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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 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.ThesisItem Open Access Genetic transformation in Artemesia annual L. for hairy root induction and enhancement of secondary metabolites(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Shaneeja, V M; KAU; Keshavachandran, RThe present study entitled “Genetic transformation in Artemesia annua L. for hairy root induction and enhancement of secondary metabolites” was carried out at the Centre for Plant Biotechnology and Molecular Biology of the College of Horticulture, Vellanikkara. The study was undertaken to standardize the in vitro regeneration protocol in Artemesia annua from different explants, to standardize the genetic transformation using A. rhizogenes, to standardize the biochemical techniques for the estimation of secondary metabolites in Artemesia annua and also to enhance the secondary metabolite production in the hairy root cultures of Artemesia annua. An efficient method for in vitro plant regeneration was developed in Artemesia annua. Different explants such as leaves, petiole, shoot tip, nodal segments, inflorescence segments and roots of Artemesia annua were used for the study. Maximum regeneration from leaf explants was in MS with 0.5 mg l-1 BAP. Shoot buds produced from leaf explant showed good multiplication in the same media. MS was found to be the best basal media for regeneration from leaf explants. The leaf originated callus produced regeneration in MS with 3 mg l-1 BAP. The shoot tip and inflorescence bits showed maximum regeneration in 0.2mg l-1 NAA and 0.5 mg l-1 BAP. Nodal segments showed maximum regeneration in MS with 0.1 mg l-1 with NAA and 0.2 mg l-1 BAP. The roots taken from in vitro rooted plantlets showed high callusing but failed to regenerate by direct organogenisis. However, a green coloured structure was obtained from root callus in MS media supplemented with BAP 3 mg l-1 and NAA 0.1 mg l-1. The shoot/ shoot buds showed good elongation in MS media with GA3 0.2 mg l-1. The shoots were successfully rooted in half MS with 0.5 mg l-1 IBA and 2 per cent sucrose. The plants were successfully hardened and transferred to large pots in the green house. Genetic transformation was carried out in A. annua. using three different A. rhizogenes strains like A4, ATCC 15834 and MTCC 2364 for inducing hairy roots. The explants such as leaf segments shoot tips and nodal segments were used for genetic transformation. Here the influence of different parameters such as type of explants, type of bacterial inoculum, co-cultivation periods and acetosyringone effects on transformation frequencies were studied. Among the three A. rhizogenes strains used, ATCC 15834 produced the highest transformation efficiency. Acetosyringone (100 M) enhanced the transformation percentages in all the strains. The hairy root cultured in hormone free basal media showed high lateral branching. Among the four liquid media tested, B5 with 3.0 per cent sucrose was found to be superior in promoting hairy root growth followed by B5 with 2.0 per cent sucrose, MS and half MS. The confirmation of transformation was obtained by opine detection and PCR. A Thin Layer Chromatographic method was employed for artemisinin estimation. Artemisinin obtained from Sigma Chemicals, USA was used as the standard in estimation studies. Silica gel60 F254 plate with hexane- diethyl ether (1:1) as the solvent system was used. The pink spot was observed by immersing the plates in a pool of freshly prepared glacial acetic acid: conc. sulphuric acid: anisaldehyde (50:1:0.5) followed by drying in a chromatographic oven at 110˚C for 15 min. No artemisinin was detected in roots taken from plants grown in the field, in vitro roots and hairy roots induced by MTCC 2364. Rooting of in vitro shoots enhanced the artemisinin content. Enhancement of secondary metabolite production was studied using techniques such as addition of osmoregulants, precursor feeding and elicitation. The artemisinin content in the hairy root biomass and the culture medium were estimated. The osmoregulant PEG (2.0 % and 5.0 %) and the precursor methionine (1 mM) and yeast extract (2.5g l-1) failed to enhance the artemisinin content. However, the biotic elicitor Aspergillus homogenate (250l /125 ml) elicited a positive influence on the biosynthesis of artemisinin in the hairy root cultures.ThesisItem Open Access Bioecology and management of pod bugs infesting vegetable cowpea (Vigna unguiculata ssp. sesquipedalis (L.) Verdcourt(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Bharathi Meena, T; KAU; Sudharma, KThe study entitled ‘genetic transformation of chilli (Capsicum annuum L.) with osmotin gene’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, from December 2003 to September 2005. The study was undertaken to standardize in vitro regeneration and Agrobacterium mediated transformation of chilli with osmotin gene. Different explants, media and hormonal combinations (auxin and cytokinin) were tried in order to standardize in vitro regeneration in chilli var.Ujwala. The best explant for in vitro regeneration was hypocotyl. Only the buds originated from hypocotyl explants showed elongation. Shoot buds formed from cotyledon and leaf segments did