as well as routine plantations. 50 clones of E. tereticornis were studied. Out of the various clones that were tested, three clones (No. 348, 360 and 2116) proved to be significantly superior at half the rotation age as compared to the conventionally raised control plants. Over 1.4 lakh plants of E. Tereticornis and about 24,000 plants of E. Camaldulensis were dispatched from MTP. Production of Dendrocalamus strictus through somatic embryogenesis progressed well. The main problem associated with D. Strictus protocol is its high sensitivity to media ingredients, subculturing time and the ability of technicians to distinguish and separate out embryogenic calli from non-embryogenic calli. To simplify the production process, a micropropagation protocol by axillary branching method using seedling was worked out. 4.1 lakh plants of P. deltoides were despatched from MTP facilities. Field results suggest that the cuttings derived from tissue cultured plants have a better root system and are more vigorous. This might be due to rejuvenation of clones under in vitro conditions. In a field trial being carried out at National Research Centre for Agroforestry, Jhansi two genotypes of A. pendula multiplied through tissue culture have shown a mean annual increment in height of over 1 m. Considering the fact that A. pendula is a very slow growing and recalcitrant species, this gain in height is very significant.
The existing productivity levels of most of our fruit crops are among the lowest in the world. This problem is severe in crops that have been perpetually propagated vegetatively and have accumulated several diseases. To improve the quality of the planting material, a number of horticultural species were taken up. Nearly 22,000 tissue culture plants of banana were distributed to various growers who are likely to be benefitted both by the disease-free nature of the planting stock as well as the superiority of the clones. The two varieties of banana presently being multiplied are Robusta and Dwarf Cavendish. Test results of tissue culture raised plants in the field indicate clonal uniformity and comparatively higher yields. Mass propagation of strawberry was undertaken. During 1997-98 over 85,000 plants were distributed to various growers for commercial use. In 1998, over 1,00,000 strawberry plants were distributed to growers from MTP. Mycorrhizal associates for strawberry were isolated. Tissue culture propagated plants are supplemented with mycorrhizae at the nursery stage.
In order to popularize tissue cultured plants, a nursery meet and a landscapers meet was organised during this year. Nearly 50 nursery men and landscapers from leading nurseries in Delhi, Noida and Faridabad participated. They were appraised of the benefits/advantages of tissue cultured plants over conventionally raised plants. During the meet, the nursery owners shared their views about the technology.
Technologies for fire forest and horticultural species were under transfer to the industry for commercialization. Training was provided to scientists, technicians and students.
TOTAL AREA COVERED UNDER FOREST TREES
TISSUE CULTURE LARGE CARDAMOM – PRODUCT PLAN
demonstration project on evaluation of the performance of tissue cultured
plantlets of large cardamon (Amomum subulatum) vis-a-vis open pollinated
seedings in planters’ field in Sikkim (52.5 ha) and West Bengal (17.5 ha)
continued. During 1998 planting season, an area of 12.25 ha in 49 units
was field planted as per details given below :
A total of about 1,24,000 tissue culture plantlets and 40,000 open pollinated (OP) seedlings are being maintained in four nurseries which will be field planted in about 140 units during 1999 planting season. Multiplication of four elite clones through tissue culture and raising of OP seedlings for field planting during the year 2000 are in progress. Three training programmes were organised for the beneficiaries of the project and also to those who have shown interest in taking up planting during the next season. More than 100 farmers participated in the above training programmes.
TISSUE CULTURE VANILLA – PRODUCT PLAN
The project envisages evaluation of the performance of tissue culture plantlets of vanilla (Vanilla planifolia) vis-a-vis stem cuttings in planters’ field over an area of 150 ha in the states of Kerala (60 ha), Karnataka (60 ha) and Tamil Nadu (30 ha). An area of 39.25 ha during the last two planting seasons – 1996 (5.50 ha) and 1997 (33.75 ha) was planted. During the 1998 season, an area of 44 ha covering 176 units was planted in three states. The details of statewise coverage is given below in the table. The balance target area in the project will be covered during the 1999 planting season. Field nurseries comprising tissue culture plantlets of MCV-1, MCV-2 and MCV-3 were established at various centres located in Kerala, Karnataka and Tamil Nadu and 56,320 tissue cultured plantlets and corresponding stem cuttings (14,080) supplied for planting in the farmers’ farms during 1998 season. Field visits were made for providing technical guidance and to record growth parameters.
of field transfer of tissue cultured vanilla plants during 1996, 1997 and
1998 planting seasons:
The observations made from the plots of 1996 and 1997 planting revealed that stem cuttings show initially better performance in vine length than tissue cultured plantlets. The number of nodes and leaves were more in tissue cultured plantlets of 1997 planting in Trivandrum, Idukki and Wynad zones of Kerala. Four training programmes on cultivation and processing of vanilla have also been conducted during the year at different zones (Idukki, Wynadu, Trivandrum and Calicut) identified in the project. In all, 216 beneficiaries/farmers participated.
BIOTECHNOLOGY – BASED PROGRAMME FOR SC/ST POPULATION
About 35 project have been implemented with a view to providing training, demonstrations and extension activities.
Aromatic and Medicinal Plants : The Itanagar branch of RRL, Jorhat implemented a project on “Promotion and cultivation of aromatic plants and production of essential oils for the benefit of tribal population of Arunachal Pradesh”. Its main objective is to motivate local people through training and demonstration to take up organised cultivation of potential aromatic plants in Arunachal Pradesh. 224 local tribal people were trained on cultivation and processing of Citronella, Lemongrass and Patchouli. The planting material of citronella has been grown in 9 ha. area and about 1.7 lakhs Citronella seedlings have been distributed to beneficiaries belonging to villages of Arunachal Pradesh. In addition, a nursery of 4000 seedlings of patcouli was raised for distributing planting material free of cost to the tribals. These efforts have resulted in cultivation of aromatic plants in more than 50 ha. area at 5 different locations. The institute is also setting up 4 processing units for essential oil extraction at 4 locations. In an another project at Krishi Vigyan Kendra (KVK), Pal, dist. Jalgaon (MS) nurseries of Citronella, Lemongrass, Palmrosa, Ashwagandha, Isabgol, Safed Musali and Costus has an area of 5 ha. and planting material/seed distributed to 270 farmers who raised crops of medicinal and aromatic plants at their 32 ha. land. The project has helped the farmers to increase their farm income 2-4 times. More than 1000 people have benefited through training, field demonstrations and cultivation of aromatic/medicinal plants. A distillation unit has also been established for Citronella and Lemongrass.
The Central Institute for Aromatic and Medicinal Plants, Lucknow introduced cultivation of rose and geranium and their distillation for commercial oils in UP hills. The institute has raised a nursery of 3000 rose and 2500 geranium plants at its Purara farm near Baijnath in Dist. Bageswar for supplying planting material to the SC/ST farmers. The institution conducted training courses and apprised 19 farmers, on economic details, the methods of cultivation, marketing etc. of rose and geranium and other supporting crops like palmrosa, tagetes, lemongrass through lectures and live demonstrations at Purara farm.
The IHBT, Palampur, has encouraged local SC/ST farmers to cultivate the Tagetes minuta and Rosa demascena and extraction of their essential oils at commercial levels. A multi-purpose direct fired essential oil distillation unit of 4 quintal capacity was installed at village Jhiri in dist. Mandi has been formally handed over to the village Panchayat Jhiri, and it is now being used by the villagers. During November, 1998, 50 kg of Tagetes minuta oil worth Rs. 1.00 lakh was produced by this unit. Another multi-purpose 1.5 quintal capacity field distillation unit for distillation of fine grade oils was fabricated and being installed at Dharamsala dist. Kangra. Chlorophytum borivilianum (Safed musli) is an important medicinal plant comparable to Korean Zinseng. Gyan Bharti Trust, Udaipur has undertaken a project for procuring musli tubes from identified sources, multiplying the same at its Tapovan Ashram premises and lay out demonstrations on fields of tribal farmers after imparting training in its cultivation practices. The Trust conducted 20 demonstrations in fields of the trained tribal farmers.
The Ayurved Gramoudyog Sodh Sansthan, Jodhpur raised Withania somnifera and Cassia angustifolia plants in 21 ha. land belonging to SC/ST farmers. It has also raised a nursery of nearly 500 plants of Commiphora wightii which would be transferred in fields in next 2-3 months. Grama Nava Nirman Samithi, Hyderabad implemented a project on cultivation and distillation of aromatic plants viz., Palmarosa, Citronella & Mentha for the benefit of SC/ST women. Navalbhau Pratisthan, Navalnagar dist. Dhule (MS) has also undertaken a project on aromatic and medicinal plants.
