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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 DNA fingerprinting of selected black pepper (piper nigrum L.) varieties.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Manjunath, Mogalayi; KAU; Nazeem, P ABlack pepper (Piper nigrum L.) famous as “Black Gold” and also known as “King of Spices” is one of the important agricultural commodities of commerce and trade in India since pre-historic period. The crop is the major source of income and employment for rural households in the predominantly pepper growing State of Kerala where more than 2.5 lakh farm families are involved in pepper cultivation. Karnataka, Tamil Nadu are the other major pepper producing States in the country. Kerala accounts for 80-90% of the total pepper production in the country. Idukki and Wynadu are the two major pepper producing districts in Kerala. Different cultivars/varieties are popular among the farmers and there phenotypic identity is not very distinct. The study entitled “DNA fingerprinting of selected black pepper (Piper nigrum L.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2009-2011. The objectives of the study were to characterize the released black pepper varieties of KAU using different molecular markers - RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected. Seven black pepper varieties (Panniyur 1 to Panniyur 7) collected from Pepper Research Station, Panniyur and maintained at CPBMB, COH were used for the study. DNA extraction was done with CTAB (Rogers and Benedich, 1994) method with slight modification. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assay. 30 RAPD, 34 ISSR and 29 SSR primers were screened with bulked DNA of black pepper varieties for amplification and those which gave reliable distinct banding pattern were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analysis were separated on 1.3 to 2 percent agarose gel and the amplification patterns recorded. The genomic DNA from each variety was amplified with 10 each of selected RAPD and ISSR primers and 8 SSR primer pairs. The amplification pattern was scored and depicted to develop fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 7.42 to 9.42 in RAPD and 5.42 to 12.28 in ISSR analysis; indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.86 to 0.90 for RAPD analysis and it was between 0.80 and 0.89 in ISSR analysis indicating the variability among the genotypes. Distinct bands were used to develop DNA fingerprint of black pepper varieties Panniyur 1 to Panniyur 7 through RAPD, ISSR and SSR analysis. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint of each variety with RAPD, ISSR and SSR data was also developed. The amplification pattern observed in RAPD, ISSR and SSR analysis was scored and analyzed for quantifying the variability among the varieties. The computer package NTSYS-Pc was used for cluster analysis. Maximum variability observed was 48 percent for the variety Panniyur 4. The varieties Panniyur 1 and Panniyur 3 having the same parentage indicated 76 percent similarity. The fingerprint developed was good enough to provide varietal identity and the analysis could reveal variability/relatedness among the seven varieties. Separate and combined fingerprints were developed for all the seven varieties through RAPD, ISSR and SSR analysis. The DNA fingerprints thus developed could be utilized for the variety registration and settling IPR issues.ThesisItem Open Access Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex Poir)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Sahna Hamsa, N H; KAU; Girija, DThe study entitled “Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex poir.)” was carried out in the Molecular Biology Laboratory of Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2009-2011. The objectives of the study included the molecular characterization of geminivirus causing yellow vein mosaic disease in pumpkin and developing a PCR based diagnostic kit. Yellow vein mosaic infected pumpkin leaf sample was collected from the field of Olericulture Department, College of Horticulture, Vellanikkara and total genomic DNA was extracted by CTAB method. Specific primers for coat protein and movement protein genes were designed based on the sequences of geminiviruses infecting vegetables, obtained from the NCBI database. PCR amplification was carried out using these primers. Amplicons of size ~900bp and ~700bp were obtained for coat protein and movement protein genes respectively. The purified PCR products were ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin layered with X-gal and IPTG. After confirmation of the inserts by colony PCR, the clones were sequenced. Sequences of 891bp and 702 bp were obtained for coat protein and movement protein genes respectively. Sequence analysis was carried out with standard bioinformatics tools. On blastn analysis both coat protein and movement protein sequences showed maximum similarity to Squash leaf curl China virus (SLCCV) from Coimbatore. For coat protein gene, full length ORF of 771bp was obtained and the ORF of