not elongate in any of the media tested. Based on the percentage regeneration in cultures and no. of shoot-buds per explant, MS medium containing the hormonal combination; BA (5.0 mg l-1) + IAA (0.3 mg l-1) was found optimum. The regenerated plantlets were transferred to pots for acclimatization so that they can sustain and survive in the natural conditions. The hardened plantlets were planted out. Agrobacterium mediated transformation protocol was optimized considering all the factors for successful transformation. Optimum inhibitory concentration of selectable marker (Kanamycin: 100mg l-1) was established. The antibiotic cefotaxime (200 mg l-1) was selected for killing the bacteria. Agrobacterium strain EHA 105 harbouring the gus reporter gene was used for the standardization of transformation. Hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (EHA 105). The inoculum density-0.1 OD600 nm, infection time-5minutes and co-cultivation period-2 days were found optimum based on GUS assay and survival rate 60 days after co-cultivation. The hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (GV2260) harbouring osmotin gene in the plasmid pGV2260 tagged with 35S CaMV promoter. The transformed explants were regenerated on the selection medium optimized for regeneration of chilli. However, none of the transformed buds were elongated in the elongation medium containing selection agent. Hence the transformed plants were transferred to elongation medium containing no antibiotics. Even in this medium, no elongation of shoots was observed even after 60 days. So, further refinement of transformation protocol using an optimal selectable marker is needed for the production of transgenic chilli. After selection of the transformants, the putative transgenics were characterized employing molecular biology techniques viz. PCR-utilizing the gene specific primers of nptII. The presence of transgene was confirmed in the transformed plants.ThesisItem Open Access Molecular characterization and development of trait related markers for aphid resistance in cowpea (Vigna unguiculate (L) Walp.)(Centre for Plant Biotechnology and Molicular Biology, College of Horticulture, Vellanikkara, 2007) Ramya, Haridas; KAU; Keshavachandran, RCowpea is one of the most important grain legumes that has been grown throughout the tropics. It is a rich source of proteins (25 percent). Their ability to fix nitrogen is of great advantage on small farms. However their production is greatly limited by many pests, especially aphids. Keeping in view of the importance of this crop, a study was conducted on ‘Molecular characterization and development of trait related markers of aphid resistance in cowpea (Vigna unguiculata (L) Walp.)’. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology and Radio Tracer Laboratory, College of Horticulture during the period 2005-2007. The objectives of the study were to identify the sources of resistance to aphids and to develop markers for aphid resistance in cowpea. The cowpea accessions which were susceptible, moderately resistant and resistant were identified based on the average count of aphids. Five susceptible (VS 1177, VS 1179, VS 1173, VS 1208, VS 1034) and five resistant (VS 1230, VS 1231, VS 1201, VS 1248, VS 1263) accessions were used for the present study. These 10 accessions were subjected to molecular characterization using RAPD and AFLP markers. For RAPD and AFLP analysis, the protocol for genomic DNA isolation from cowpea was standardized. The protocol suggested by Doyle and Doyle (1987) was found to be the most appropriate. Thirty random primers were screened and ten were selected for RAPD profiling of cowpea accessions. The primer OPA 4 was found to have the highest resolving power. A total of 75 scorable amplification products were generated by 10 random primers of which 48 bands were polymorphic. Specific bands were generated for the resistant accessions VS 1230 and VS 1201 with OPA 2, VS 1248 and VS 1263 using OPA 3 and VS 1248 alone with OPA 10. In the dendrogram, the 10 accessions were grouped into two major clusters of 8 and 2 accessions each. The resistant accessions VS 1230 and VS 1231 were the most closely related with 90 percent similarity. Another two resistant accessions VS 1248 and VS 1263 were also grouped together in the dendrogram. The cowpea accessions were subjected to AFLP analysis with 5 primer combinations of EcoR1 and Mse1. A total of 237 scorable amplification products were produced by the primer pairs of which 93 bands were polymorphic. The highest resolving power was obtained for EACT+MCAA. Specific bands were produced for EAAG+MCAA in resistant accessions VS 1230, VS 1263 and VS 1248. The dendrogram obtained for AFLP showed that VS 1177 and VS 1179 were most closely related at 91 percent similarity. Similarly the resistant accessions VS 1263 and VS 1248, VS 1230 and VS 1201 were grouped in different sub clusters. Thus RAPD and AFLP markers were utilized to characterize cowpea accessions for aphid resistance. The specific bands identified in the resistant accessions could be treated as trait related markers for aphid resistance. Further studies should be conducted on screening the cowpea accessions with more number of primers to develop more specific markers with reference to the aphid resistance. The studies