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Kerala Agricultural University, Thrissur
The history of agricultural education in Kerala can be traced back to the year 1896 when a scheme was evolved in the erstwhile Travancore State to train a few young men in scientific agriculture at the Demonstration Farm, Karamana, Thiruvananthapuram, presently, the Cropping Systems Research Centre under Kerala Agricultural University. Agriculture was introduced as an optional subject in the middle school classes in the State in 1922 when an Agricultural Middle School was started at Aluva, Ernakulam District. The popularity and usefulness of this school led to the starting of similar institutions at Kottarakkara and Konni in 1928 and 1931 respectively.
Agriculture was later introduced as an optional subject for Intermediate Course in 1953. In 1955, the erstwhile Government of Travancore-Cochin started the Agricultural College and Research Institute at Vellayani, Thiruvananthapuram and the College of Veterinary and Animal Sciences at Mannuthy, Thrissur for imparting higher education in agricultural and veterinary sciences, respectively. These institutions were brought under the direct administrative control of the Department of Agriculture and the Department of Animal Husbandry, respectively. With the formation of Kerala State in 1956, these two colleges were affiliated to the University of Kerala. The post-graduate programmes leading to M.Sc. (Ag), M.V.Sc. and Ph.D. degrees were started in 1961, 1962 and 1965 respectively.
On the recommendation of the Second National Education Commission (1964-66) headed by Dr. D.S. Kothari, the then Chairman of the University Grants Commission, one Agricultural University in each State was established. The State Agricultural Universities (SAUs) were established in India as an integral part of the National Agricultural Research System to give the much needed impetus to Agriculture Education and Research in the Country. As a result the Kerala Agricultural University (KAU) was established on 24th February 1971 by virtue of the Act 33 of 1971 and started functioning on 1st February 1972. The Kerala Agricultural University is the 15th in the series of the SAUs.
In accordance with the provisions of KAU Act of 1971, the Agricultural College and Research Institute at Vellayani, and the College of Veterinary and Animal Sciences, Mannuthy, were brought under the Kerala Agricultural University. In addition, twenty one agricultural and animal husbandry research stations were also transferred to the KAU for taking up research and extension programmes on various crops, animals, birds, etc.
During 2011, Kerala Agricultural University was trifurcated into Kerala Veterinary and Animal Sciences University (KVASU), Kerala University of Fisheries and Ocean Studies (KUFOS) and Kerala Agricultural University (KAU).
Now the University has seven colleges (four Agriculture, one Agricultural Engineering, one Forestry, one Co-operation Banking & Management), six RARSs, seven KVKs, 15 Research Stations and 16 Research and Extension Units under the faculties of Agriculture, Agricultural Engineering and Forestry. In addition, one Academy on Climate Change Adaptation and one Institute of Agricultural Technology offering M.Sc. (Integrated) Climate Change Adaptation and Diploma in Agricultural Sciences respectively are also functioning in Kerala Agricultural University.
