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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 Characterisation of Pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Agatha Shiny, A; KAU; Deepu MathewCowpea (Family: Fabaceae) is an important pulse cum vegetable crop of suitable for the tropical and sub-tropical regions of the world. The grain type cowpeas better tolerates the biotic and abiotic stresses against the vegetable types. Under humid conditions, vegetable types, especially the pole types are susceptible to many diseases and among them, anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Br. and Cav. is very severe. In Kerala, complete yield loss in vegetable cowpea is reported due to anthracnose during monsoons. The study entitled “Characterization of pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.” was carried out with objective to develop the protein profiles of resistant and susceptible bush and pole genotypes through SDS-PAGE analysis at different time intervals of infection and to characterize the differentially expressed proteins by MALDI-TOF followed by in-silico analyses. Two bush type varieties Pusa Komal and Kanakamony, the former reported to be highly susceptible and the latter immune to anthracnose and two pole type varieties Lola and Arimbra Local, of which the former susceptible and the latter resistant were used in the study. Pure culture of the pathogenic fungus was developed and maintained on selective medium (Neopeptone-Glucose-Agar) at the Dept. of Plant Pathology. The identity of Colletotrichum lindemuthianum has been established from the spore characteristics observed under phase contrast microscope and the pathogenicity was confirmed through artificial inoculation under controlled conditions. The pot culture experiment was conducted with 50 pots per variety. Artificial inoculation of pathogenic fungus was done and the leaf samples were collected at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144,168 and 192 hours after artificial inoculation. The total protein was extracted using Tris-HCl buffer (pH-7.5), quantified using spectrophotometer and analyzed by SDS-PAGE method. The defense enzymes like peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were assayed. By artificial inoculation, disease responses for anthracnose were confirmed to be highly susceptible in Pusa Komal and Lola; highly resistant in Arimbra Local and immune in Kanakamony. Protein expression was found to be higher from the initial hours in resistant varieties whereas in susceptible varieties, the expression was reduced immediately after infection then peaked at 18hr and gradually decreased later on. Two prominent and differentially expressed protein bands at 56 kD and 14 kD were sequenced in MALDI-TOF to obtain the peptide mass fingerprint. Through in-silico analyses using Mascot server software, they were identified to be the large and small subunits of the chloroplastic enzyme RuBisCo. Thus the capability of a variety to maintain high levels of RuBisCo was found to be the deciding factor for anthracnose disease resistance. Further, protein profiles developed after purification of proteins by dialysis have clearly identified the differentially expressed band at 29 kD in the resistant varieties which is in the size range of already reported PR proteins. PO and PAL activities were proportionate to the resistance behavior, with the peak values at 18 and 24 hr after inoculation. With the results of this study, these defense enzymes are recommended as biochemical markers for identifying the resistance in the accessions. Capability to maintain higher levels of RuBisCo, PO and PAL enzymes is the characteristic of anthracnose resistant vegetable cowpeas and the future breeding programmes could be oriented in this directionThesisItem Open Access Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Jusna Mariya, P L; KAU; Keshavachandran, RBanana is one of the important fruit crops of India. Banana is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production is Fusarium wilt or Panama wilt caused by Fusarium oxysporum f. sp. cubense. Control of the pathogen is difficult and mainly involves the use of disease free suckers. Although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult due to its triploid nature and sterility factors of banana. The study entitled "Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2009-2013 with an objective to identify differentially expressed genes in disease resistant genotype of banana, Palayankodan using the molecular technique called suppression subtractive hybridization (SSH). Total RNA and mRNA were isolated from healthy and inoculated plants (with Fusarium oxysporum f.sp. cubense) and were used respectively as 'driver' and 'tester' in SSH reaction. The reactions were performed utilizing the PCR select" cDNA subtraction kit provided by CLONTECH, USA. Control subtraction was carried out first using PCR