Floriculture: The Central Agricultural University, Imphal implemented a project for training SC/ST youths of Manipur in orchid production. It has developed a protocol for micropropagation of popular tropical Dendrobium spp. And trained about 80 unemployed SC/ST youth for 10 days on commercial cultivation of hybrid orchids. The university developed 5 hybrids involving local spp. Renanthera, Vanda and Cymbidium. Efforts are underway to develop hybrids from the local orchid spp. Which have qualities of cutflower orchids and are at the verge of extinction.
Guwahati University took up project on application of in vitro propagation techniques in multiplication of cultivars of Chrysanthemum and Gladiolus for large scale planting, training of SC/ST rural youth and field demonstration to demonstrate floriculture as a source of self employment and income generation.
Mushroom Cultivation : The M.S. Swaminathan Research Foundation Community Agrobiodiversity Centre, Puthoorvaya in Wayanad distt. in Kerala surveyed the district for the distribution pattern of mushrooms and trained 25 SC/ST youths of the selected villages on mushroom cultivation, post harvest technology and development of value added products. The trainees were also taken to nearby agricultural research institutes and imparted skill in spawn production. The center also collected few mushrooms used in traditional medicinal system for treatment of Jaundice, ear ache, inflamation, dehydration etc.
The Centre for Rural Development and Technology IIT, New Delhi imparted training in growing edible mushroom to about 100 persons from two villages in Gurgaon and Rohtak distt. of Haryana. During the last two seasons, about 1000 Kg mushroom was produced by the beneficiaries as a kitchen garden activity for their consumption and as a cottage industry on co-operative basis for marketing. In a project on Demonstration of mushroom cultivation for the benefit of SC/ST in Eastern U.P., 90 persons from 3 selected villages of distt. Faizabad were involved in mushroom cultivation and they were acquainted with the economics and nutritional value of mushrooms and recipe preparations. Most of them started cultivation of Pleurotus sajor caju in their own houses.
Vermiculture and Vermicomposting: The Gandhigram Rural Institute, Gandhigram (TN) made efforts to popularize vermicomposting alongwith sericulture (moriculture & silkworm rearing) through field demonstration and training as revenue generation programme. The institute trained 120 persons from 25 villages. These persons have already set up 10 vermiculutre and 6 sericulture units, which are functioning successfully and also encouraged a large of farmers to venture in this area. Krishi Vigyan Kendra (KVK), Nanded (MS) has trained 100 farmers from nearby 10 villages on the vermicompost production through bioconversion of agro-residues, and its use in agricultural and horticultural crops. Most of these farmers were reported to produce and use vermicompost in their fields. A vermicomposting biofertilizer productioon-cum-training project is also being implemented at Kohima in Nagaland. Under this project, 100 unemployed youths were trained on different aspects of vermicompost production, and its application demonstrated in potato crop.
Quail Farming : The Regional Research Laboratory (RRL) Jammu continued its demonstration project on disease free and better quality quail broiler production for meat for the benefit of tribals to help the poor tribal masses. Around 360 persons belonging to 3 distts. Namely Jammu, Kathua and Udhampur were trained on quail production covering brooding, rearing, feeding schedule, fabrication of cheaper cages and litter system from locally available raw material. The project resulted in the establishment of 32 small production units (100-200 birds each) by the beneficiaries in 14 villages in the 3 distts. From these small units each beneficiary got a profit of Rs. 400-Rs.800/- per lot and training for better care and management of Japanese quails (Coturnix coturnix japonica).
Biological Control of Plant Pest and Diseases : The Tamil Nadu Agricultural University trained 130 farmers in pest and disease identification, collection of crop pests for the production of biopesticides, laboratory host Corcyra rearing for the production of egg parasitoid, Trichogramma chilonis, production of botanical pesticides, antiviral principles and nuclear polyhedrosis viruses of Spodoptera litura and Helicoverpa armigera. In addition, 174 farmers were trained in a one day programme to create awareness on the hazards of chemical pesticides and benefits of biopesticides in Integrated Pest Management (IPM). The project covered a total of 7 villages of Madurai and Virubhunager distts. In Tamil Nadu.
Botanical pesticides from neem, Pongamia, Vitex, Ipomea, Jatropha, sps. Etc. were used in brinjal, bhindi, tomato and redgram for the control of pests and diseases. In rice fields, sprays were given with non-edible oils like neem, Pongamia and Madhuca at 3% individually and in combination at reduced rates, and compared with one insecticide and untreated control. The rice diseases and pests controlled by these methods were demonstrated.
NPVs B.t and fungal pathogens of insect pests, viz. Beauveria bassiana, Paecilomyces spp., Verticillium lecanni, etc. were used in cotton, vegetables, pulses and oilseeds. The egg parasitoid, Trichogramma chilonis was used in the management of lepidopteran pests of cotton, vegetables, oilseeds, etc. Pheromone traps were used for monitoring and control of Helico-erpa armigera and Spodoptera litura in cotton, vegetables and pulses. The TNAU covered a total of 22 ha. crop area of tomato, brinjal, bhindi, redgram and groundnut under the project for field demonstration on the use of various biological control agents and biopesticides.
KVK, Parbhani (MS) produced biological control agents viz., Trichogramma chilonis (parasite), Chrysoperla carneia (predator) and Nuclear Polyhedrosis Virus (antomopathogen) on laboratory scale. A total of 82 lt. HaNPV, 55 lakhs Trichogramma and 10 lakhs Chrysoperla were produced and used in IPM of pigeonpea, cotton as well as chickpea. KVK has conducted field demonstrations on the use of various bioagents covering HaNPV on 82 ha., Trichogramma on 37 ha. and Chrysoperla on 20 ha., A one month training was conducted for 9 persons on the production biological control agents. Shri Dhaneswari Manav Vikas Mandel, Kalamb distt. Osmanabad (MS) also has trained around 600 persons from the targeted groups on the production of HaNPV, Trichogramma card production, production of Cryptolemus, neem powder and urea coating with neem product. It has conducted field demonstrations on the use of these biological agents in IPM in Jawar & Cotton crops at 12 farmers’ plots which were visited by about 1000 farmers from nearby areas.
Aquaculture: Oriental Research Foundation, Bhopal implemented an aquaculture project to increase employment and improving economic status of weaker sections in Hoshangabad dist. In M.P. The facilities for fish seed rearing available with Central Institute of Fisheries Education, Powarkheda were utilized under the project. Seeds of major carps were reared upto the size of 50 mm fingerling at Chandrapura and Powarkheda farms, and healthy free seed supplied to 20 co-operative societies working at various perennial and seasonal tanks in different distts. of the state. The seed was also stocked in Khoripura, Dewas and Goharganj tanks. A total of 1,64,000 fingerlings were stocked in these tanks and about 16 tonnes of fish harvested and 50 people were benefited as each of them earned about Rs. 6,600 to Rs. 10,000 per year. KVK, Karda dist. Washim (MS) continued its demonstration project on aquaculture in the tribal areas. It procured fish seed of Indian major carp, grass carp, silver carp and common carp, and stocked in the nursery pond at Karda for transferring to 10 selected seasonal and penile ponds. In a series of training courses conducted on different aspects of the fish and prawn culture, 80 persons were trained. Assistance was provided to the persons trained in the previous year to start their fish farming activities. With these KVK efforts fish production could be raised upto the level of 3000-3500 kg/ha/yr. With the application of scientific and technical know-how, against the yield of 500-600 kg/ha/yr. With traditional practices. The St. Anthony College, Shillong took up a project on the production of fish seed and rearing of major carps and catfish. The project will cover induced breeding, rearing and training. It is expected that the project would help in improving the financial status of the fish farmers of the region.
Health Care: All India Inst. Of Medical Sciences, Sir Ganga Ram Hospital, New Delhi with the District Collectorate, Department of Tribal Development and Health, Wayanad and the Foundation of Fetal Research, Channai initiated a control programme for sickle cell disease in tribal population of Kerala. The first phase of the screening of the tribals was started in September, 1998 with daily visits to the villages, and haemoglobin electrophoresis at two base laboratories are currently in progress. About 15,000 subjects were covered. The ultimate goal of the project is to use biotechnological tools to develop and execute a community control programme for this dreaded disease.
Indian Women Scientists’ Association and Institute of Immuno-haematology, Mumbai continued working on the project for training and education of tribal women for improving their health and economic standards. 10 tribal villages of Ahmednagar district, each having the population of 500-1500 tribals were selected, and 10 semi-illiterate women one each from the villages were trained in workshops held at Rajur and Bhandardhara. In the workshops, information on aspects such as gastro-intestinal diseases, skin diseases, respiratory infection, tuberculosis, tetanus, malaria, vitamin deficiencies, snake bite, purification of drinking water, immunisation, nutrition etc. was given in simple language with the help of posters. Vivekananda Institute of Medical Science, Calcutta continued the study on the use of biotechnology dietery derived supplements in improving health status of SC population of three villages in West Bengal. The study has covered 3 adjacent villages of South 24-pragna distt. of West Bengal. During the surveys, it was found that 62 children out of total 229 and 79 mothers out of 151 had sign and symptoms of Vitamin-A deficiency. Of them, 14 children and 26 mothers were found with histological evidence of Vitamin-A deficiency as evident from examination of their conjuctival swabs. It was observed that in case of children after giving Spirulina preparation (500 mg twice daily x one month) the specimen of their conjuctival swabs became normal, and in case of mothers also the swab reverted to normal after one course of Spirulina (1 g twice daily X one month). Vivekananda Institute of Medical Sciences has also taken up recently another project on studies of genetic disorders to provide counselling with special reference to hemoglobinopathies in the tribal population of Tripura. The project is focussed on screening of the population to assess the extent of genetic diseases (HbE betathelisinia) among the tribals and educate them in gene related diseases.