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ThesisItem Open Access Isolation and Characterization of cDNA encoding chalcone synthase from flower buds of orchid Dendrobium variety sonia 17(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2007) Anjana, G R; KAU; Soni, K BThe study entitled “Isolation and characterization of cDNA encoding chalcone synthase gene from the flower buds of orchid Dendrobium variety Sonia 17” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2005 to 2007 with an objective of studying the isolation and characterization of cDNA encoding chalcone synthase gene involved in anthocyanin pigmentation in orchid flower buds. . Heterologous forward and reverse primers were designed based on the gene sequences of Oryza sativa, Fragaria ananasa and Phalaneopsis orchid using primer3 software. Total RNA was isolated from immature floral tissues using hot phenol method which gave an yield of 80 - 200 μg g -1 of the tissue and a A260/A280 ratio ranging between 1.6 –2.0. Messenger RNA was purified from the total RNA using the mRNA purification kit from GENEI (Bangalore). Reverse transcription-polymerase chain reaction was carried out to study the expression of gene. The RT-PCR amplified products representing chalcone synthase (CHS) gene was eluted and purified. The product was sequenced and studied the similarity of the same using homology search. All sequenced regions were subjected to BLASTN and BLASTX similarity search. Rice chalcone synthase specific primer produced an amplified sequence of 460 bp long and showed maximum similarity to the cDNA clone 5', mRNA sequence of. flower bud of Phalaenopsis violacea and flower bud of Phalaenopsis equestris Lambda ZapII cDNA Library in BLASTN similarity search.BLASTX analysis of the sequence showed similarity to maturase K protein of Aerangis kirki. The cDNA amplified with strawberry chs specific primer showed maximum similarity to the cDNA clone 5’, mRNA sequence of Phalaenopsis violacea flower bud and flower bud of Phalaenopsis equestris in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to LFY-like protein of Serapias lingua. The results of the nucleotide to nucleotide search (BLASTN) of the cDNA of orchid, amplified using chalcone synthase specific primer from orchid showed similarity to cDNA 5', mRNA sequence of Ipomoea batatas in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to retrotransposon protein of Oryza sativa (japonica cultivar-group). The result of the sequences obtained from the study shows similarity with the genes involved in the biosynthetic pathway of Phalaenopsis orchid flower fragrance.ThesisItem Open Access Management of biodegradable plant tissue culture lab wastes through biomethanogenesis(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Abdulla Fayas, T; KAU; Rajendran, P CGenerating renewable source of energy from tissue culture laboratory waste by the process of biomethanogenesis is the focal theme of present investigation. Unlike developed countries, the developing countries are hesitant to establish more number of biotechnology/ tissue culture laboratories due to financial constraints. Easy and regular availability of biogas from TC wastes will be a boon to establish self-sustainable TC laboratory in view of present energy The biogas experimental units required for the study was designed and various treatments were employed for the biodegradation of tissue culture waste, using the methanogenic bacteria Methanobacterium ruminatium, Methanobacterium formicicum; Methanosarcina barkeri, Bactereoides ruminicola, Selenomonas ruminatium, Eubacterium tortuosum and Clostridium butyricum. Treatment involving TC waste and cow dung was also conducted for biomethanation in the present study. Quantity of gas production and its combustibility was noticed for various treatments. In bacterial treatments the quantity of gas generation was highest for Clostridium butyricum. Only treatments involving cow dung produced combustible gas. Molecular characterization, of methanogenic bacterial cultures was also done for finding the genetic similarity between them. RAPD followed by scoring . of the bands by UPGA routine showed maximum similarity between bacterial cultures of Methanobacterium ruminatium and Methanobacterium jormicicum with Methanosarcina barkeri. Physio-chemical characters like CIN ratio of the TC wastes, pH and temperature of medium and Hydraulic retention time was also observed for the various treatments. The CIN ratio of the TC wastes was found to be very low and nowhere near the optimum CIN ratio of 20-30 required for gas production. Other parameters like pH of the treatments and Hydraulic retention time was also. • noticed. The pH of the treatments involving bacterial cultures was very low, considering the normal pH of 6.8 to 7.5 required in biogas generation. The main constraints in the biogas generation were found out to be the low CIN ratio of the TC waste and the low pH of the medium. The present study indicated the possibility ofbio-gas generation from TC waste through fortification using various supplements like coconut water and coir pith which have higher CIN ratio.ThesisItem Open Access In vitro shoot regeneration and micrografting in nutmeg (Myristice fragrans houtt.