movement protein was partial. Primers MP1F and MP1R with expected amplicon size of 1363bp were designed to get full length ORF (846bp) of movement protein gene. The technique was validated with DNA from 15 PYVM infected and 4 healthy pumpkin leaf samples collected from Palakkad, Thrissur and Malappuaram districts. The virus was detected only in the diseased samples. Hence these primers could be used in developing a molecular diagnostic tool to detect the virus. PCR amplifications were carried out in weed plants like Emilia sonchifolia, Ageratum conyzoides, Hibiscus surattensis and Synedrella nodiflora and crop plants like okra and ash gourd with yellow vein symptom to check whether these plants serve as the collateral hosts of the virus. PCR amplification was also performed in bitter gourd with distortion mosaic symptom. No amplifications were obtained in plants other than pumpkin. Using the primers PYVMV was detected in apparently healthy mature leaves of infected pumpkin. Hence, these primers could be used to detect latent infection. Sequence and phylogenetic analysis of coat protein and movement protein gene sequences showed maximum similarity to bipartite Squash leaf curl china virus (SLCCV) from Coimbatore. Hence Pumpkin yellow vein mosaic virus (PYVMV) from Kerala can be considered as a strain of SLCCV.ThesisItem Open Access Isolation and characterization of water stress activated protein kinase gene from black pepper(Piper nigrum L.) var. Kalluvally.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Bankar Ashok, Dnyandeo; KAU; Nazeem, P VWater stress is identified as one of the main constraint for enhancing the productivity in black pepper. To survive stress, plants employ a complex set of distinct signaling pathways that trigger stress-specific tolerance or avoidance in the organism as a whole. An important biochemical mechanism for regulating such pathways is reversible protein phosphorylation which is mediated by protein kinases. Gaining an understanding of the mechanisms that regulate the expression of these genes and functional annotation of their transcripts will be necessary for the genetic improvement of plants cultivated in extreme environments. Genotypic variations for drought tolerance have been reported in black pepper and the variety Kalluvally is one of the drought tolerant genotype of black pepper (Thankamani, 2003). In the previous studies at the Centre, water stress specific cDNA library of Kalluvally was constructed by suppression subtractive hybridization (SSH) (Kushwah, 2008). Several protein kinase genes were found to be up-regulated during the study. The present investigation was undertaken to obtain full length coding sequence information on the partial clones of protein kinase genes available in the cDNA library by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The clones named PNK21 and PNK49 were selected from the library. The clones were revived on LB/ampicillin media and subjected to full length sequencing. Degenerate primers were designed based on protein kinase gene sequences of various crops, obtained from NCBI database to amplify the unknown ends of the transcripts by RT- PCR. Plants raised under greenhouse were screened for revival after water stress with different interval and frequency under open condition. The total RNA was isolated using TRI® reagent from stressed plants and subsequently cDNA was synthesized. The PCR amplification was carried out using degenerate primers designed for all three clones. Amplicons of size of ~600 bp for PNK21 clone and ~600 bp for PNK49 clone were obtained. Direct sequencing of PCR product of PNK21 clone was done. The sequence data obtained was merged and analysed using bioinformatics tools. Blastn analysis revealed ~50 per cent coverage with the cDNA sequences for protein kinase from database. So, further primers were designed to amplify the full length cDNA sequence by RNA ligase mediated-RACE. The 3’ end of the PNK21 cDNA was successfully amplified using RLM-RACE PCR with amplicon size of ~400 bp. The purified PCR product was ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin with X-gal and IPTG. After confirmation of the insert by colony PCR, the clones were sequenced. The finally enriched sequence was analysed using bioinformatics tools. Blastn and Blastx revealed maximum similarity with Ricinus communis APK1B protein kinase. The sequence indicated open reading frame and conserved domains for protein kinase and polyadenylation signal site TATAAA was found just upstream of the polyA tail at 3’ end when analysed with different Bioinformatics tools.ThesisItem Open Access Development of a molecular marker for bacterial wilt resistance in brinjal ( Solanum melongene L.) varieties Surya and Swetha.