select" cDNA subtraction kit, which gave satisfactory and expected results. For experimental subtraction, the double stranded cDNAs synthesized from Zug mRNA from normal 'driver' and treated 'tester' were digested with RsaI enzyme. Two tester populations were created and each ligated to two different adaptors. This was followed by two hybridization reactions and finally a selective PCR amplification. Only differentially expressed cDNAs were amplified exponentially. This was confirmed by analyzing the PCR products on agarose gel, which showed a smear ranging from 0.9 to 1.3 kb in the subtracted sample and was different from smear pattern of unsubtracted ones. The cDNA fragments from subtracted sample were cloned in pJET and pGEMT vectors and sequenced. Fifty clones were sequenced and analysed after vector and adaptor editing. In silica analysis using bioinformatics tools revealed that some of the cloned sequences showed similarity with known sequences which play important roles during disease resistance conditions directly or indirectly. These included resistance gene candidate NBS type protein, mitogen activated protein kinase, phytoene desaturase, glycerol 3-phosphate dehydrogenase, neutral invertase, 1- aminocyclopropane-l-carboxylase synthase, superoxide dismutase, MADS-box protein, ubiquitin 2, actin, NADPH oxidase, phytoene synthase, ACC synthase, sucrose phosphate synthase, phosphatidic acid phosphatase-like protein, ORF III like polyprotein, bHLH transcription factor like protein, cytochrome oxidase, isochorismatase hydrolase, basic helix-loop-helix family protein, constitutive triple response I-like protein, granule bound starch synthase, alpha amylase precursor, rop protein, GTPase family protein, S-adenosyl-L-methionine synthase protein, ADP-glucose pyrophosphorylase glucose-l-phosphate adenylyl trans, ethylene signal transduction factor and ribosomal protein. Clones were classified into 6 major groups based on function of protein. Sequences had conserved domains for the above mentioned proteins. Genes involved in defense, signal transduction, metabolism, hypothetical protein, transcription factor and translation. For further exploitation of these sequences it is necessary to clone full length cDNA. ESTs thus generated in the present study will be of great use in future for further downstream applications.ThesisItem Open Access Identification of AFLP marker linked with bacterial wilt resistance in chilli (Capsicum annum L)(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Thakur Pranita, Prabhakarrao; KAU; Deepu MathewChilli is one of the most important condiments in India and our country is the largest producer, contributing 25% of the world production. During 2010-11, India produced 0.8 mt of dry chilli from an area of 0.93 mha. Bacterial wilt (caused by Ralstonia solanacearum) is a major reason for the lower productivity of this crop, causing up to complete losses under severe infections. The study entitled “Identification of AFLP marker linked with bacterial wilt resistance in chilli (Capsicum annuum L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture during the period 2011-2013. The objective of the study was to identify AFLP marker linked with bacterial wilt resistance in chilli (Capsicum annuum L.). Three chilli genotypes Ujwala, Anugraha and Pusa Jwala and their progenies were used in the study. Anugraha is a chilli variety with resistance to bacterial wilt disease. Pusa Jwala is the near isogenic line (NIL) of Anugraha, both differing only in the gene for resistance to bacterial wilt. Ujwala was the donor parent for resistance while developing Anugraha from Pusa Jwala through back crossing programme. Pusa Jwala and Anugraha varieties were screened in open field with artificial inoculation to confirm the disease reaction. Highly resistant Anugraha plant was crossed with the pollen from most susceptible Pusa Jwala plant. The F1 seeds were harvested and this generation was field screened to observe the disease reaction. Previous report on bacterial wilt resistance points to monogenic homozygous recessive condition for resistance. Accordingly, all the F1 plants are supposed to be susceptible; but our screening had shown the resistance in F1 generation to be nearly 50 per cent. The reason for the deviation from the expected ratio is attributed to the selection of a heterozygous (Rr) plant as male parent. Further the F1 plants were raised in pots, selfed seeds were harvested and further the pots were infected with bacteria and F2 seeds harvested from susceptible F1s were used for raising the F2 population. F2 population was used as segregating population for generating the susceptible and resistant bulks in bulk segregant analysis (BSA) using AFLP method. Screening of 200 F2 plants was done along with parents Anugraha and Pusa Jwala using leaf cutting and pin pricking methods. F2 segregating plants have shown 69.5 per cent wilt incidence, pointing to ~3:1 