BIOTECHNOLOGY BASED PROGRAMME FOR WOMEN AND RURAL DEVELOPMENT
The Task Force me twice recommended fifteen new projects and reviewed the ongoing ones.
Food Processing: Under The Project proposal entitled “Storage and preservation technologies for agriculture produce of U.P. hills”, plants for horticultural importance were identified keeping in mind the agro-climatic data of area, land specific information, people based information, present availability of fruits and vegetables, list of conventional and non-conventional fruits, seasonal availability of different fruits and traditional method of preservation of fruits/vegetables. Tow training programmes were organised and around fifty women were trained for setting up their units for processed food products using the local horticultural produce.
In another project entitled “Empowering women through entrepreneurship development training”, the first training programmes was conducted for 15 unemployed women graduates. The intensive training course included lectures on entrepreneurship development, marketing, setting up a small enterprise, procurement of working, capital, loan from bank and other agencies, raw materials for food processing industry, aspects of finance, steps on setting up of industry, legal aspects, hygiene at work place & maintenance of equipments. The hands on training were given for two weeks. These trained women were taken to various units. Some of them are in the process of setting up their own enterprises.
Waste Land development : To cultivate Emblica in `Usar’ soil of U.P. seeds of Emblica officinalis and training were provided to the beneficiaries. The beneficiaries were allotted the nursery trees to keep the plants viable and growing. Eighteen training programmes were arranged including one major camp. The trainees have also been taught about the various properties of `Usar’ soil.
Waste Management : In Vermicomposting and Mushroom cultivation to utilize waste material, selected rural women/families were trained at their respective places on different aspects of two technologies through lectures and demonstrations, site visits and distribution of popular literatures. The families are being provided the requisite material and guidance for technology implementation and marketing of the produce. Six public contact programmes on Vermicomposting and Pleurotus (Mushroom) cultivation were organised.
Floriculture : Two projects were supported on tissue culture assisted floriculture in selected village panchayats of Kerala at TBGRI and Federation of Floriculturist of India (FIF), Trivandrum and “Micropropagation of ornamental plants of Attipra panchayat” at Kerala University. These projects deal with tissue culture of Orchids, Anthurium and other ornamental flowers. TBGRI has already trained 25 beneficiaries from Venganoor and 10 from Sreekaryam Panchayts. Orchid and Anthurium cultures from selected hybrids of commercial value were initiated and multiplied. About 50 plants are already established in the green house. Two training programmes have been organized by Kerala University twenty women were trained in micropropagation of ornamental flowers.
Sericulture : During the first phase of project, techniques for rearing of silkworms species 5dfls of CB (PM x KA) and two hybrids KA x NB4D2 OR NB7 x NB18 were perfected. Beneficiaries from non-traditional areas were identified. Demonstration units at beneficiaries’ land are being set up.
Medicinal Plants : To prepare an inventory on medicinal herbs of Garhwal Himalayas, a project is being supported for collecting and compilation of information on herbs in the villages of three districts Dehradun, Chamoli and Rudraprayag. Information has been compiled on the villagers, market value of different herbs, abundance of herbs available in the region in different seasons. Based on market demand, nursery preparation for Mentha arvensis (Pudina), Withania somnifera (Ashwagandha), Rawolfia serpentina (Sarpgandha), Potentilla fulgeum (Bajradanti), Sarpetina (Sarpgandha), Piccrorhiza cusava (Kutki), Centella asiatica (Brahmi), Euphorbia thymifolia (Chopalu) has been started.
Human Health, Genetic Disorders and Counselling : The project on “Role of Biotechnology awareness in prevention of genetic disorders among rural women”, is under operation at Sanjay Gandhi Post-Graduate Institute (SGPGI), Lucknow. Surveys have been conducted to identify genetic disorders in the villages. Blood samples were collected and being analyzed by trained nurses/mid-wives and counselling to the affected families are being provided.
Biotechnology Based Product and Process Development : Development and transfer of the process know-how for mosquitoes and household pests repellents/herbal formulations for women welfare” is being pursued by CIMAP, Lucknow. The products are floor mopping emulsion, mosquito repellent agarbatti & dhup batti. Unemployed women and youth of 3 blocks of Bakshi Ka Talab have been selected for training in cultivation and product development.
Environment Protection and Awareness : One project is being implemented to provide training to the rural women in the environmental protection through bio-monitoring of water pollution and other associated problems. The Biosensor technology developed at Vishvabharti University, West Bengal was adopted for rural application. Regular visits to the villages, were also conducted to develop awareness and motivation for the use of bio-technological tool for monitoring the level of contamination through pesticides in their agriculture land and water bodies.
Golden Jubilee Women Biotechnology Park : “Golden Jubilee Women Biotechnology Park” at Kelambakkam, Chennai was inaugurated by Honorable President of India on July 29, 1998 for celebrating Golden Jubilee Year of country’s independence.
The park aims to provide opportunities for professionaly qualified women to take a career of remunerative self-employment through the organization of environment friendly biotechnological enterprises. The park will be designed on the principle of decentralised production supported by appropriate centralised services to promote a series of high tech biotechnology based enterprises aiming to capture a number of niche markets in the areas of Agri-biotech, Food biotech, Medical biotech etc. The R&D institutions, the corporate sector and the financial institutions would assist the women entrepreneurs in achieving the objectives of the Park. The Park will serve as a model to foster the technological and economic empowerment of women. Twenty acres of land have been provided by the Tamil Nadu Government at Kelambakkam, Chennai for the park, Eight prospective entrepreneurs have been selected to begin with for producing ornamental fish, Collagen, Vitamin and Mineral Premixes, Entero rapid biochemical testing system, Tissue culture/Micropropagation of Plants, Bioconversion of waste into organic manure, Spices mixture and Spirulina.
“A Colloquium On Biotechnology for Women : A Vision for 21st Century”: To celebrate International Women Year a Colloquium was organised at NII, New Delhi 7th November, 1998, was inaugurated by Prof. Archana Sharma. The 7th November, 1998 being the birth day of Madam Curie, the first women scientist who bagged two Nobel Prizes was selected. About 130 scientists and technologists actively participated and deliberated in the areas like agriculture, health, environment, industry, basic sciences and human resource development. Important views were expressed on technological empowerment of women using suitable biotechnology.
Model Biovillage Project (Mocha-Gorser, Porbandar Gujarat) : A model Biovillage project was formally announced by the Hon’ble Minister for Science & Technology Dr. M.M. Joshi during the inauguration of S&T exhibition on 3rd August 1998 at the National Stadium, New Delhi. This was one of the events planned by the Department of Biotechnology to commemorate the Golden Jubilee of India’s Independence. The selection of the site was done keeping in view the significance that this is the birthplace of the Father of the Nation. The lead agency Central Salt and Marine Chemical Research Institute, Bhavnagar coordinated with various other agencies to launch the program. The other collaborative agency for this project are Meteorological Department, Porbandar; Gujarat Agricultural University, Junagarh; National Research Center for Groundnut, Junagarh; Regional Centers of Central Leather Research Institute, Ahmedabad; National Environmental Engineering Research Institute, Ahmedabad; Birla VXL Industrial Ltd., Porbandar, Distt. Panchayat. The activities undertaken at the site are managed through the Panchayat of Mocha-Gorser.
The Project has progressed well and sites were selected for desalination plant, wasteland plantation, carcass disposal-cum-training center and marine algae cultivation. Nursery for waste-land plantation (Salvadora, Jojoba, Jatropha) has been raised and ready for plantation in wasteland plot of the project village; Desalination plant is ready and installed in January 1999 at project village. Blue-Print for carcass disposal-cum-training center is ready. Farmers Training was organised for use and production of Biofertilizers and Biopesticides. About 30 farmers took training at Gujarat Agricultural University Campus, Junagarh for 2 days (10-11 Dec. 1998).
BIOTECH PROCESS ENGINEERING AND INDUSTRIAL BIOTECHNOLOGY
A number of projects directed towards product and process development have been supported.