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Liffey Zachariah, Antony; KAU; Valsala, P ANutmeg (Myristica fragrans Houtt.), is dioecious and dimorphic in branching habit with erect growing orthotropic and horizontally growing plagiotropic shoots. The long gestation period and dioecious nature of the crop causes difficulty in the production of quality planting materials of known sex. Vegetative propagation, budding and grafting with orthotropic scion material produces erect growing tree with upright tree architecture. In vegetative propagation, scarcity of orthotropic scion material is a limiting factor in large scale production of planting materials. So the programme “In vitro shoot regeneration and micrografting in nutmeg (Myristica fragrans Houtt.)” was taken up. The objectives of the study were: (1) To identify the culture conditions for multiple shoot production from orthotrops of gynoecious plants of nutmeg through enhanced release of axillary buds and shoot tip culture and (2) To standardize micrografting technique with in vitro and in vivo shoots as scion. The work was done at CPBMB, College of Horticulture, Vellanikkara. SH medium (Schenk and Hildebrandt, 1972) was found to be the best basal medium for in vitro culture establishment of nodal segments of nutmeg compared to ½ MS (Murashige and Skoog, 1962) and WPM (Lloyd and Mc Cown, 1980). Surface sterilization of nodal segments by soaking in (0.1%) carbendazim for 10 minutes followed by (0.1%) HgCl2 treatment for six minutes and sterile water wash, recorded 33% survival of cultures. The best explant for culture initiation was nodal segments. The best season for culture establishment was summer months (April-May) compared to rainy season (June- July). Loss of cultures was due to fungal contamination and necrosis of tissues. The media combination SH + Thidiazuron (TDZ) 0.03mg l-1 + Activated Charcoal (A.C.) 0.5% recorded bud expansion in 50% of the cultures within a period of nine days. Nodal segments are superior to shoot tips in culture establishment. Culture condition for culture establishment was 26 ± 20C at a light intensity of 1000 lux. The carbon source; 2% Sucrose + 1% glucose or 5% sucrose supported bud elongation and leaf expansion Refinement of culture establishment media was attempted with organic supplements; Coconut water (5, 10, 15 and 20% v/v), Casein hydrolysate (10, 25 and 50mg l-1) and Brassinolide (0.05, 0.1 and 0.2mg l-1) with nodal segments from juvenile seedlings, regenerants from coppiced trees and mature trees. In explants from juvenile seedlings and mature trees, 5 to 15%coconut water supported culture establishment. In juvenile explants, shoot elongation was also observed at 10% coconut water. Casein hydrolysate supported bud expansion in juvenile and mature tree explants at 10 to 50 mg l-1. Bud elongation and leaf expansion was observed at 25 mg l-1 concentration. Brassinolide (0.2 mg l-1) supported bud expansion in juvenile explants. The suggested media for explants from juvenile as well as coppiced trees for culture establishment of nodal segments of nutmeg is SH + 0.03 mg l-1 TDZ + 25 mg l-1 Casein hydrolysate + 2% sucrose + 1% glucose + 0.5% A.C. Casein hydrolysate concentration for explants from mature trees could be 50 mg l-1. In vitro seed germination was observed in mature seeds in presterilized bottles with water soaked cotton/ little water. Somatic embryos were formed at the cut portion of six month old seeds in the medium of ½ MS + 2% Sucrose + A.C. 0.5%. Proliferation of callus and somatic embryos was observed with the medium B5 + 0.1 mg l-1 Kin + 0.01 mg l-1 NAA + 0.01 mg l-1 GA3 + 10.0 mg l-1 Casein hydrolysate + . 0.5% A.C. The response was obtained two and a half months after inoculation. Three days old in vivo germinated seedlings did not established under in vitro condition even though surface sterilization treatment with 0.1% Emissan for thirty minutes followed by 0.1% HgCl2 for six minutes was given. Feasibility of grafting in juvenile plants was studied with epicotyl grafting and got 80% success. Grafting was done on twenty days old seedling with scion material from different seedling. In vitro epicotyl micrografting was done with in vitro raised scion and root stocks. It was also done on in vivo germinated seedlings after surface sterilization. Scion shoot of 2.5 cm length was grafted on twenty days old root stock. Graft was cultured in liquid nutrient medium and survived for two weeks. Later fungal contamination destroyed the cultures. Grafting with in vitro shoots on