ratio, confirming that the resistance is governed by homozygous recessive condition. DNA extraction was done from parents and all the 200 F2 plants and Ujwala by CTAB method (Rogers and Bendich, 1994). Good quality of DNA with UV absorbance ratio (A260/280) ranged 1.8- 2.0 were used for further AFLP analysis. DNA from 9 highly susceptible and 9 highly resistant F2 plants was separately pooled for developing bulk. BSA was carried out with AFLP analysis the DNA using resistant parent (Anugraha), susceptible parent (Pusa Jwala), resistance donor (Ujwala), F2 resistant bulk and F2 susceptible bulk. The AFLP was performed using the standard kit provided by Chromus Biotech, Bangalore, following the restriction digestions using frequent and rare cutters, adapter ligation, pre-amplification and selective amplification using the primer combination EcoACT+ MseCAC. The amplified PCR products were separated by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer along with GeneScan™ 500 LIZ® size standard and data generated were collected by Data Collection 2.1 software. Totally, 124 bands ranging 50-500bp were amplified. Among them, three polymorphic bands with 103, 118 and 161 bp linked with the resistance allele and three polymorphic bands with 183, 296 and 319 bp linked with susceptible allele were identified. Due to the repeatable nature of AFLP, these 6 markers could be directly employed in MAS breeding programmes.ThesisItem Open Access Proteome analysis of induced systemic resistance medicated by plant growth promoting Rhizobacteria (PGPR) in rice for biotic stress(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Shinde Subhashini, Ganesh; KAU; Abida, P SRice (Oryza sativa L.), is vulnerable to a number of pest and diseases. Among them sheath blight disease caused by Rhizoctonia solani, and insect pest, brown planthopper (BPH) (Nilapavata lugens) are the most devastating agents and major challenge to rice cultivation. Approximately 30 per cent yield loss has been reported due to above said disease and pest. In this context, the present study entitled „proteome analysis of induced systemic resistance mediated by plant growth promoting rhizobacteria (PGPR) in rice for biotic stress‟ was carry out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2011-2013, with the objective to identify and characterize the interacting proteome in inducing systemic resistance in rice mediated by PGPR during pathogen and insect attack. Plant growth-promoting rhizobacteria (PGPR) are associated with plant roots and augment plant productivity and immunity. However, recent work by several groups shows that PGPR elicit physical or chemical changes related to plant defense, a process referred as „induced systemic resistance‟ (ISR). ISR induced by PGPR has suppressed plant diseases caused by a range of pathogens in both greenhouse and field (Yang et al., 2009). Jyothi (PTB -39), a popular rice variety of Kerala and susceptible to blight and BPH was used as the experimental material. The pot culture experiment was carried out by direct sowing. The biometric observations were taken on control plants and the plants treated with Pseudomonas fluorescens Pf1 (KAU culture) 30 days after sowing. Significant increase was observed in shoot length, root length, number of tillers, fresh weight and dry weight in Pf primed rice plants. Both R. solani and BPH were inoculated forty five days after sowing, to check the efficacy of Pf strain against the pathogen and insect. Protein analysis was carried out to study the molecular mechanisms operating behind the PGPR mediated pest and disease resistance and growth promotion. Total Proteins were isolated from rice plants treated with and without Pf and challenged with pest and pathogen at 0, 6, 24, 48, 72, and 96 hours after inoculation (HAI). The protein samples were analyzed by SDS-PAGE gel system. The comparison analysis of relative abundances of protein bands between inoculated and non inoculated samples were carried out. At 24, 48, and 72h, 16 proteins were expressed in Pf treated and control plants challenged with R. solani. A twenty nine kDa protein was up regulated in all the Pf treated plants challenged with R. solani and this was selected for sequencing. In all the Pf treated and BPH challenged plants 3 different proteins of 19, 23 and 30 kDa were expressed. Two protein bands of 19 kDa from 48h and 30 kDa from 96h were selected for MALDI-TOF sequencing. These three proteins bands were sequenced by MS MALDI-TOF in Sandor Proteomics, Hyderabad. Protein band 1 of 29 kDa, showed, 98 per cent homology to chloroplastic aldolase, fructose-bisphosphate aldolase and 81 per cent homology to peroxidase. The protein band 2 of 19 kDa, showed 84 and 86 per cent homology to 2- cys peroxiredoxin bas and 2-cys peroxiredoxin bas1, respectively. The Protein band 3 of 30 kDa, showed 99 per cent homology to small subunit of ribulose-1, 5-bisphosphate carboxylase and 100 per cent similarity