should also be focused on converting these RAPD and AFLP markers linked to aphid resistance to a more specific amplification, a technique called Sequence Characterized Amplified Regions (SCAR).ThesisItem Open Access Molecular detection of viruses infecting vanilla (Vanilla planifolia Andrews)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Nisha, G; KAU; Girija, DVanilla, often quoted as the ‘Princess of Spices’, owing to its unique flavour and aroma is the second most expensive spice on the world market and a valuable source of foreign exchange for several countries. Viruses are known to cause significant yield reduction in many vanilla growing countries of the world. Vanilla mosaic disease was first reported by Wisler et al. (1987) from French Polynesia. Recently mosaic disease has been reported from South India (Thomas, 2002). The virus was identified to be a potyvirus and is sap transmissible. It is known that viruses can be readily transmitted through tissue culture process. To produce virus free plantlets, the source plants for tissue culture must be completely free of virus. Considering the nature of spread and damage that can be caused by the virus, it is essential that we should take effective control measures in the initial stage itself. Recent advances in biotechnology and molecular biology have played a significant role in development of rapid, specific and sensitive assays for detection of plant viruses. RT-PCR and PCR are popular techniques for detection and identification of RNA and DNA plant viruses respectively. The procedures are extremely sensitive, fairly inexpensive and require minimal skill to perform and it has the potential to be an extremely sensitive alternative to ELISA. In the present study an attempt was made to develop a reverse transcription polymerase chain reaction assay to detect vanilla mosaic virus (VanMV) infecting vanilla. Mosaic and necrosis infected vanilla plants collected from different locations of Kerala were used to inoculate the test plants viz., healthy tissue culture-derived vanilla. Biological indexing of the virus on petunia (Petunia hybrida) and cowpea (Vigna ungiculata subsp. sesquipedalis) seedlings, which are the indicator plants of potyvirus and cucumber mosaic virus (CMV) respectively, clearly indicated that the infection is due to potyvirus, not due to CMV. RNA was isolated from mosaic infected plants collected from different locations of Kerala, artificially inoculated vanilla samples and also from healthy tissue culture-derived vanilla plants and this was used for cDNA synthesis with oligo dT primers using MMuLV RT-PCR kit. A set of degenerate primers was designed. Of these two sets were designed based on published data (RKJ3 and HRP52; PotyF1 and PotyR1) and the other one (PVF1 and PVR1) was based on the conserved sequence of coat protein genes of potyviruses, and these were used in PCR. Primer pair RKJ3 and HRP52 yielded a product of lower size [approximately 300 bp than expected (850 bp)]. On sequencing the amplified product gave information on 240 bp and sequence analysis using blastn revealed identify with chloroplast genome. This could be due to nonspecific amplification. Hence based on the present study this primer pair cannot be recommended for detection of poty viruses infecting vanilla. Attempts made to obtain amplification with primers PotyF1 and PotyR1 did not prove successful. Hence this primer pair was not used for further experiments. Amplification of two bands (approximately 350 bp and 149 bp) could be observed with primer combination PVF1 and PVR1 invariably with all the isolates collected from different locations. The similar bands were even yielded with all the artificially inoculated vanilla samples, confirming that it was the coat protein gene of potyvirus that was amplified. It was not found with the analysis of healthy control suggesting that the characteristic bands are of virus coat protein gene. Two bands yielded may be due to binding of primers at an internal site of potyvirus sequence. Reamplification of the upper band gave two bands of 350 bp and 149 bp, corresponding to the bands obtained in PCR analysis and the lower band gave an intact band of 149 bp. Hence it could be concluded that the amplified product is a part of coat protein gene of vanilla mosaic potyvirus. Therefore, RT-PCR assay using PVF1 and PVR1 primers could be used for rapid and easy detection of potyviruses infecting vanilla. However, sequence information of the amplified fragment is necessary before this method is recommending on a large scale for the detection of potyviruses infecting vanillaThesisItem Open Access Cloning of genes encoding insecticidal proteins (cry/vip genes ) of Bacillus thuringiensis from Western Ghats of Kerala(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Neema, P M; KAU; Girija, DThe study entitled ‘Cloning of genes encoding insecticidal proteins (cry/vip genes) of Bacillus thuringiensis from Western Ghats of Kerala’ was carried out in the Molecular Biology Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2005- 2007. The crystal protein genes (cry/vip) of B. thuringiensis possess insecticidal activity against larvae of insect orders Lepidoptera, Diptera and Coleoptera. In the present study, an attempt was made to isolate and clone cry genes of