in vivo raised root stocks did not succeedThesisItem Open Access Exploitation of invitro cultures of Indian Madder(Rubia cordifolia.Linn) for anticancerous compounds(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Labade, Dinesh Sitaram; KAU; Asha Sankar, MThe present investigation on “Exploitation of in vitro cultures of Indian Madder (Rubia cordifolia L.) for anticancerous compounds” was carried out at the Plant Tissue Culture Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara and Amala Cancer Research Centre, Thrissur during the period 2006-2008. The study was undertaken with the objective to standardize the in vitro techniques for initiation and proliferation of static and suspension cultures of Rubia cordifolia and to screen the in vitro cultures for synthesis of naphthoquinone and quantify it. It was also envisaged to enhance the level of product synthesis in in vitro cultures and to assess the anticancerous activity of in vitro and in vivo extracts in terms of cytotoxicity, antioxidant and prooxidant activities in vitro. Leaf, nodal and root derived callus cultures of Rubia cordifolia were established in vitro. Explants were pre treated with the fungicide, Bavistin 2.5 per cent for 15 minutes. Surface sterilization with mercuric chloride (HgCl2) at 0.1 per cent for 1 min and 30 sec was effective for yielding healthy, contamination free cultures from nodal segments and leaves, respectively. MS medium at full strength, supplemented with NAA at 2 mg l-1 along with BA at 0.5 mg l-1 was observed ideal for initiation and proliferation of calli. The auxin synergist phloroglucinol, when supplemented to the medium, did not not yield encouraging results, with respect to callusing in the experimental species. Root derived cultures were inferior with respect to callus initiation and proliferation, registering low values for all the parameters studied. Incubating in vitro cultures under illuminated condition at 26 ± 2 C was superior to dark incubation, with respect to callus initiation and proliferation. Chloroform – methanol at 8.5 :1.5 ratio was indentified as the appropriate solvent system for detection of naphthoquinone on thin layer chromatograms in the test extracts of the experimental species, with alcoholic KOH (10 per cent) as the spray reagent. Ms medium at full strength, fortified with NAA and BA at 2.0 mg l-1 and 0.5 mg l-1 respectively, which recorded maximum naphthoquinone synthesis, was standardized as the production medium. Enhancing concentration of sucrose to 5 per cent in the production medium, did not elicit a positive response on naphthoquinone production in vitro. Reducing nitrate concentration of the production medium, to half and one fourth the original concentration, resulted in enhanced in vitro synthesis of the target compound. Supplementing the production medium with yeast extract (1 per cent and 2 per cent) as well as precursor feeding with phenyl alanine and tyrosine each at levels of 50 mg l-1, 100 mg l-1 and 150 mg l-1 exerted a favourable influence on synthesis of naphthoquonines, in vitro. Incubation in dark resulted in marginal increase in in vitro production of naphthoquinones. Incorporation of autoclaved mycelia of Pythium aphanidermeatum at levels of 2.0 per cent and 5.0 per cent resulted in enhanced in vitro production of naphthoquinone. The abiotic elicitor, salicylic acid at concentration of 10 μM and 100 μM resulted in maximum synthesis of naphthoquinones in in vitro root cultures (8.76 units g -1 calli) of Rubia cordifolia. Immobilization of test calli with sodium alginate – calcium chloride complex as well as subjecting the in vitro cultures to stress conditions, as imposed by sorbitol failed to bring about an enhancement in the in vitro production of naphthoquinones. None of the explants employed in the study induced hairy roots, when co- cultured with the Agrobacterium rhizogenes strains, MTCC 2364 and MTCC 532. Based on cell count, subculturing intervals of leafs, nodal and root derived suspension were fixed as 24, 27 and 27 days respectively with the respective packed cell volume as 0.93 per cent, 0.83 per cent and 0.80 per cent. Naphthoquinone was detected, in ex vitro and in vitro test extracts at all levels of maturity tested. Both ex vitro and in vitro root extracts exihibited maximum cytotoxicity, as revealed by the percentage of cell death on DLA and EAC cell lines as well as their IC50 values. As compared to whole plant extract, in vitro systems of the experimental species exhibited least antioxidant action. Extent of pro-oxidant activity was higher in in vitro root extract of the experimental species.ThesisItem Open Access Induction of genetic variability in ginger (zingiber officinale rosc.) through in vitro fertilization(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Rethidevi, A; KAU; Valsala, P AInvestigations on ‘Induction of genetic variability in ginger (Zingiber