to hypothetical proteins Os12g0291400 and OsI_38046. Western blot analysis was also carried out to further confirm the presence of PR protein chitinase. The analysis confirmed the presence of chitinase of molecular weight 17 kDa, 18 kDa, and 35 kDa in PGPR primed plant challenged with R.solani. The PO assay was also carried out to check the activity of peroxidase enzyme in both control and PGPR primed plants challenged with BPH and pathogen. PO activity was increased from 6 to 96 h after challenge inoculation in Pf primed plants. There was 43.62 and 21.9 per cent increase in PO activity over control plants in pathogen inoculated and BPH challenged Pf primed plants at 96hours. The result obtained gave information for further elucidation of candidate genes operating in signal transduction pathways mediated by PGPR during ISR to biotic stress.ThesisItem Open Access DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc)(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Pujaita Ghosh; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.), one of the widely cultivated and consumed spices worldwide, is well known for its medicinal properties also. As the natural variability stands limited in the crop, induction of variability through tissue culture techniques was attempted at College of Horticulture, Vellanikkara from 1996 onwards. After indepth investigations on the somaclones regenerated, two varieties viz., “Athira” and “Karthika” were released during 2010 and four clones viz. B3, 292R, 88R and 478R were selected as superior somaclones. For the newly released ginger varieties and selected superior somaclones in pipeline for release, no fingerprint data are available for genotype identification and protecting the plant varieties / clones. The investigations on “DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc.)” were carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during the period from January 2012 to March 2013. The objectives of the study were to characterize two released varieties and four selected superior somaclones using molecular markers and to develop a DNA fingerprint specific to each variety / somaclone. Morphological characters like growth habit and size and shape of the rhizomes were found to vary in the varieties / somaclones studied. The somaclone 292R could be distinguished based on its dwarf plant stature and dark green leaves. The variety Athira has bold and flat rhizomes while the variety Karthika has medium bold and round rhizomes. Quantitative clustering for vegetative and rhizome characters attempted as per Mahalanobis D2 analysis could group the varieties and somaclones into three separate clusters. Of the seven vegetative characters analysed, plant height and number of tillers showed more divergence. The number of fingers, girth of primary and secondary fingers, thickness of flesh and inner core were the characters which exhibited more divergence for the rhizome characters. For molecular characterization, good quality genomic DNA extracted from ginger varieties / somaclones using CTAB (Rogers and Bendich, 1994) method was used. Thirty five RAPD and thirty ISSR primers were screened for amplification of genomic DNA and ten RAPD and eleven ISSR primers were selected based on the amplification pattern. DNA fingerprints of the varieties / somaclones were developed utilizing the clear, distinct bands generated in RAPD and ISSR profiles and size of the bands. Different colour codes were assigned for sharing of bands between varieties / clones to generate specific fingerprints. The RAPD marker system could bring out unique bands in the variety Karthika and somaclones B3, 292R and 478R. The RAPD primer, OPA 12 produced unique band in Karthika and B3, the primer OPA 04 in 292R and the primer OPA 28 in 478R. ISSR marker system could also bring out unique band in the variety Athira with primer ISSR 06. The RAPD, ISSR and combined fingerprints developed for each variety / somaclone were unique. Variability in the somaclones and the extent of variability from source parent cultivars were analysed using cluster analysis. The dendrogram seperated Maran and Rio-de-Janeiro somaclones in two separate clusters. Somaclones derived from cultivar Maran exhibited more variability than somaclones from Rio-de- Janeiro. The variety Athira was more diverse from the source parent cultivar Maran. Similarly, the somaclone 292R was more diverse from the source parent cultivar Rio-de-Janeiro. The Resolving Power (Rp) of RAPD and ISSR primers ranged from 6.00 to 16.25, indicating the ability of the selected primers to distinguish the varieties / clones most efficiently. The Polymorphic Information Content (PIC) ranged from 0.67 to 0.88, indicating the suitability of the selected primers for DNA fingerprinting. RAPD, ISSR and combined fingerprints developed specific for the ginger varieties / somaclones could be utilized for registration, documentation of varieties and