B. thuringiensis from the Western Ghats of Kerala. Bacillus thuringiensis strains were isolated from soil samples collected from different locations of the Western Ghats of Kerala. The pure colonies obtained were stab inoculated and stored under refrigerated conditions. Variability among the isolates were studied by various cultural, morphological and biochemical tests. The insecticidal activity of the isolates was determined by bioassay against the major lepidopteran pest of cucurbitaceous vegetables, the pumpkin caterpillar. The information on cry1A gene sequences of different species of Bacillus thuringiensis available in the public domain NCBI was collected and subjected to multiple sequence alignment to detect conserved boxes of the gene among species. Based on the data, one pair of gene specific primer was designed for amplification of partial cry1A gene fragment of about 800bp in B. thuringiensis isolates. Total DNA was isolated from the B. thuringiensis strains of Western Ghats of Kerala. Profiling of cry1 and cry4 genes of bacterial isolates were done using universal primers for cry1 and cry4. Amplification was obtained with cry1 gene for seven isolates and with cry4 gene primer for two isolates. The amplicons obtained with universal cry1 primer from two isolates and with cry4 primer from one isolate were used for cloning. The amplicons obtained with cry1 and cry4 primers were eluted, cloned in pGEMT vector and transformed into competent cells. High level of recombination was observed on blue-white screening. Recombination of the insert was confirmed by PCR of the plasmid isolated from white colonies. The cloned fragments were sequenced. The amplicon obtained with cry4 primer in the second isolate was sequenced after purifying the PCR product. The cry1ky5 and cry1em11 sequences when subjected to Blast search revealed significant levels of homology with cry1 genes reported from other B. thuringiensis strains deposited in the public domain. The cry4em10 sequence when subjected to Blast search, showed high level of similarity with cry4 genes from B. thuringiensis. The cry4ky1 sequence showed similarity with cry genes of different species of B. thuringiensis. The sequences were also subjected to various sequence analysis using bioinformatics tools which include ORF finder, SOPMA, GENSCAN, AASTAT and TCAG tools of Biology Workbench and Interproscan. PCR of all the isolates found positive in cry1 gene profiling, was done with cry1A primer designed during this study. Amplification was obtained with cry1A primer in two isolates. The amplicon obtained in one isolate was subjected to sequencing after purifying the PCR product. The cry1Aky3 sequence showed similarity with other cry genes of Bacillus thuringiensis present in the NCBI databank. 1500 bp long variable region of cry1 was amplified in two isolates using specific primers. Future research works should be focused on the isolation of B. thuringiensis from completely undisturbed ecological niches. Novelty of cry genes can be detected by restriction digestion of the genes. Characterization of novel full-length cry genes and its expression in transgenic crops will help to develop resistant varieties thereby reducing insecticide applications and resistance development in insect pest populations.ThesisItem Open Access Identification of molecular markers for developing breeding strategies in rose(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Cinu Sebastian; KAU; Rajendran, PRose, one of the most important flowering ornamentals is a favourite for landscaping and an important commercial cut flower. Breeders are always looking for new and novel varieties to meet the ever increasing demand of consumers. However, many years are required to develop a new variety through conventional methods. Many desirable roses are female sterile and hence pose a real limitation to breeding. Developing a molecular marker that can readily identify a female parent can go a long way to avoid unproductive hybrids. Premature abortion of developing embryos resulting in few or no viable seeds is another major set back. The present investigation entitled ‘Identification of molecular markers for developing breeding strategies in rose’ was held out at this context at the Centre of Plant Biotechnology and Molecular Biology (CPBMB) with the aim of determining a molecular marker and attempting embryo rescue of rose. Fifty rose varieties were selected based on morphological characters viz., seed setting ability. Variations in foliar characters of plants were recorded. Genomic DNA extraction from tender leaves of rose plants using Roger and Bendich’s method (1994) with slight modification was found to be the best. Out of fifty-one primers screened four primers belonging to OPAA 2 , C 4, C 15 and C19 were identified as the best. Molecular characterization by RAPD assay generated a total of 331 amplification products. Some bands were specific or prominent to the group. The clustering of sterile and fertile varieties mostly into two separate clusters indicated their similarity at the genetic level. Further studies have to be conducted by increasing the number of primers used, for identification of fertility status of more varieties. In view of the problems faced by breeders regarding unproductive hybrids, an attempt was made for