officinale Rosc.) through in vitro fertilization’ were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2004-2005. Molecular characterization studies of the eight ginger cultivars -Z-0-78 (V1) Kodakara Local (V3), Kuruppampady (V4), Mahima (V5), Maran (V6), Rejatha (V7), Rio-de-Janeiro (V8) and Varadha (V9)- by RAPD using nine random primers revealed that the genetic variability among the cultivars very narrow in the range of 5 to 19 per cent. The cultivar Rio-de-Janeiro was distinct with maximum variability. The variability among the released varieties from IISR ranged from 5 to 10 per cent. The flowering season in ginger extended from August to November. The cultivars showed variability with respect to duration for initiation of flowering and flowering duration. The mean pollen fertility and viability among the ginger cultivars were 35.76 per cent and 8.11 per cent respectively. The autotetraploids had high pollen fertility and viability compared to diploids. The range of pollen viability in autotetraploids was from 12.03 to 15.68 (Z-0-78) and in diploids it was from 3.68 to 8.90 per cent. Among the diploids the released varieties Varadha, Mahima and Rejatha had more pollen fertility and viability. Attempts were made to refine the in vitro pollination technique with respect to basal media combination, growth regulators, vitamins and gelling agent. Studies conducted in the cross Varadha X Z-0-78 using placental pollination technique. The basal medium of half MS with 2X vitamins was superior to B5 medium. As gelling agent phytagel 0.18 per cent was superior to agar 0.65 per cent. Among the various plant growth regulator combinations tried the combination of picloram and BA was found to be best. The medium of half MS with 2X vitamins + 3 per cent sucrose + BA 2.5 mgl-1+ picloram 0.2 mgl-1 + 0.18 per cent phytagel was the best with respect to maximum ovule swelling, percentage of cultures with ovule development and percentage setting per culture. Seed set and seed development in various crosses involving autotetraploids and diploids were assessed. The parental combination between autotetraploids (Z-0-78 X Z-0-86) produced seeds with very good swelling in maximum number of cultures with maximum setting per culture. The parental combination Z-078 X Varadha and its reciprocal also favoured very good ovule swelling. The parental combination Z-0-86 X Varadha as well as Varadha X Mahima and their reciprocals produced seeds with good swelling. The size increase of the developing ovules after in vitro pollination was monitored from the day of pollination to 60 DAP. The size increase upto 15 DAP was rapid. At 60 DAP the ovules attained four-fold increase in size. The pollinated ovules cultured, in the course of maturation turned pink and later blackened. The seeds showed complete endosperm filling at 10 DAP. The soft and jelly like endosperm became hard by 20 DAP. Viability test with tetrazolium showed that the seeds were viable up to 50 DAP. Embryo was found to be seated towards the chalazal end and endosperm constitutes the major portion of the seed. Germination studies were conducted in a number of plant growth regulator combinations. The 60 days old seed derived from the parental combination of Kuruppampady X Z-0-78 showed radicle emergence in the medium of half MS + 3 per cent sucrose + 0.65 per cent agar + IBA 3.0 mg l-1. The seeds of parental combination Z-0-78 X Varadha germinated through somatic embryogenesis in the medium of B5 + 3 per cent sucrose + 0.18 per cent phytagel + 2,4-D 0.2 mg l-1 + BA 2.5 mg l-1 105 days after in vitro pollination. The germination was 10 per cent It was found that the seed coat was pushed apart by the callusing embryo and it developed globular somatic embryoids. Embryoids developed root and shoot poles in the same medium. The somatic embryoid is in the initial stage of development. Embryo culture studies with embryos excised from seeds of 15 to 60 DAP in various media combinations did not give positive response. Prolonged culturing may give positive results.ThesisItem Open Access Response of immature inflorescence for in vitro regeneration on coconut (cocos nucifera L)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2006) Siny, C V; KAU; Nazeem, P AInvestigations on 'Response of immature inflorescence for in vitro regeneration in coconut (Cocos nucifera L.)' were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2005 to 2006. Y3 medium was found to be the best basal medium for in vitro culture of immature inflorescence of coconut. Inflorescence of length 40 to 50 cm was found to posses male flowers at pollen mother cell stage of microsporogenesis. Wiping the spathe with 70 per cent ethyl alcohol before excising the immature inflorescence parts could effectively control contamination with 100 per cent culture establishment. The young inflorescence parts could survive up to 12 minutes treatment with 0.1 per cent HgCl2. Among the different explants tried, anthers at premeiotic stage and immature rachillae were found to be the best for callus induction and embryo formation. When the explants were inoculated the exudation of polyphenols from the explants adversely affected their survival. Polyphenol exudation was checked by incorporating PVP 0.1 per cent and activated charcoal 0.2 per cent in the medium and by incubation under dark condition. 