for settling IPR issues.ThesisItem Open Access Variability analysis in ginger (Zingiber Officinale Rosc) somaclones using molecular markers(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Gavande Sharda, Shivaji; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.), an important spice crop grown in India, is much valued for its flavour and medicinal properties. As natural variability available in the crop is limited, somaclonal variation is being utilized for crop improvement programmes. Currently, molecular marker techniques are widely employed to detect and assess somaclonal variation in several crop species as they are stable, detectable in all tissues and are not confounded by environment, pleiotropic and epistatic effects. The present investigations on “variability analysis in ginger (Zingiber officinale Rosc.) somaclones using molecular markers” were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Kerala Agricultural University during February 2012 to May 2013. The objectives of the investigations were to assess somaclonal variation in ginger at molecular level, to study the influence of genotype and mode of regeneration on somaclonal variation, to assess the extent of variability in somaclones from the original source parent cultivars and to select the variants. Two molecular marker systems viz. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were utilized for the analyses. Ginger somaclones (180 Nos.) regenerated through various modes of regeneration viz. bud culture, indirect organogenesis / embryogenesis and in vitro mutagenesis, along with two source parent cultivars (Maran and Rio-de-Janeiro) were used for the present study. The genomic DNA was extracted from somaclones using CTAB method (Rogers and Bendich, 1994) and Sigma’s GenEluteTM Plant Genomic DNA Miniprep kit. The somaclones were grouped as per genotype and mode of regeneration. DNA extracted from individual somaclones was bulked as per the procedure reported by Dulson (1998). Bulked DNA samples of the thirteen groups of somaclones along with two source parent cultivars were subjected to RAPD and ISSR analyses with selected primers. Of the 35 RAPD primers screened, twelve gave good amplification. RAPD analysis using selected primers produced 129 amplicons, 44 were polymorphic with an average of 3.66 polymorphic bands / primer and a polymorphism percentage of 34.10. In ISSR assay, twelve selected primers produced 122 amplicons, 32 were polymorphic with an average of 2.66 polymorphic bands / primer and a polymorphism percentage of 26.23. The study could identify certain specific RAPD and ISSR primers for identification of Maran and Rio-de-Janeiro cultivars and irradiated mutants from non- irradiated somaclones. The dendrograms generated based on RAPD and ISSR profiles grouped the somaclones into two separate clusters, with somaclones of Maran in first subcluster of cluster I and somaclones of Rio-de-Janeiro in second subcluster of cluster I. The regenerants from Rio-de-Janeiro calli irradiated with 20 Gy and somatic embryogenic calli irradiated with 10 Gy formed the second cluster. RAPD and ISSR marker systems showed that somaclones derived from cultivar Maran exhibited more variability than Rio-de-Janeiro. RAPD marker system was more effective for bringing out variability. The variability observed in RAPD assay was 28 per cent while in ISSR assay it was 21 per cent and in the combined it was 25 per cent. In groupwise variability analysis using bulked DNA, the groups RC20 Gy and RSe10 Gy recorded higher variability from source parent cultivar. The variability exhibited in plantwise analysis using three selected primers (OPA 28, S11 and ISSR 05) was found very high (39%) as compared to groupwise analysis (25%). The somaclone RC2Kr1031 of the callus regenerants and RSe1Kr1052 of the somatic embryo regenerants showed more variability exhibiting 59 and 53 per cent variability respectively from the source parent cultivar Rio-de-Janeiro. The extent of variability in ginger somaclones could be assessed using molecular markers and in vitro mutagenesis could be employed to widen the genetic base in ginger.ThesisItem Open Access DNA Fingerprinting of brinjal (Solanum melongena L) varieties and related species(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Vikhe, Parimal Laxman; KAU; Nazeem, P ABrinjal (Solanum melongena L.) also known as aubergine or eggplant, is a member of the family Solanaceae. It is an important vegetable in central, southern and south-east Asia, and in a number of African countries. In India, it is one of the most common, popular and principal vegetable crop grown throughout the country except in higher altitudes. The study entitled “DNA fingerprinting of brinjal (Solanum melongena L.) variet ies and related species” was carried out at the Center for Plant Biotechnology and Molecular Biology, College of Horticulture Vellanikkara during the period 20112013. The objective of study was