Role of interfacial concentrations in reverse micellar biotechnology: Working on the role of the interfacial concentrations in reverse micellar, the investigating group at IICT, Hyderabad observed that the interfacial basicities of cationic reversed micelles are remarkably less that that of normal aqueous cationic micelles. Such interfacial basicities of reversed micelles are likely to be one of the key factors controlling the catalytic efficiencies of biocatalysts located in the interfacial region of the reversed micelles such as lipase. An experimental protocol for probing the local molar concentrations of water and bromide counter-ions present inside the water pool of a cationic reversed micellar system was developed. A novel chemical method was developed for sensing the remarkable exo-surface basicities of cationic liposomes.
Microbial production of plant growth regulators : The significant achievements in the project at NCL, Pune are isolation of actino-mycetes and azotobacter strains producing plant growth regulators. Extraction methods for isolation of cytokinin-like products from culture broth were standardized. Bioassay methods for detection of cytokinin-like activity in crude broth, crude extract and prep TLC fractions were also standardized. It is observed that a considerable number of actinomycetes and azotobacter isolates are found to produce cytokinin-like activity in minute quantities.
Scale up of A.rhizogenes mediated transformed hairy root cultures: Experiments conducted at CIMAP, Lucknow in bench top fermentor showed that the stagnation or clumping of hairy root mass at certain regions of the fermentor resulted in inadequate mixing which limited the growth of hairy roots. Experiments conducted on the culture of hairy roots in the fermentor led to the conclusion that the bio-reactor needs certain modifications. These modifications in the bio-reactor yielded 1.65 to 1.78 times higher bio-mass when compared with the yield of previous experiments.
Extraction of Penicillin-G from fermentation broth : The work was carried out at NCL, Pune. The solvent extraction of Penicillin-G from aqueous broth was optimized by using amine extractants. The extraction was optimized for pH 7.0 and high recoveries got. The back extraction of solvents, which initially posed a big problem was optimized in a single stage recovery. Other conditions optimized for the amine extractions were pH, temperature, ionic concentration and relative concentrations of the penicillin, amine extractant. The penicillin was extracted from aqueous phase using a membrane extraction device, developed at NCL, Pune. More than 70% extraction efficiency was obtained using a recycle operation.
Characterization of human breast tumor associated antigen : Human breast tumor associated antigen (BTAA) was purified from 28 breast tumor tissues at NCL, Calcutta. Three DEAE fractions (HF1, HF2 and HF3) were obtained and presence of the tumor associated antigen in the fraction was determined by screening the breast cancer patients sera against the DEAE fractions. It was observed that the female breast cancer patients presented antibodies against HF1 whereas male breast cancer patients had antibodies against HF3. BTAA purified from the HF1 by HPLC was of 85 kDa and HF3 was 72 kDa protein. The BTAA is being used for screening of sera samples obtained from breast cancer patients.
Optimization of methods of biosynthesis of plant lipoxygenase based bioactive lipids : In a study at the University of Hyderabad, Lipoxygenase (LOX) activity levels were measured in different plant parts of varied species. Highest LOX activity was observed in rapidly growing seedlings of green gram, followed by pigeon peas and other species of leguminaceae. Potato tubers also recorded significant production of LOX. LOXs from pigeonpeas, greengram and potato tubers were purified by employing conventional chromatographic as well as HPLC techniques. Different substrates resulted in different lipoxygenase product profiles in green gram/pigeon pea. The purified lipoxygenase products were tested for their anti-fungal effects on the wilt fungus. Preliminary reuslts indicated that the LOX metabolites are highly effective in inhibition of growth of the fungus.
Scale up of hairy root cultures in specially designed bioreactor and down stream processing for the production of high value phytochemicals: Hairy root cultures of Beta vulgaris for production of betalaines, Tagetes patula for production of thiophene were established at CFTRI, Mysore and experiments conducted to study the influence of polyamines and microbial elicitors on the growth of hairy root cultures. Several kinetic parameters which influence large scale cultivation in bioreactors were studied at shake flask level focussing mainly on the aspects such as growth curve, bio-mass maxima, kinetics of nutrient depletion, effect of inoculum density, effect of larger volumes and product accumulation. A prototype bioreactor was designed.
Bioreactor design: role of hydrodynamic shear on enzymes: Investigations were taken up at UDCT, Mumbai on the measurement of turbulent flow in stirred bio-reactor using Laser Doppler Anemometers (LDA), design of impellers, Computational Fluid Dynamics (CFD), etc. About 40 different impellers have been fabricated and the fields generated by these impellers measured using LDA. A combination of experimental LDA measurements and CFD simulations have been enabled prediction of the impeller characteristics like flow numbers and power numbers offer different stresses even when compared on the basis of equal power consumption per unit mass. A combination of LDA measurements and CFD simulations thus offer a possibility to compare different impeller designs which may lead to development of optimum impeller geometry which would offer less hydrodynamic shear, yet maintain good oxygen transfer rates.
Mass inoculum production of Ceriporiopsis subvermispora and Phanerochaete chrysosporium for bio-bleaching and bio-pulping: The work at University of Delhi involved morphological characterization of C.subvermispora on different media, effect of physical factors on sporulation in minimal medium, effect of nutritional factors on sporulation etc. Based on the results, conditions were standardized for inducing maximum sporulation in C.subvermispora. Methods were also standardized for separation of viable spores.
Development of high flex-life polyurethane for use in cardiovascular devices: The main objective of the investigation at SCT, Trivandrum is to develop blood compatible and biodurable (high flex-life ) poluurethane for use in cardiovascular devices. Hydrophobic poluurethane-urea HFL 18-PU9 was prepared using discyclohexyl methane diisocyanate, hydroxy terminated polybutadience and hexamethylene diamine and subjected to accelerated chemical degradation and accelerated flexural fatigue tests. Accelerated chemical degradation tests, weight loss determination tests, accelerated flexural fatigue tests were conducted. It was found that the candidate polyurethane HFL 18-PU9 survived 720 million cycles of flexing without weight loss, which is equivalent to 18 years of survival in human being. The studies reveal that the polyurethane HFL 18-PU9 has the potential application for use in cardiovascular devices.
Value addition to foods, development and presentation technologies and packaging of foods, biosafety have been the areas of focus.
Production of low cholesterol egg: At CFTRI, Mysore supercritical carbon dioxide extraction technique was followed to reduce the cholesterol in liquid egg and spray dried egg powder. Isopropanol treatment helped in breaking the lipoprotein binding and facilitated extraction of cholesterol in liquid egg by supercritical carbon dioxide technique. This method was found to remove cholesterol from liquid egg. The recovery of cholesterol was high (90.0%) in spray dried whole egg power (without isopropanol treatment). Phospholipids were not observed to be affected.
Biodegradable ecofriendly packaging films: In vivo biodegradation studies chitosan and cross-linked chitosan films was carried out at CFTRI, Mysore in soil collected from four different locations and at different RH values. The soil samples were cleaned and films of size 2X$ cms. were weighed and buried inside the soil at both constant and varying RH values. From the percent weight recorded regularly, type –4 soil showed 22 and 49% weight loss, respectively for chitosan and cross-linked chitosan films after 7 days. After 2 weeks, complete disintegration of films in soil types 1 and 4 was observed but C in soil types 2 and 3 it was moderate.
At RH of 25% and above, crosslinked chitosan film was observed to be degraded completely, whereas the chitosan film was only partially (12.5%) degraded. 50% RH it was found to be completely degraded. Cross-linked chitosan film required comparatively less moisture to undergo degradation in vivo in the soil. Both cellulose and starch based films being very hydrophilic, were observed to be degraded and disintegrated in less than 48 hr. Significant shelf life extension at ambient and cold storage temperature was observed by use of coating formulations in various fruits.
Culture and cultivation of seaweed for food : Studies were conducted at CSMCRI, Bhavanagar. The germplam of Porphyra (nine species) and Monostroma (one species) occurring on the Indian coast was studies and maintained in laboratory cultures which were optimized for growing all the species of Porphyra and Monostroma. The studies on the life cycle of all seaweeds were completed in the laboratory cultures. The biochemical analyses (ash, protein, carbohydrates, total carbon and nitrogen) including mineral (Na, Ca and K) and trace elemental constituents of all the species, which occur abundantly, were carried out. Studies on organic nutrients like amino acids, lipids and fatty acids are in progress. Attempts are under way to conduct field cultivation of above-mentioned seaweeds on pilot scale. It is expected to develop cultivation technology package as well as process for making seaweed foods from Indian plants.