embryo rescue. Surface sterilization of hips with 0.1 per cent mercuric chloride was standardized. The pollen fertility was assessed by acetocarmine staining and mean fertility was observed to be 74.6 per cent. The poor response of germination observed for achenes was due to both physical and chemical restriction on the embryo. Effect of media type and combination of growth regulators were assessed. A high germination rate was observed in cultures incubated for two weeks in dark and subsequently transferred to light. Inoculation media with BA and IAA and subculturing media with BA and NAA combined with low salt concentration (half MS) was found to give maximum response. Further trials can lead to the identification of a definite protocol for regeneration.ThesisItem Open Access Biochemical characterization and cell immobilization of costus pictus D. Don with special reference to antidiabetic property(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Eliza lincy, T; KAU; Augustian, AKerala, a state known for its indigenous knowledge and traditional healing practices, is endowed with thousands of medicinal plants. More than 1200 species of plants are being used in the indigenous system of medicine in the state. The use of synthetic chemicals in modern medicine has been causing several side effects. Hence, more and more scientific and commercial activities are now directed towards plant-based medicines. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2004-2006. The objective of the study was the biochemical characterization and cell immobilization of Costus pictus with special reference to antidiabetic property.The hypoglycemic properties of the plant has been reported both in streptozotocin and alloxan induced diabetes in animals. The toxicity study of crude extract of the plant was also studied (Benny, 2004a; Benny et al., 2004a & b) and reported a positive sign of non-toxicity. Based on the above facts the biochemical characterisation and cell immobilization of Costus pictus was carried out and this was compared with antidiabetic plants like ginger, gymnema, adhatoda, neem and tulsi. Molisch's test, Iodine test, fehling's test, benedict's test and seliwanoff's test confirmed the presence of carbohydrates in Costus pictus and in all selected plants with varying intensities. Carbohydrate tests indicated the presence of monosaccharides of ketohexoses origin in all selected plants including Costus pictus. xanthoproteic reaction, glyoxylic reaction for tryptophan and modified million’s test confirmed the presence of proteins having aromatic amino acids in Costus pictus and in all selected crops. TLC also confirmed the presence of - amino acids by giving pink colour with ninhydrin. The best separation of phenolic spots occurred on acid hydrolysis. The phenols appeared as dark absorbing spots in 253 nm and fluorescent spots in 366 nm. Phenol spots appeared as blue with folins reagent. Four compounds were isolated in TLC from Costus pictus. Ethanol was used as the solvent for the extraction of flavonoids from leaf samples. There was only one spot (Rf value 70) at 253nm in Costus pictus. The red colour production observed in neem and tulsi leaf samples confirmed the presence of condensed tannins. The presence of four hydrolysable tannin in Costus pictus was confirmed. The TLC for terpenoids, Costus pictus and ginger expressed same compound at Rfx100 value 71.4. Gymnema, adhatoda and neem had different spots. In TLC the carotenoids expresed different coloured spots such as green-chlorophyll b, blue green-chlorophyll a, yellow green-xanthphyll and yellow-β-carotene in ascending order under visible light. TLC for plant acids, Costus pictus only gave spot only similar to that of oxalic acid. Mayer’s test, dragendorff’s test and wagner’s test showed positive result for alkaloid in all the plants studied. All the above antidiabetic plants for comparison were subjected to electrophoresis. Protein extraction was carried out using Tris-HCl, Tris-HCl + ingredients and Phosphate buffers. The Tris-HCl +ingredients buffer was found to be more appropriate on account of discrete and distinct banding pattern. Protein-banding pattern in SDS denatured gel conditions and isozyme (peroxidase) banding in non denaturing gels were also studied. The 10% gel gave good separation of bands. The bands were clear in coomassive brilliant blue stain than in the silver staining. Costus pictus exhibited four bands. Isozyme analysis was carried out using peroxidase enzyme system. Costus pictus exhibited four bands in which three are similar to that of ginger. The cells of Costus pictus were immobilized using sodium alginate. Immobilized cells kept for secondary metabolite production in ½ MS liquid media showed the presence of phenolics, terpenoids different Rf values and plant acids, indicating the utility of the technique of producing secondary metabolites from Costus pictus in vitro. Considering the presence of similar hexoketoses, free amino acids and / or soluble protein and phenolic compounds in gymnema and Costus pictus cannot be ruled out for the medicinal use of Costus pictus as an antidiabetic plant. Detailed study on Costus pictus with respect to protein and oxalic acid are needed to obtain more information of the antidiabetic property and toxicity to biological system.