to characterize the four brinjal varieties released by Kerala Agricultural University using molecular markers like ISSR and SSR and to develop a DNA fingerprint specific to each variety. Three superior accessions and two wild relatives were also characterized to detect the level of variability. The brinjal varieties/accessions selected include three Solanum melongena varieties- Surya, Swetha, Haritha and the hybrid Neelima; three accessions- SM 116, SM 396, SM 397 and the two wild relatives S. melongena var. insanum and S. macrocarpon. Breeder seeds were obtained from the Department of Olericulture, College of Horticulture, Kerala Agricultural University and maintained at CPBMB, COH and further used for the study. Morphological characters were recorded for six vegetative and five reproductive characters according to the descriptor of NBPGR. The data recorded was used to validate the genotypes which was used for fingerprinting. DNA extraction was done according to the method described by Rogers and Bendich 1994. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (OD 260 /OD ) 1.80 – 1.89 was used for further analysis. 280 Thirty six ISSR primers and sixty one SSR primer pairs were screened with DNA of brinjal genotypes for amplification and those which gave reliable distinct banding pattern were selected for further amplification/fingerprinting. The PCR product obtained from ISSR and SSR analysis were separated on 2 per cent agarose gel and amplification patterns recorded. The genomic DNA from each genotype was amplified with 10 each of selected ISSR and SSR primers. The amplification pattern was scored and depicted to develop fingerprint for each brinjal genotype. The Resolving power (Rp) of the ISSR primers was calculated and the values ranged between 9.9 and 28.44 indicating the capacity of the primers selected to distinguish the genotype. The Polymorphic Information Content (PIC) was also calculated and it ranged between 0.83 and 0.96 for ISSR primers, further indicating the suitability of primers to detect polymorphism. The PIC value for SSR primers were zero and not suitable to detect polymorphism. Distinct amplicons developed through ISSR and SSR analysis were used to develop the DNA fingerprint of brinjal genotypes. Sharing of amplicons for each primer with other genotypes was also analyzed and demarcated with different colour codes in the fingerprint developed. Most of the amplicons were found shared among the genotypes indicating their genetic uniformity. However, the pattern of sharing was different and good enough to separate out the varieties and distinct amplicons were observed in the genotypes. The ISSR and SSR banding pattern was scored and analysed for their uniformity/variability using the software NTSYS pc (version 2.02i). Similarity coefficient ranged from 0.51 to 0.84. The highest similarity (84%) was observed between the brinjal hybrid Neelima and its maternal parent Surya. The S. macrocarpon was the most distinct one from other brinjal genotypes with 49 per cent variability. Separate fingerprint were developed for all the four varieties, three accessions and two wild relatives through ISSR and SSR analysis. The DNA fingerprints thus developed could be utilised for the variety registration and settling IPR issues.ThesisItem Open Access Molecular characterisation of east Indian Lemongrass (Cymbopogon flexuosus stapf} Germplasm Accessions(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Bansode Ravindra, Dinkar; KAU; Abida, P SEast Indian lemongrass (Cymbopogon flexuosus Stapf.) is commonly known as Malabar or Cochin grass. It is an aromatic crop cultivated for its commercial essential oil and oil exported from India is known as “Cochin oil” in the world trade. The major component of this oil is citral which is widely used in pharmaceutical and perfumery industry. The Aromatic Medicinal plants Research Station, Odakkali has maintained around 400 accessions of East Indian lemongrass. Among them twenty five elite accessions good in oil and citral content were selected for the molecular characterization. The study elucidated genetic variation among the selected accessions. The present investigations on “Molecular characterization of East Indian lemongrass (Cymbopogon flexuosus Stapf.) germplasm accessions” were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Kerala Agricultural University during the period 2011-2013. The objective of the investigation was to characterize twenty five accessions of East Indian lemongrass using molecular markers, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR). For molecular characterization, good quality genomic DNA was isolated from East Indian lemongrass accessions using CTAB method (Rogers and Benedich, 1994). Thirty five RAPD and thirty one ISSR primers were screened for amplification of genomic DNA and ten RAPD and ten ISSR primers were selected for further analysis based on the amplification pattern. RAPD