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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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 C
Generating 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.
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 A
Nutmeg (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 succeed
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, M
The 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.
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 A
Cultivated 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.
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 C
Generating 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.
Open Access
Morphomolecular charecterisation of the variants of piper nigrum L. variety panniyur -1
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2008) Smitha Bhasi; KAU; Swapana Alex
The study entitled “Morphomolecular characterization of variants of Piper nigrum L. variety Panniyur-1” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and in the Block V of Panniyur-1 at the Regional Agricultural Research Station (RARS), Ambalavayal during the year 2006-2007 with an objective of characterizing the variants of black pepper variety Panniyur-1 based on morphological traits and RAPD profiles. Black pepper often referred to as the ‘King of spices’ is the most important spice in the world. The first ever hybrid of black pepper, Panniyur-1 (Uthirankotta x Cheriyakaniyakadan) is the most popular pepper variety grown in India and also in Kerala. In black pepper, propagation through cuttings is being practiced for decades for producing true-to type plants. However, contrary to this belief, there are reports for the existence of variability. Variability was reported even at the intraclonal level. The first such report in black pepper was in the local variety Karimunda (Ratnambal et al., 1985). According to Pradeepkumar et al. (1999), there exists intra-clonal variability in yield among the hybrid clone Panniyur-1 at the RARS, Ambalavayal. Such reports deserve serious concern and in depth analysis as pepper is a leading commercial crop of India, important in the domestic as well as international markets. The present study was taken up in this context utilising the progeny of the forty variant plants reported by Pradeepkumar et al. (2003) from the RARS, Ambalavayal. The objective was to assess the extent of variability with respect to morphological traits including yield parameters as well as the molecular analysis of genetic variability. On morphological analysis of the forty plants, considerable variation was observed. The maximum variation was observed in number of berries per spike followed by drying percentage. The analysis of the dendrogram showed that none of the plants were 100 per cent similar at a distance of 1.0. At a distance of 2.0 the clones can be grouped into five clusters. At a distance of 10, the plants can be grouped into two clusters comprising a major group with twenty-nine plants and a minor group with eleven plants. Molecular analysis also revealed variability, accounting for 66.34 per cent polymorphism. In the dendrogram at the similarity index 0.70 the plants grouped into two major clusters indicating thirty per cent dissimilarity. None of the plants were showed 100 per cent similarity. All the forty plants under study formed individual clusters at a similarity index 0.91 except V36 and V37. Ninety percent similarity was observed between the plants V20 and V30. At a similarity index below 0.70 the dendrogram showed a cluster including all the plants except V14. The present findings need further confirmation with more number of primers and other molecular markers like ISSR, AFLP etc. The occurrence of variability among the clones of Panniyur-1 in other major pepper growing tracts also needs to be investigated in detail.
Open Access
Molecular analysis of spike branching observed in black pepper (Piper nigrum L.) type from Idukki
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2009) Vimarsha, H S; KAU; Rajmohan, K
The study entitled “Molecular analysis of spike branching observed in black pepper (Piper nigrum L.) type from Idukki” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2006-2008. The objective of the study was to analyse the sequence homology for the inflorescence architecture gene (TFL1) and flowering gene (FT) in spike branching black pepper type from Idukki, using polymerase chain reaction technique. Spike branching in black pepper is very rare phenomenon. Profuse spike branching was observed in an uncharacterized black pepper type from Idukki. Single spike had three to four times more berry yield, compared to improved varieties. This type needed to be characterized at morphological and molecular levels to know its parental descendent and possible involvement of gene regulation responsible for spike branching. As an initial approach, molecular analysis was done to find out the presence of TFL1 and FT genes, which have been reported to be involved in inflorescence branching in modal plants. TFL1 primer pair (TFL-F1 and TFL-R1), designed for the fourth exonic sequence of TFL1 gene in Arabidopsis thaliana, could amplify a 700 bp DNA sequence in the spike branching type, as well as in Karimunda and Vellamundi, indicating homology for the gene sequence and possible conserved exonic sequence in black pepper. Presence of sequence homology for the gene TFL1 indicated the possible involvement of TFL1 gene, which had been reported to be associated with inflorescence branching in Arabidopsis thaliana, in the spike branching trait of the black pepper type. This result is significant, as five out of the eight cultivars tested did not give any positive response for the primer pair designed based on TFL1 gene. However, two non-spike branching varieties including Karimunda and Vellamundi have also shown amplification for the TFL1 primer pair. As an initial step towards characterisation of the spike branching type, RAPD analysis of it along with seven other cultivars/varieties was done. This analysis revealed variability, accounting for 83 per cent polymorphism. In the dendrogram, at a similarity index of 0.43 the plants grouped into two big clusters, indicating 67 per cent dissimilarity. All the eight plants under study formed individual clusters at similarity index 0.74, except the spike branching type and Vellamundi. Results of gene specific PCR which yielded single amplicon can be hypothecated for the presence of sequence homology for the gene TFL1 and has conserved fourth exon in black pepper cultivars and varieties. Further transcript level and expression level studies are essential to find the specific role of the TFL1 gene in spike branching black pepper.
Open Access
Isolation and characterisation of cDNA encoding dihydroflavonol 4- reductase gene from Orchid Dendrobium variety Sonia17
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2009) Saritha, V S; KAU; Soni, K B
The thesis entitled “Isolation and characterization of cDNA encoding dihydroflavonol 4-reductase gene from Dendrobium orchid variety Sonia 17” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2006-2008 with an objective of characterizing one of the key genes involved in anthocyanin biosynthesis, viz., dihydroflavonol 4-reductase from flower buds of Dendrobium Sonia 17 orchid. Reverse Transcription-PCR technique was used in the present study for the amplification of cDNA encoding dfr gene. Heterologous forward and reverse primers were designed based on the gene sequences of Cymbidium orchid and Oryza sativa using Primer 3 software. Total RNA isolated from the flower buds using TRIZOL reagent yielded good quality intact RNA, with no genomic DNA contamination and showed an A260/A280 ratio of 1.6-2.0. The cDNA synthesized from purified mRNA was amplified using gene specific primers designed. All the primers included in the study yielded reproducible amplification. The primer sets A (Af and Ar) and C (Cf and Cr) which were designed based on the dfr gene sequence of Cymbidium orchid yielded amplified fragments of size approximately 400-500 bp and 100 bp respectively. Primer set B (Bf and Br) synthesized from Oryza sativa dfr gene sequences yielded an amplified product of size nearly 300 bp. The cDNA amplified with primer set A was cloned in pGEM-T Easy Vector and multiplied in competent E. Coli strain DH5α for sequencing. The transformed E. Coli colonies were grown on L.B agar plates and selected by blue – white screening. Three white colonies were randomly selected and plasmid DNA was isolated from transformed clones. The presence of insert was checked by PCR analysis of the plasmid DNA with gene specific primer (primer set A). The agarose gel electrophoresis showed the presence of amplified fragment. Sequencing of two clones at Chromous Biotech Pvt. Ltd. using T7 and SP6 primer failed to amplify the plasmid DNA. This work was an initial step towards isolation and characterization of dihydroflavonol 4-reductase gene in Dendrobium variety Sonia 17. The primers designed in this study could successfully amplify the cDNA encoding dfr. PCR reactions gave good results but the sequencing was not satisfactory. For the better understanding of the dfr sequences, the sequencing reaction needs to be repeated with other clones.
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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 C
Generating 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.

Kerala Agricultural University, Thrissur

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