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Somya, P P; KAU; Valsala, P AThe study entitled ‘Development of a molecular marker for bacterial wilt resistance in brinjal (Solanum melongena L.) varieties Surya and Swetha’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is one of the important problems of brinjal cultivation in warm humid tropics. The loss due to this varies from 30-100 per cent. Use of chemicals and field sanitation are not sufficient for controlling the disease. Worldwide approach is to use resistant varieties. KAU has developed and released bacterial wilt resistant brinjal varieties for cultivation. The Surya and Swetha are two among them and have received bacterial wilt resistance from SM-6 an Annamalai collection. This investigation was taken up to develop a molecular marker for bacterial wilt resistance in Surya by RAPD through bulk segregant analysis as reported by Michelmore et al (1991). It also aimed to test the suitability of the same for identifying bacterial wilt resistance trait of resistant variety Swetha. The genotypes used for the study were Surya, Pusa Purple Long (susceptible variety released from IARI), Swetha and F2 population of the cross between Surya and Pusa Purple Long. To raise segregating F2 population F1 was raised by controlled crossing of Surya with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population. Two different methods viz., stem puncturing and soil drenching with root wounding were compared for the delivery of inoculum of R. solanacearum for bacterial wilt incidence and stem puncture method was found as the best. So stem puncturing method was used for phenotyping of genotypes for bacterial wilt incidence. The F2 population along with Surya, Pusa Purple Long and F1 were phenotyped for bacterial wilt incidence. This was done through artificial inoculation with Ralstonia solanacearum by stem puncture method. Confirmation was done by ooze test. The genotypes were classified according to classification of Mew and Ho (1976). The variety Surya was resistant and Pusa Purple Long was susceptible. F1 population showed 90 per cent susceptibility while F2 population showed 83 per cent susceptibility. They were classified as susceptible. Five resistant and five susceptible genotypes from F2 were selected for bulk segregant analysis. Genomic DNA for RAPD analysis was isolated by Rogers and Bendich method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis.PCR reaction mixtures and conditions for DNA amplification were standardised. Ninty two, 10-12 bp primers belonging to OPA, OPB, OPC, OPF, OPE, OPU, OPH, OPAH, OPAG, OPL, OPM, RY, RN, RA, SC, RF, AG 8, WG 44, GLE11, RF, R10, R6, and PUC101 were initially screened with resistant genotype Surya and susceptible genotype Pusa Purple Long to select primers with polymorphism and good amplification. Out of ninty two primers tested thirty were reported as bacterial wilt specific. The PCR products were electrophoresed and twenty two primers were selected for BSA based on amplification power and polymorphism. They were RY 01, RY 02, RY 03, RN 19, OPF 5, OPL 04, OPA 04, OPA 6, OPA 9, OPA 24, OPA 26, OPA 34, OPA 36, OPS 9, OPS 10, OPS 16, OPS 17, PUC 101, RA 12-41, and RF. Among these only the primer RY 02 recorded polymorphism between resistant and susceptible variety with an amplicon of 1.2kb. In bulk segregant analysis DNA of Surya, Swetha, Pusa Purple Long and bulk DNA from resistant genotypes and susceptible genotypes were amplified with selected primers and products were electrophoresed. All primers produced only monomorphic band. None has produced unique polymorphic bands capable of differentiating resistant and susceptible genotypes. This may be due to low polymorphism at genomic level.ThesisItem Open Access Detection and quantification of piperine from in vitro cultures of black pepper (Piper nigrum L.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Achintya Kumar, Dolui; KAU; Sujatha, RThe present study on “Detection and quantification of piperine from in vitro cultures of black pepper (Piper nigrum L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, during the period 2009-2011. The objective of the study was to standardize the protocol for in vitro synthesis of piperine in black pepper and to detect and quantify the in vitro synthesized piperine. Callus cultures from tender, pale green leaf from the variety Panniyur 5 and immature berry and epicarp of berry from the variety Panniyur 2 were established successfully. Surface sterilization with mercuric chloride (0.1 %) for 5 min was effective in initiating healthy, contamination free callus from leaf and 10 min were effective in initiating healthy callus from berry explants. It was observed that half strength MS (Murashige and Skoog, 1962) medium supplemented with IAA (1.0 mg l-1) along with BA (1.0 mg l-1) was suitable for initiation and proliferation of calli. Leaf derived cultures were superior with respect to callus initiation and proliferation. Leaf calli were sub cultured into five different modifications of the culture initiation medium viz., (I) increased sucrose concentration (5 different concentrations), (II) 1/4th nitrate and phosphate concentration in the MS stock solution, (III) different hormone combinations (2, 4-D and BA in four combinations), (IV) stress inducing agent (mannitol) and (V) precursor of piperine (L-lysine) in three different concentrations. The effect of these modified media on piperine