analysis using selected primers produced 101 amplicons, 58 were polymorphic with an average of 5.8 polymorphic bands/primer and polymorphism percentage was 57. In ISSR, selected primers produced 117 amplicons, 48 were polymorphic with average of 4.8 polymorphic bands/primer and a polymorphism percentage was 41. The RAPD markers found to be more effective to bring out variability among the accessions. The RAPD primer OPC 2 gave maximum polymorphism i.e. 77 per cent. In ISSR, the primer ISSR 5 gave maximum polymorphism i.e. 58 per cent. The polymorphic information content and resolving power of ISSR primers was higher than RAPD primers. The dendrogram generated based on RAPD and ISSR profiles grouped twenty five accessions of East Indian lemongrass into 3 main clusters. The first cluster grouped 21 accessions of lemongrass together. This cluster includes the accessions OD-11, OD-2, OD-28, OD-320, OD-9, OD-10, OD-4, OD-39, OD-24, OD-15, OD-40, OD-16, OD-21, OD-18, OD-8, OD-14, OD-20, OD-12, OD-29, OD-25 and OD-29. In this cluster, the accessions OD-4 and OD-39 were genetically similar which yielded 86.40 to 83.70 per cent citral content and these accessions were collected form Kollam district of Kerala state. This indicates that accessions of same geographic origin are genetically and biochemically similar. These 2 accessions were similar in terms of same leaf sheath colour. This first cluster formed one subcluster with accession OD-13 showed only 5 per cent variation with first cluster. The second cluster involved 2 accessions i.e. OD-7 and OD-17 showed 75 per cent similarity with each other with 84.40 and 83.20 percent citral content. The third cluster involved only one accession i.e. OD-23 with high citral content i.e. 88.50 percent was collected from Dehradun. The information generated will be useful in designing future breeding programmes involving the selected accessions.ThesisItem Open Access Induction of Somaclones in vetiver (Chrysopogon zizanioides (L) Roberty)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Resmi, S K; KAU; Kesavachandran, RThe present investigation on “Induction of somaclones in vetiver “[Chrysopogon zizanioides(L.)Roberty]” was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2010-2012 with an aim to induce variation in vetiver through callus mediated culture and evaluation of the same using morphological, molecular and biochemical markers. The explants collected from AMPRS, Odakkali was used for the study. Callus formation was induced in full MS media with growth regulator combinations of 0.5mgl-1 2,4-D, 0.5mgl-1 2,4-D+0.5mgl-1 KIN, 1.0mgl-1 2,4-D, 1mgl-1 2,4-D+1.0mgl-1 KIN, 1.0mgl-1 2,4-D+1.0 mgl-1 KIN +1.0 mgl-1 BA, 1.5mgl-1 2,4-D and 1.5mgl-1 2,4-D+1.5mgl-1 KIN. The regeneration and rooting were done in full MS media with various combinations of BA and IAA respectively. Hardening of the rooted plantlets were done in paper cups filled with sand under poly house. The hardened plantlets were established into pots containing potting mixture. Standardisation of DNA extraction was done with the CTAB method. Optimum PCR conditions for RAPD were standardised with various quantities of DNA, dNTPs, MgCl2, primers and Taq polymerase. Initially 40 RAPD primers were screened against genomic DNA of the mother plant for their ability to amplify DNA fragments. Of these, 10 RAPD primers were selected for further detailed RAPD profiling. All selected primers produced robust amplification patterns. The PCR products obtained were separated on agarose gel stained with ethidium bromide. A total of 92 RAPDs were generated of which average percentage of polymorphism was 7.4. The scored data based on RAPD banding was used to construct a dendrogram using NTSYS pc (ver. 2.02i). The RAPD assay confirmed the existence of considerable variation at the DNA level in the somaclones obtained.. MSAP analysis was done with 250 ng of template DNA for each reaction with four primers. The results showed that cytosine methylation varied from 10.1 per cent to 17.0 per cent. It also revealed that there was considerable variation between the somaclones The oleoresin extraction was done using solvent extraction method by reflexion and condensation. Highly volatile acetone and less volatile hexane were used for extraction. The oleoresin obtained was light yellow in colour. The content varied from 0.3 (SC 7) to 2.67 (SC 4) percentage. GC Analysis was carried out using DB-5 column. The chromatographic profiles varied for all the somaclones and the mother plant. More number of components was obtained for SC 6 and the least number for SC 2. SC 4 has shown more variation from the mother plant. In all the analysis, there is variation reported between the mother palnt and somaclones and also in between the somaclones. The oil extraction can be done further at 18 months of age of somaclones and the GC analysis result obtaining can be correlate with the molecular analysis and morphological analysis.