Development of process for flavours & food additives: Various strains of Trichoderma viride were screened for 6 penty 1 –alpha-pyrone (6pp) which is a secondary metabolite at CFTRI, Mysore. Optimization of cultural and nutritional parameters was carried out under submerged fermentation conditions. The highest level of 6 pp obtained was in the range of 400-500mg/I. Solid substrate like defatted coconut gratings, wheat bran etc., though supported good mycelia growth showed predominance of methyl ketone production with no production of 6pp. A process at the lab scale level has been developed for the production of 6-penty 1 –alpha-pyrone. Procedures were standardized for producing flavour nucleotides by the in-vivo digestion of nucleic acid contents of the food grade yeast by exogenously added enzyme. Reaction conditions have also been optimized for the conversion of d-lemonene to recemic carvy1 acetate with 65% yield using microbial lipases and for the enzymatic preparation of important terpene esters.
Development of a process for Xanthan gum by fermentation: The bench scale data obtained in shake flasks were successfully scaled-up to 50-L scale in a pilot fermenter at CFTRI, Mysore. Xanthan yield of 60-70g/100 g of sugar supplied was obtained in 72 hours. The media was optimized to replace the expensive organic nitrogen source with inexpensive ammonium nitrate and corn steep liquor. The complex problem of mass transfer and agitation of high viscosity was tackled by appropriate impeller design. The xanthan product obtained compared favorably with the commercial sample. The product was applied successfully in selected foods such as fruit squashes, extruded products, traditional foods like, Idli etc. Toxicity testing studies showed no adverse effects. The process has been demonstrated and an Indian industry has agreed to be a partner for the development towards commercialization of the process know-how.
Microbial Bio-transformation of Terpenes of High valued Food Flavours : Two fungal cultures, Penicillium Sp. and Aspergillus Sp. showing potential for biotransformation of terpenoids to high valued food flavorings were selected after screening at CFTRI, Mysore. The Penicillium sp. was observed to convert with high efficiency, limonene to carveol under the optimized for conversion of alpha-pinene to verbenol and verbenone by Aspergillus sp. The yield observed was 1.2 mg off verbenol from 20 mg of alpha pinene.
Strain improvement attempts were made by subjecting the fungal cultures to UV irradiation and treatment with ethyl methane sulphonate and colchicine. Some of the variants showed marked morphological differences which were reflected in the product yield. An Aspergillus variant caused biotransformation of alpha-pinene with a yield of 4.56 mg% of verbenol and a UV derived Penicillium variant caused formation of 3.7 mg% verbenol which amounted to about 15 and 8 fold increases respectively in the yield of products. Based on the results of laboratory studies, large scale trials on the verbenol formation by Aspergillus sp. and its UV variant were carried out in 2 litre fermentors and a yield of 3 mg% and 5.3%, respectively was obtained.
Biotechnology research for food safety : The project jointly supported by the Department of Biotechnology and Ministry of Food Processing Industries at CFTRI has obtained significant leads in the following areas:
(a) Safety analysis of genetically engineered plants of food value : Genetically engineered Brassica was evaluated for safety of the oil. The animal experiments for 90 days by feeding with normal and genetically engineered Brassica oil at 5 and 7.5% did not induce any mortality or abnormal changes in albino rats. The toxicity studies (both acute and subacute) with Brassica seed oil cake are underway.
(b) Development of ELISA based detection kit for pesticides of importance in foods: Validation of thiram kit for poultry feed raw materials and poultry feed was done by spiking maize, bengal gram, green gram, ragi and jowar with thiram. No interference occurred in any one of the above extracts and thus further clean up of the sample was not required. However, the locally available poultry feed samples spiked with thiram and extracted with extraction solvent of the kit, interfered with thiram analysis. The extract was passed through an adsorbent system to remove interfering substances before thiram analysis.
(c) Development of rapid detection techniques for Aflatoxin B1 based on ELISA: Several maize samples were analyzed for Aflatoxin estimation in the range of 10-200 ppb by immuno-dot blot method. The values were found to be in agreement with the TLC method of estimation, a conventional method widely used.
(d) Development of a biosensor for the detection of organophosphorous (OP) pesticide: Studies were conducted by using both spectrophotometer and biosensor methods to find the optimum pH for the immobilized AchE enzyme activity. Studies revealed that the immobilized enzyme exhibited maximum activity at pH 7.5. Studies were conducted to optimize the molarity of buffer with immoblized enzyme for maximum activity and it was found that percentage inhibition did not change with the molarity of the buffer used. A signal conditioning and data acquisition system is under way to analyze OP-pesticides. The response range was kept between 0-10 volts. Validation of the biosensor for detection of OP pesticides in food samples is underway.
(e) Biomarkers of toxicity : Metal induced testicular toxicity was studies in rat; gerbil and mice under metal (Cd, Ni, and Pb) induced lipid peroxidation. Rats were found to be the most susceptible followed by gerbil and mice. Modulatory effect of taurine, quercetin on cadmium induced lipid peroxidation was studies in vitro. Both taurine and quercetin significantly abolished metal induced lipid peroxidation (LPO) with quercetin being more effective than taurine. Various hydroxy1 scavengers viz., mannito1, histidine, sodium azide reduced the degree of LPO significantly suggesting involvement of hydroxy1 radicals. Cadmium induced lipid peroxidation in testicular cells was studies, Cd was found to induce concentration related increase in lipid peroxidation. Mercaptosuccinate (glutathione peroxidase inhibitor) and 3 – aminotriazole ( catalase inhibitor) resulted in a marked increase in LPO.
Processing juices from exotic & minor fruits: The work taken up at UDCT, Mumbai. Fruits were analyzed for proximate constituents acidity, pH, ascorbic acid, anthocyanin pigments, tannin & pectin contents, Analysis of the flavor profiles of the fruits is under process. Parameters for inactivation of major enzymes like polyphenol oxidase, peroxidase & catalase from these fruits have been optimized. Assays of enzymes important to fruit processing viz. Polygalacturonase, polymethyl galacturonatelyase, pectinmethyl – esterase, polygalacturonase, polymethyl galacturonase, cellulase, xylanase & amylase were standardized.
Deseeding of amla seeds was conducted using traditional method & compared with enzymatic deseeding. Pectinex 3XL was found to be the best with respect to deseeding, vitamin C retention, & juice yield.
Large scale production of betalains by hairy root cultures of Beta vulgaris: Hairy root cultures of Beta vulgaris were established by Transformation of B. Vulgaris using Agrobacterium rhizogenes clone TR 105 at CIMAP, Lucknow. From the established clones, three elite were slected on the basis of their pigment production, rapid growth in phytohormone free media and stability of biomass and pigment production for 62 generation (15 days as one generation). One clone produced predominantly yellow colored betaxanthins, while the other predominantly the red violet betacyanins and the third one orange red color (mixture of betaxanthin and betacyanins). The pigment content was observed to be increased three folds in a suitable medium.
Enhancement of Yields and Diversification of Growth Substrates for Production of Pleuroutus (Oyster Mushroom) : A single step oil seed cake supplementation of the rice straw substrate after complete colonization by the mushroom mycelium was observed to double the mushroom yield and its protein content at CFTRI, Mysore. The OSC – M (mushrooms raised on rice straw supplemented with oil seed cake) decreased the carbohydrate content by 50% of RS – M (mushroom raised on rice straw). There was a desired softening of the OSC – M accompanied by reduction in total dietary fiber (TDF) by 45%. These produce nutritionally upgraded modified mushrooms with intense flavor and desired softening of texture.
Confirmatory trials on the use of coffee pulp as an effective growth substrate for production of oyster mushroom were conducted. The yields were observed to be at par with that on rice straw. These were found to be rich in proteins and free amino acids. Production of oyster mushrooms on coir waste and sunflower plant residue was standardized and is relevant as a rural based technology.
Screening and identification of edible tribal belt mushroom species of Madhya Pradesh and protocols for large scale production: The study was taken up at R.D. University, Jabalpur. Extensive periodical survey of various tribal and forest belts of Madhya Pradesh, resulted in the collection of 140 mushroom specimens including 33 edible forms. Ethnomycological studies conducted during survey revealed that certain species of mushrooms are used for medicinal and edible purposes by making various types of recipes. Further studies on in-vitro cultivation of selected mushroom species are underway.
Biotechnological development of shelf stable probiotic cultures containing dairy food: These studies were undertaken at Gugarat Agriculture University, Anand. Promising cultures of lactobacilli and bifidobacteria of human and other origin were isolated; low cost buttermilk/whey-cereal blends with good nutritional value and acceptable organoleptic quality were formulated. Comparison of milk, buttermilk and whey blends was done and finally six blends each from different groups were selected for spray drying.
Genetics of amylase production in thermophilic fungi and strain improvement: Thermomyces lanuginosus a thermophilic fungus of immense biotechnological importance for the production of thermostable amylases was taken up at GND University, Amritsar for strain improvement. Through repeated mutation, protoplast fusion and transformation a strain that was catabolite repression resistant, partially constitutive and hyperamylase producting was developed. A total of 10,300 mutants were derived by mutagenesis of aleurospores/protoplasts of T. lanuginosus and screened for amylase strains were observed to be stable. Further optimization of the production parameters and strain validation is being carried out at the fermentor level.