2,4-D at 15 to 30 mgl-1 was found to be the most effective auxin for callus induction and proliferation. Y3 basal medium with growth regulator combinations of 15 mg l-1 2,4-D, 0.5 mg l-1 picloram, 1mg l-1 NAA and 0.1 mg l-1 TDZ was identified as the best medium for callus induction and embryogenesis of immature anther. Sucrose at 5 per cent concentration was identified as the optimum concentration for callus induction. Pretreatment of inflorescence at 4°C for 24 hrs or 30 hrs doubled the callus induction and reduced the browning of explant. Callus induction was observed from rachillae tissue in Y3 medium containing15 mg l-1 2,4-D, 1 mg l-1 picloram, 1 mg l-1 IAA and 0.1 mg l-1 TDZ.ThesisItem Open Access Real-time PCR assay for p-1,3- glucanase gene expression in black pepper (Piper nigrum L.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2009) Dukare, Kiran Shankar; KAU; Nazeem, P ACultivated black pepper {Piper nigrum) varieties are susceptible to Phytophthora rot, caused by Phytophthora capsici, resulting in yield losses upto 50 per cent. Since conventional breeding programmes for this perennial spice crop are complex and time consuming, an attempt was made to unravel the mechanism for disease tolerance at the molecular level.(3-1,3-glucanases are the pathogenesis-related proteins reported in black pepper. The present study was carried out to confirm the role of |3-l,3-glucanases in the defence mechanism and to unravel the mode of expression in comparison with the resistant genotype, Piper colubrinum. Real-time PCR (polymerase chain reaction) assay and northern blotting were performed to determine the expression pattern of the gene encoding (3-1,3- glucanases in both susceptible and resistant genotypes after infection with the fungus Phytophthora capsici. The data was normalized with a stably expressing housekeeping gene. Southern hybridization was carried along with real-time PCR to determine the gene copy number in both P. nigrum and P. colubrinum. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the reference gene (endogenous control), since it was more stable than 18S rRNA and actin genes studied. The real-time PCR assay indicated marked difference in (3-1,3-glucanase at transcript level in the genotypes studied, when the data was normalized with endogenous control. The rate of increase in transcript level after infection with the pathogen was low and gradual in P. nigrum while it was much higher and faster in P. colubrinum. The glucanase enzyme activity at healthy stage in P. colubrinum was 88.00 unit/mg protein/10 min, while it was only 26.29 unit/mg protein/ 10 min in P. nigrum. Upon inoculation with the fungus, the gene expression was elevated over four times within two hours after inoculation in P. colubrinum while it was much lower in P. nigrum. The northern blot analysis alsoindicated differential expression of the gene. Southern hybridization indicated that the gene copy number varied in two species, while real-time PCR assay confirmed that it was almost double in P. colubrinum than in P. nigrum. The results clearly indicate the positive role of |3-l,3-glucanases in the defence mechanism and highlight the differential gene expression in susceptible and resistant genotypes.ThesisItem Open Access Management biodegradable plant tissue culture lab wastes through biomethanogenesisof(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2008) Abdulla Fayas, T; KAU; Rajendran, P CGenerating renewable source of energy from tissue culture laboratory waste by the process of biomethanogenesis is the focal theme of present investigation. Unlike developed countries, the developing countries are hesitant to establish more number of biotechnology/ tissue culture laboratories due to financial constraints. Easy and regular availability of biogas from TC wastes will be a boon to establish self-sustainable TC laboratory in view of present energy crisis. The biogas experimental units required for the study was designed and various treatments were employed for the biodegradation of tissue culture waste, using the methanogenic bacteria Methanobacterium ruminatium, Methanobacterium formicicum, Methcmosarcina barkeri, Bactereoides ruminicola, Selenomonas ruminatium, Eubacterium tortuosum and Clostridium butyricum. Treatment involving TC waste and cow dung was also conducted for biomethanation in the present study. Quantity of gas production and its combustibility was noticed for various