production was studied by detecting the presence of piperine and estimating the quantity by High Performance Liquid Chromatography in the respective calli, callus growing in the culture initiation medium (as control) and the calli growing in these modified media. Piperine was extracted from 1.0 g callus by grinding in 5 ml methanol and the centrifuged crude extract was used for High Performance Liquid Chromatography analysis (Shimadzu, Japan). Gradient elution system using 0.1% ortho-phosphoric acid and 90 % acetonitrile in 0.1% ortho-phosphoric acid was employed for detection of piperine. The study revealed that increasing the concentration of sucrose up to 7 per cent in the callus initiation medium increased the piperine content from 0.018 % to 0.135% in leaf derived calli whereas, reducing the nitrate nitrogen and phosphorus content of the basal medium to 1/4th of its original strength exerted an inhibitory effect on piperine production (0.003%). Similarly, supplementing the callus initiation medium with mannitol at 4 per cent along with 3 per cent sucrose, resulted in enhanced in vitro synthesis of the target compound (0.107 %). Similarly the medium with 2, 4-D (3.0 mgl-1) enhanced the in vitro synthesis of piperine to 0.042 per cent in berry calli. Precursor feeding with L-lysine at higher concentration (100 mgl-1) also exerted a favorable influence on piperine (0.066 %) synthesis in comparison to other concentration (5 and50 mgl-1). Among the various explants studied, young leaves were found to be the most suitable for callus induction in black pepper. In vitro piperine production from leaf calli (0.135 %) was higher than that in the leaf used as explant (0.01%). Berry calli produced very less amount of piperine (0.042 %) compared to that of fresh berry used as explant (2.7%). The results indicated that in vitro synthesis of piperine may be exploited by further modifications of the culture media.ThesisItem Open Access Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Choudhary Rakeshkumar, Sheshrao; KAU; Kesavachandran, RThe present investigation on “Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers” was undertaken in the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2009-2011 with an aim to characterize the variability in plantain ecotypes of banana (Musa spp.) using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Twelve plantain ecotypes collected from BRS, Kannara was used for the study. Standardisation of DNA was done with the CTAB method. Optimum PCR conditions for both RAPD and ISSR were standardised with various quantities of DNA, dNTPs, MgCl2, primers and Taq polymerase. Initially 60 RAPD and 40 ISSR primers were screened against genomic DNA of two plantain ecotypes (Big Ebanga and Njockkon) for their ability to amplify DNA fragments. Of these, 16 RAPD and 14 ISSR primers were selected for further detailed RAPD and ISSR profiling. All selected primers produced robust amplification patterns. The PCR products obtained were separated on 1.4 per cent and 1.6 per cent agarose gel respectively stained with ethidium bromide. A total of 138 bands were obtained by using 16 RAPD primers. The number of bands produced by the primers varied from 5 (OPS 40) to 14 (OPS 31 and 37) and the molecular weight of bands varied from 2.876 to 0.564 Kb. The average number of bands was 8.63 and average percentage of polymorphism was 3.25. The total percentage of polymorphism was 37.68. The Polymorphic Information Content (PIC) value for 16 primers varied between 0.79 (OPS 40) and 0.92 (OPS 37) with mean of 0.87. The Resolving power (Rp) of the random primers ranged between 9.33 (OPS 12) and 24.33 (OPS 37) with an average of 15.49. The Marker Indices (MI) of primers varied from 0.83 (OPS 7) to 7.28 (OPS 31) with a mean of 2.86. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Njockkon and Changalikodan, occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. A total of 111 bands were obtained by using 14 ISSR primers. The number of bands produced by the primers varied from 5 (UBC 835, 820) and 11 (UBC 857) and the molecular weight of bands varied from 1.584 to 0.564 Kb. The average number of bands was 7.93 and average percentage of polymorphism was 4.14. The total percentage of polymorphism was 52.25. The PIC value for 14 primers varied between 0.77 (UBC 835) and 0.90 (ISSR 6) with an average of 0.85. The resolving power of the ISSR primers ranged between 7.83 (UBC 820) and 16.83 (ISSR 6) with an average of 12.49. The Marker Indices (MI) of primers ranged from 0.77 (UBC 835) to 8.01 (UBC 857) with a mean of 3.55. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Changalikodan and Zanzibar; Manjeri Nendran (a) and Manjeri Nendran (b) occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. The combined dendrogram was also derived from pooled data from RAPD and ISSR analysis and morphological data. The dendrogram generated revealed grouping of plantain ecotypes into clusters more or similar to earlier dendrogram with a few exceptions. The study revealed that variability exists among the plantain ecotypes of banana.