Development of low cost nutritious food supplement(s) using biotechnological approaches: Four low cost food supplements (a) soft chikki (b) suruchi meetha (c) nutro-crispo and (d) sweet and salty providing more that 400 K cal and costing less than Rs. 5/- per 100 g packets wee developed by NDDB, Anand at CFTRI, Mysore and which were validated by NIN, Hyderabad for their organoleptic & nuritional properties. These products were released by Honorable Prime Minister Atal Behari Vajpayee to the school going children on 14th November 1998.
Restoration of Coal and Manganese Mine Spoil Dump through an Integrated Biotechnological Approach: An Integrated Biotechnological Approach (IBA) developed at NEERI involves design of suitable pit size, amendment of mine spoil with organic material (pressmud, ETP sludge) and soil, isolation and multiplication of site specific strains of Azotobacter and Rhizobium and mycorrhizal fungi, screening of most suitable plant species, inoculation of selected species with specialised cultures of endomycorrhizal fungi and nitrogen fixing bacteria like Azotobacter for nonleguminous and Rhizobium for leguminous plant species. Biorestoration studies were carried out at Durgapur coal mine under Western Coalfields Limited (WCL) and Gumgaon and Dongribuzurg mines under Manganese Ore India (MOIL) on 1 hectare area at each site with plantation of about 2,500 trees of high ecological and economical importance. 7,500 trees at three sites included high value timber trees like teak, shishum, shiwan, neem, fruit trees like mango, chickoo, amala, custard, apple, ber and other ecologically important species like bamboo, cassia, acacia etc. The overall impact of IBA is that it provides food, fodder, timber wood and clean environment which improves the living conditions and socio-economic status of the local population.
Studies of the proteases of Bacillus thuringiensis: At Osmania University, Hyderabad, the endogenous protease activated crystal toxin from Bacillus thuringiensis subsp. Kurstaki was purified. The purified toxin was homogenous, as demonstrated by two-dimensional polyacrylamide gel electrophoresis and contained 1.38 mmoles neutral sugar and 9 nmoles sialic acid per mg protein. Amino terminal amino acid sequence data revealed that the toxin is a cleavage product of 132 kDa protoxin with glutamic acid-30 of the deduced amino acid sequence of the crystal protein.
The intracellular proteases in sporulated Bacillus thuringiensis subsp. Kurstaki were studies to indentify the endogenous proteases involved in the activation of protoxin. The proteases obtained with 30% ammonium sulphate saturation were analysed by both gelatine zymography and azocasein hydrolysis. The three proteases were heat-stable at 65°C, while the 69kDa proteases were active up to 75°C. Intracellular protease-deficient mutants (ethyl methanesulfonate mutagenesis) could not generate the active toxin suggesting the existence of a specific enzyme affecting the conversion of protoxin to toxin.
Cloning and expression of haemoglobin gene: Cephamycin C belongs to betalactam group, produced by Streptomyces clavuligerus and is used as a basic drug for the synthesis of cefoxitin, cefmetazole, cefotetan etc., which are extensively used in preventing post-operative infections caused by resistant bacteria. The project at Osmania University, Hyderabad aimed at genetic manipulation of S.clavuligerus to improve the efficiency of trapping (Sequester) oxygen under reduced oxygen tension by cloning and expression of heterolo-gous bacterial hemoglobin gene from vitreoscilla in cephamycin C producer strain of S. Clavuligerus.
Two promising isolates were obtained which belong to Vitreoscilla stercoraria and exhibit interesting features with respect to heme protein expression. The two promising isolates were designated as PS 15 and PS 24. The two isolates have shown higher cellular heme protein and its expression was dependent on oxygen levels. Under oxygen limiting or oxygen poor conditions (hypoxic conditions) the production of heme protein was significant that the culture grown under highly aerated conditions.
Cloning and molecular characterization of thermostable inulinase gene from thermophilic actinomycetes: The studies were undertaken at GND University at Amritsar. In order to search for novel microbial sources of thermostable inulinase soil and root samples from rhizosphere of dehlia (Dehlia pinnata) and chicory (Cichorium intybus) plants collected from 15 different locations in Punjab were screened for inulin hydrolysing activity. Of the forty strains isolated, two actinomycetes and two fungal strains viz., Aspergillus fumigatus and Pencillium purpurogenum showed considerable extracellular inulinase activity. The two fungal strains were deposited with MTCC, IMTECH, Chandigarh.
Biochemical, genetic and molecular analysis of nitrophenols degradation by microorganisms: Several organisms capable of utilizing p-nitrophenol (PNP) as the sole source of carbon energy were isolated by enrichment technique at IMTECH, Chandigarh. Following biochemical characterization, two organisms, viz. Arthrobacter protophornase RKJ100 and Pseudomonas cepacia RKJ200 were chosen for further work. Both organisms degrade PNP via an oxidative route as indicated by the accumulation of nitrite molecules in the culture medium. Studies indicated that degradation occur via hydroquinone formation as shown by TLC and GC; hydroquinone is further degraded via the b-ketoadipate pathway. Results have demonstrated that a plasmid of approximately 50 kilobase pairs is responsible for carrying genes for PNP degradation in P. cepacia RKJ200.
The P. cepacia RKJ200 is also capable of utilising 4-nitrocatechol as sole source of carbon, nitrogen and energy and is degraded via an oxidative route.
Bio-treatment of refractory gold ores and concentrates : The main focus of the research at IISc., Bangalore has been the design and development of a laboratory scale continuous reactor system for biotreatment. Among four different samples investigated, G.R. Halli concentrate, which found to be highly sulphidic (50% pyrite & arsenopyrite) was chosen for continuous trials. A strain of Thiobacillus ferrooxidans TfH6 isolated, characterized and activated in previous studies was used. A 17L bioreactor system has been designed and fabricated. Several experiments on bioleaching of the concentrate have been conducted to develop a blueprint for design and operation of the demonstration plant at Hutti. The bioleaching of concentrate was carried out at 20% pulp density and it was found that the microorganisms were not completely active at this density. Therefore, experiments were conducted with microbial culture, grown for 48 hours in 9K medium. The steady state was found to reach after 8 days and 30% of the iron and 50% of the sulfur present initially in the concentrate was found to be oxidized. The gold extraction efficiency was 70-75%. The untreated concentrate showed only about 40% gold extraction efficiency. These results clearly indicate the beneficial effects of biotreatment of refractory gold ores. Efforts are being made to enhance the extraction.
Anti-microbial drugs from Cyanobacteria : At the University of Madras, a marine cyanobacterium (Spirulina subsalsa BDU 40303) with potent antibacterial compound was identified which can be used even against Pseudomonas. A marine cyanobacterium (Oscillatoria laetivitens BDU 20801) with anti-Candida activity was identified and the partially purified compound was highly effective. Purification and identification of the active compound is in final stages. A marine cyanobacterium (Phormidium valderianum BDU 30501) with potent antiviral activity against HBV, HSV and HIV has been identified. Partial purification resulted in enhanced activity against different viruses with different fractions noted, indicating the presence of more than one antiviral compound.
A survey of North Andaman Islands conducted by Bharathidasan University has resulted in the enumeration of 70 species of marine Cyanobnacteria belonging to 25 genera and 10 families. This reveals the rich biodiversity of marine cyanobacteria in this area. The centre now has marine cyanobacteria of 268 strains belonging to 70 species 25 genera, and 10 families. Phormidium valderianum BDU 20041 was identified to be a good biotransformant. It has transformed ferulic acid (a product of lignin degradation) to vanillic acid (an aromatic alcohol used as food additives. This was confirmed by GC-MS studies. Likewise, cholesterol transformation to several products has been worked out in P. valderianum BDU 30501 and O. Subtilissima. BDU 92183. Identification of these by products in underway. A surfactant producing strain was identified. Its stability was found to be better than commercial surfactants such as sodium dodecyl sulphate, (SDS) and Tween 80. The mode of chromium uptake by cyanobacteria was found to be an active process. The metal gets detoxicated to trivalent chromium and enzyme glutathione-S-tranferase also aids in reducing the toxicity. This finding helps to improve the bioaccumulation capacity of organism and its use in effluent treatment is being attempted. Study on the effect of stabilisers with P. valderianum BDU 30501 has aided in the identification of the enzyme as a cephalosporinase.
Molecular breeding of polycyclic aromatic hydrocarbon degrading bacteria: A bacterial strain Alcaligenes odorans, capable of degrading a wide range of PAH compounds was selected at TERI, New Delhi. The carbon starved cells of the selected bacterium A. odorans could take up about 13% of total [14C] – Pyrene, of which 11% [14 C] – Pyrene were retained within the cell biomass and 3% [14 C] – pyrene was liberated as 14CO2 in 120 minutes.