treatments. In bacterial treatments the quantity of gas generation was highest for Clostridium butyricum. Only treatments involving cow dung produced combustible gas. Molecular characterization of methanogenic bacterial cultures was also done for finding the genetic similarity between them. RAPD followed by scoring of the bands by UPGA routine showed maximum similarity between bacterial cultures of Methanobacterium ruminatium and Methanobacterium formicicum with Methanosarcina barkeri. Physio-chemical characters like C/N ratio of the TC wastes, pH and temperature of medium and Hydraulic retention time was also observed for the various treatments. The C/N ratio of the TC wastes was found to be very low and nowhere near the optimum C/N ratio of 20-30 required for gas production. Other parameters like pH of the treatments and Hydraulic retention time was also noticed. The pH of the treatments involving bacterial cultures was very low, considering the normal pH of 6.8 to 7.5 required in biogas generation. The main constraints in the bio gas generation were found out to be the low C/N ratio of the TC waste and the low pH of the medium. The present study indicated the possibility of bio-gas generation from TC waste through fortification using various supplements like coconut water and coir pith which have higher C/N ratio.ThesisItem Open Access Molecular characterization of tomato (Solatium lycopersicum L.) with special reference to tomato leaf curl virus (ToLCV) resistance(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2007) Anjali, Divakaran; KAU; Nazeem, MTomato (Solarium lycopersicum L.) is one of the major vegetable crops in the world. India ranks sixth in the production of tomatoes worldwide with a total area of 0.50 million hectares and productivity of 17.4 MT per hectare. Tomato leaf curl disease caused by the Tomato Leaf Curl Virus (ToLCV) and transmitted by whiteflies (Bemisia tabaci) is one of the most important diseases affecting this crop. The disease causes losses in yield to the tune of 70 to 100 per cent. ToLCV is severe under conditions prevalent in Kerala also. Identification of resistant sources of the disease and development of trait-related markers from these sources would be an important approach to overcome the problem of ToLCV. With this objective in mind, an investigation was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara from the year 2005 to 2007 to characterize the reaction of tomato genotypes to ToLCV under conditions prevalent in the area and to identify molecular markers (RAPD and AFLP) linked to disease resistance.Fifteen genotypes were raised in sterile soil in earthen pots and field during the peak season of ToLCV infection (December - February) and their reaction to the disease was categorized based on the coefficient of infection. Out of 15 genotypes, eight were observed to be highly resistant to ToLCV under both pot culture and field experiments. Observations of biometric characters of the genotypes grown in pots and field were made. All genotypes showed significant difference in all the characters observed both in pot culture experiments and field study. Plant height was the most striking character of difference observed in the two different culture conditions. Genotypes were subjected to molecular characterization using RAPD and AFLP markers. Genomic DNA required for these assays was isolated by two protocols. The protocol suggested by Rogers and Bendich (1994) with modifications was found to be most appropriate for DNA isolation from tomato leaves. Forty random decamer primers were screened for RAPD assay. Thirty-six of these were used for further RAPD profiling of the tomato genotypes. Out of this, 12 primers displaying good and reproducible patterns were selected for molecular characterization. The primer OPS 8 recorded the highest resolving power. A total of 116 amplicons were generated by the 12 selected primers of which 71 were polymorphic. The dendrogram constructed separated the genotypes into two groups. ToLCV resistant genotypes Anagha and H-24 with 92 per cent similarity were found to be most related. RAPD analysis did not reveal any trait- related marker in the present study. AFLP assay was carried out with five combinations of Eco&l and Msel based primers. A total of 241 amplicons were detected, out of which 122 were polymorphic. Three markers linked to ToLCV susceptibility were obtained using the primer combination EAAG/MCAC. All genotypes studied showed genetic uniformity in RAPD and AFLP assay except with respect to a few primers. Trait-related marker was detected in a single primer pair in AFLP assay, while RAPD assay did not give any clear demarcation with respect to ToLCV resistance/susceptibility. The markers identified could be further exploited for obtaining nucleotide sequence information and level of specific gene expression in susceptible/resistant genotypes.