Assays for the know enzymes involved in PAH degradation revealed that Alcaligenes odorans also has catechol 2,3 dioxygenase, an enzyme primarily responsible for ring cleavage in aromatic degradation. The observation clearly shows that A. odorans cleaves each of the four rings that are present in pyrene to generate catechol, which can thus be channelised into degradation pathway that generate intermediates for the Kreb’s cycle – the principal energy generating pathway.
Having obtained an efficient PAH degrader, which also has the capacity to degrade a variety of alkanes, the larger objective of the project has been to develop a technology that can effectively reclaim crude oil/oily sludge. In order to monitor the survivability of the bacterial isolate that are released into contaminated soil, molecular markers have been developed using bacterial repeat sequences. These repeat sequences yield highly reproducible and species specific fingerprints. Experimental trials are underway wherein Alcaligenes odorans are isolated and identified from crude oil contaminated microcosm using PCR fingerprinting.
Development of GE Phosphate solubilising Pseudomonas: Pseudomonas fluorescens is a well-characterised plant growth promotion rhizobacterium that can effectively colonise a wide variety of plants under diverse environmental conditions. The objective of the project at M.S. University, Baroda is to genetically modify this organism so as to enable it to solubilize mineral phosphates, thus imparting yet another property beneficial to the plants that it colonises.
Normally P. fluorescens strains do not secrete organic acids in sufficient quantities to release phosphorous from soil phosphates. Analysis of acid production using methyl red indicator plates with different concentrations of buffer showed that this organism fails to acidify the medium when buffered Tris HCL pH 8.0 at 70mM or above is added. By transforming P.fluorescens with a vector carrying the ppc gene encoding phosphoenol pyruvate carboxylase from Anacystis nidulans, clones obtained show acid production at 100 mM buffer concentration. These modified strains can grow on medium containing rock phosphate as the sole phosphorous source and bring about reduction of pH from 8.0 to 5.0. Results indicate the transformant to be more effective at phosphorous solubilization than the parent strain. The efficacy of the modified strains in plant growth enhancement is under study.
Application of thermophillic microbes for improved leaching of copper: The studies were taken up at IICB, Calcutta. The copper ores of eastern region, mainly of the Ghatshila area of Bihar, are normally composed of chalcopyrite, and as such are not good substrates for bioleaching by mesophilic bacteria. However, leaching of these ores with acidic ferric sulfate solution in the presence of silver ion as catalyst suggests that these ores are amenable to hydrometallurgical processes, and can be extracted through application of microbes. Studies on the chalcopyrite rich copper ores of Ghatshila region with thermoacidophilic archaeal strains show that leaching may be achieved with an A. brierleyi strain which could leach more that 16% of copper with 30 days from 5% (w/v) suspensions ores at 65%C. Leaching of copper increased steadily after 15 days of incubation, and the increasing tendency of leaching was maintained even after one month. The results suggest that leaching should be continued for a longer period of time to get more dissolved copper from ores. Apart from A. brierleyi, other archaeal strains were reported to have bioextractive properties. A surda concentrate was tested for amenability to leaching with the A. brierleyi. Strain. After 45 days, the strain could leach as much as 3.85% of total copper from a 5% (w/v) concentrate suspension.
Cloning of dehydrogenase gene and construction of genetically engineered organism for L-ascorbic acid production: A breakthrough has been achieved at RRL, Jammu. Gluconobacter oxydans block mutant capable of producing free gluconic acid has been developed by Tn5 transpogenesis and the mutant has potential commercial importance. Glucose dehydrogenase a gene from G. Oxydans was transferred in Pseudomonas species through conjugation using pML 122 as the vector. A fast growing clone showing high specific activities of GDH and GADH enzymes was selected. Fermentation studies on the clone revealed 40% increase in the productivity of 2-keto-gluconate- a penultimate intermediate of isoascorbic acid (Isovitamin C) used as antioxidant for the preservation of food products.
Process development of alkaline proteases: The investigations were carried out at IMTECH, Chandigarh. The isolated alkalophilic Bacillus sphaericus was observed to produce an alkaline protease, using a relatively less expensive production medium, which seems to have good industrial potential for a variety of applications. This enzyme was observed to be optimally active at 50-55 °C and a pH of 10.5. The optimum temperature was found to increase to 60 °C in presence of Ca++. The enzyme was found to be quite stable at a wide range of pH values (7.5-11.0). Salt precipitated powdered enzyme preparation was found to be quite stable for 18 months at 37°C.
Iron Control in Hydrometalurgical Leach Liquor using Thiobacillus: Iron oxidation studies were carried out at RRL, Bhubaneshwar in a specially designed bioreactor. Fe(II) concentration was varied from 1 to 10 gpl in the growth medium. The leg phase continued from 8-10 hours and at 10 gpl the oxidation rate was maximum i.e. 500 mg/l.hr. Fe(III) concentration was varied from 0.5 to 4 gpl. It was observed that, higher ferric concentration had a negative effect on the growth and activity of Thiobacillus ferrooxidans. The medium of the pH was varied from 1.25 to 2.5. An increase in the oxidation rate was observed from pH 1.25 to pH 2.0 and thereafter it decreased. At pH 2.0 the maximum rate of oxidation was observed to be 132 mg/l/hr. Biomass concentration was varied from 103 to 103 cells/ml. At maximum biomass concentration the rate of oxidation was maximum i.e. 477 mg/l/hr. Generation time was observed to very from 2 to 16 hours depending on the oxidation parameter.
Fermentative production of Lipase and Immobilization of the Pure enzyme : Studies were undertaken at Jadhavpur University, Calcutta. A lipase producing Corynebacterium strain was isolated from spoiled coconut. A high-yielding strain m13 was isolated by induced mutation using N.T.G. Optimum assay conditions for enzyme (substrate olive oil emulsion) were temperature 60°C, time 10 min, pH 8.0, CaCl2, Conc. 110 mM.
Microbial Synthesis of Inulinases: Inulin sources have recently received interest as renewable raw-material for the production of fructose syrup and ethanol. The study was taken up at RRL, Trivandrum. Eighty four strains were isolated from rhizosphere soil samples around the dahlia tubers on synthetic medium containing inulin as a carbon source, for the production of extracellular mesophilic cultures were selected for further studies and finally one of them was taken for detailed characterisation. This was identified as Staphylococcus sp. The microbial culture synthesised inulinase that was active on inulin and was extra-cellular in nature. Solid state fermentation was carried out for the production of inulinase from a bacterial strain of Staphylococcus sp. RRL1 and a yeast strain of Kluyveromyces marxianus ATCC 52466. Both the cultures grew well in a wheat bran medium.
Novel Alkaline Thermostable Lipase: Purification, Characterization and Gene Cloning: An extracellular thermostable alkaline lipase was produced by a thermophilic Bacillus strain J33. An agrowaste byproduct (Wheat bran 1%) enhances the lipase production by 3-4 folds in comparison to the nutrient broth (1%). The enzyme was purified to 175 folds with 15.6% recovery by ammonium sulphate & Phenyl Sepharose column chromatography. Polyclonal antibody was raised against this lipase with other lipases from thermophilic and mesophilic microorganisms. For cloning the gene, a genomic library has been constructed in cosmid vector (pHC 79). The colonies (positive clones) started giving clear zones after 15 days incubation 4°C. Two positive clones have been isolated and checked further by western blotting. After confirmation the clones are being used for subcloning the lipase gene.
Microbial production of Biodegradable plastics: At RRL, Jorhat over hundred bacterial strains were isolated from soil samples and tested for their ability to accumulate PHA. Three strains isolated from an oil Contaminated site were found to accumulate considerable PHB (Polyhydroxybutyrate) under nitrogen limitation condition. Effect of temperature, carbon and nitrogen sources on PHB production were studies and PHB isolation procedure standardised. Isolate number RLJ 071 when cultivated at 42 °C for 28 hours in 10% sucrose containing nitrogen limitation medium, produced cells having 68% PHB content. PHB isolated by sodium hypochlorite-chloroform dispersion method was in white crystalline power form having a melting point of 180 °C.
Development of Bioabsorble Polymers: The objective of the project at IIT Delhi is to develop biosoluble materials for clinical application. The most important biomedical applications of biodegradable polymers are in the areas of controlled drug delivery systems; implants for fracture repairs, ligament reconstruction, surgical dressings, dental repairs, contact lenses, vascular graft and organ regeneration. Thus, a variety of homopolymers having these repeat units was initially synthesized in different molecular weight range. Their chemical structure and physical properties were evaluated. A new series of block copolymers were prepared from polylysine and polyaspartic acid which were subsequently characterized. Long term animal studies in several different species are required for further utilization of these materials as class III and/or implant grade materials.
Degradation of Pesticides by facultative anaerobes: Two strains of Micrococcus sp. harbouring plasmids (5 kb, 62 Kb) degraded malathion and chlorpyriphos. Degradation studies with plasmid cured derivatives of the strains suggested involvement of plasmid to be degradation of malathion and chlorpyriphos. Interspecies protoplast fusion between the strains resulted in cointegrate formation of the two plasmids as evident from comparison of electrophoretic mobility of plasmid DNA of the fusant and the parent strains. The resulting fusant was resistant to 3,5-dinitrosalicylic acid and sodium azide (300 and 200 ppm respectively) against which cells of parent strains were sensitive. Enhancement of endosulfan degradation by the fusant was also observed (about 10-12%).
Production of thermostable Proteases and Lipases in thermophiles: A gram negative spore forming rod was isolated at Punjab University and was identified to be Bacillus sp. IS-20. This was subjected to substrate improvement with chemical and physical mutagenic agents. One of the mutants SS-3 gave increased enzyme yield and was selected for subsequent studies. The enzyme was found to be quite stable at broad pH and temperature range and was compatible with various detergent preparations.
Another isolate Bacillius sp. RSJ-1 giving the best lipase at a pH of 9.0 and a temperature of 50°C was selected. The organism was subjected to mutagenesis by Ultraviolet irradiation and ethyl methyl sulphonate and one of the mutants with enhanced enzyme yield was selected. The lipase production was optimized by using various carbon nitrogen source as well as environmental parameters.
Enhancement of microbial protein production through genetically engineered Bacterial Hemoglobin: The study was undertaken at IMTECH Chandigarh. In response to oxygen deprivation, a unique hemoglobin like protein (VHb), displaying relatively high rate of oxygen dissociation, is produced by a gram-negative, aerobic bacterium, vitreoscilla. Gene (vgb) encoding for this bacterial hemoglobin has been cloned and overexpressed in E. coli under the native oxygen-sensitive promoter of vgb gene. Four site-directed mutants of VHb were generated by introducing specific changes at E7 residue of VHb through vgb gene manipulations. SDS-PAGE analysis of recombinant E.coli carrying the mutant VHb indicated that Hb constitutes 5-8% of total cellular protein. To get primary indication in the oxygen binding characteristics of these mutants, relative change of oxygenated form of various VHb mutants into reduced form with respect to time was determined. These mutants were found to have significant differences with respect to their oxygen binding kinetics as compared to wild type VHb protein.
Genetic Transformation of Filamentous Fungi : The work was carried out at IISc Bangalore. A fungus has been demonstrated to be capable of secreting up to 20-30 grams of protein per liter of culture medium. The fungus Neursopora crassa is an attractive model organism because background information in its genetics and physiology is available. A noteworthy aspect of this work was the preparation of enzyme for isolation of protolplasts. Soil samples, amended with fungal cell wall material, were incubated in moist conditions for several days to enrich the resident population of organisms capable of utilizing fungal cell wall as carbon source. A species of Trichoderma secreted cell wall lytic activity when grown in optimized media. The "home-made preparations of the lytic enzyme released protoplasts from germinating conidia of wild-type and several auxotrophic strains N. crassa; the protoplast releasing activity being comparable to that of the expensive, commercial anzymes. The transformants were selected on the basis of complementation of recessive auxotropic mutant genes. The uni-nucleate microconidia could serve as recipient cells for transforming DNA, circumventing the difficulty of purifying the transformed nucleus. Even when multinucleate protoplasts were used for transformation, prior incorporation of the mcm gene into the host strain (by crossing) allowed the primary (heterokaryotic) transformant to bud out microconidia which could be plated on selective medium to recover the individual transformation products. The transformants were selected in a medium supplemented with inositol alone. Colonies of primary transformants were grown to conidiation. The multinucleate (heter-okaryotic) conidia, containing mixtures of untransformed (his) and transformed (his+) nuclei, were allowed to germinate in shake cultures at 22°C. Under these conditions the mcm gene was expressed and the culture selectively produced microconidia by microcycle microconidiogenesis. Southern analysis of transformants showed that the plasmid sequences were integrated randomly in different nuclei.
Microbial Diversity : A national network R&D programme is being launched for biotechnological exploitation of microbes including extremophiles for industrial applications, wherein microbial diversity aspect shall also be dealt with. The programme would envisage exploring the microbial flora of the country from different regions, their collection, identification and indexing. A number of centres are to be established in different locations which would be networked so that scientists Could have access to the database of culture collections available at all the centres. This national endeavor is important keeping in new that India has acceded to PCT from 8th Dec., 1998 and is now member of the Paris Convention “Union”.
INTELLECTUAL PROPERTY RIGHTS, BIOPRODUCTS AND BIOSAFETY
Intellectual Property Rights
The department this year reconstituted the Patent Screening Committee to facilitate researchers to file their patents in India and abroad for the processes/technologies emanating out of biotechnology research. As per the recommendations of the Scientific Advisory Committee, the department is bearing the entire expenditure for patenting of research leads submitted by the applicants whether the project has been funded by the Department or the lead has been expenditure for patenting of research leads submitted by the applicants whether the project has been funded by the Department or the lead has been obtained through their institutional or other grants. During the year the Patent Screening Committee cleared 8 applications. Applications from research institutions from CSIR were also allowed to file joint patent applications with DBT through CSIR mechanism. NRDC has been helping in the process of patenting in India and abroad. So far, 48 applications have been received by the department out of which 31 applications have been filed in India and abroad. This year 3 Indian patents and 2 US patents have been sealed for DBT in joint collaboration with the host institutions. The other applications are under various stages of processing or for consideration by the Patent Screening Committee.
The department, in close collaboration with Department of Electronics has funded two projects on electroporation equipment development and on microwave disinfection of hospital wastes. Projects are also being funded for the development of projects and services in association with the industry. In the joint projects with the Department of electronics, industry has also participated in the development of technologies. The project on technology perfection and transfer of aggultination based detection of HIV-I & II antibodies in human blood has reached a stage when the prototype kit would be ready for multi-centric trials in comparison with other imported HIV detection kits. The packaging material for the kit has been standardised and efforts are on for the preparation of indigenous standard sera panels. The technology for the monoconal antibodies for phage coat proteins gIIIp and gVIIIp, which were transferred to M/s Pharmacia Biotech, USA have been launched formally by M/S Amersham Pharmacia Biotech in December, 1998. The products are HRP/anti-M13 monoclonal conjugates. These antibodies would be useful to the researchers using Phase Display Technology worldwide.
The Department of Biotechnology actively implemented the rules and procedures contained in the Notification No. 1037(E) dated 5.12.1989 under the Environment Protection Act (EPA) 1986, issued by the Ministry of Environment & Forests, to monitor the Recombinant DNA Research work in India. In accordance with the Notification, the Department had brought out "Revised Guidelines for Research in Transgenic Plants & Guidelines for Toxicity and Allergenicity Evaluation of Transgenic Seeds, Plants and Plant Parts" in August, 1998. The Guidelines include a complete design of a contained green house suitable for conducting research with transgenic plants. Besides, it provides the basis for generating food safety in formation on transgenic plants and plant parts. In addition to the above, the Department has been nominating its representatives Institutional Biosafety Committees (IBSCs) to have vigilance on the research activities in Genetically Modified Organisms (GMOs) and guiding research institutions on the safety aspects of GMOs. In order to bring about public awareness on the safety issues emanating from the use of GMOs, the Department had conducted seminars at five places at Delhi, Calcutta, Hyderabad, Pune and Bangalore on Biosafety Guidelines to widely disseminate information on the safety and benefits among the scientists who are working in this area.
(i) Institutional Biosafety Committees : Currently, 125 IBSCs are functional at research institutions, university departments and at private industries in the country where recombinant DNA work is being carried out.
Review Committee on Genetic Manipulation : The Department had reconstituted
the RCGM by inducting more members from diverse areas of applications including
agriculture, drugs & pharmaceuticals, industry and environment management.
The members inducted have been raised to 16 from the earlier 9 members.
During the year, two meetings of the newly constituted committee were convened
and several field experiments in small plots were permitted for generation
of data on transgenic plants in different agro-climatic conditions in the
country. The target transgenic crops were brassica, cotton, tobacco, brinjal,
cabbage, cauliflower and tomato. The scope of activity of the RCGM members
was also enlarged within the provisions of the law by creating a Monitoring-cum-Evaluation
Committee to independently work and assist the RCGM in technical evaluation
of limited field trials on transgenic plants in open environment. This
technical committee met twice and guidelines for evaluation of the transgenic
plants in open environment are being developed. On the basis of the evaluation
of research conducted by M/s Bharat Biotech India Ltd., Hyderabad on recombinant
Hepatitis B vaccine produced in yeast, as examined by its IBSC and the
research results scrutinised by the RCGM, the latter had recommended the
proposal to the Genetic Engineering Approval Committee (GEAC) of the Ministry
of Environment & Forests for clearance of the product under EPA for
manufacture and use in the country. Subsequently on approval, the product
was launched by the Company.