Kerala Agricultural University, Thrissur

The history of agricultural education in Kerala can be traced back to the year 1896 when a scheme was evolved in the erstwhile Travancore State to train a few young men in scientific agriculture at the Demonstration Farm, Karamana, Thiruvananthapuram, presently, the Cropping Systems Research Centre under Kerala Agricultural University. Agriculture was introduced as an optional subject in the middle school classes in the State in 1922 when an Agricultural Middle School was started at Aluva, Ernakulam District. The popularity and usefulness of this school led to the starting of similar institutions at Kottarakkara and Konni in 1928 and 1931 respectively. Agriculture was later introduced as an optional subject for Intermediate Course in 1953. In 1955, the erstwhile Government of Travancore-Cochin started the Agricultural College and Research Institute at Vellayani, Thiruvananthapuram and the College of Veterinary and Animal Sciences at Mannuthy, Thrissur for imparting higher education in agricultural and veterinary sciences, respectively. These institutions were brought under the direct administrative control of the Department of Agriculture and the Department of Animal Husbandry, respectively. With the formation of Kerala State in 1956, these two colleges were affiliated to the University of Kerala. The post-graduate programmes leading to M.Sc. (Ag), M.V.Sc. and Ph.D. degrees were started in 1961, 1962 and 1965 respectively. On the recommendation of the Second National Education Commission (1964-66) headed by Dr. D.S. Kothari, the then Chairman of the University Grants Commission, one Agricultural University in each State was established. The State Agricultural Universities (SAUs) were established in India as an integral part of the National Agricultural Research System to give the much needed impetus to Agriculture Education and Research in the Country. As a result the Kerala Agricultural University (KAU) was established on 24th February 1971 by virtue of the Act 33 of 1971 and started functioning on 1st February 1972. The Kerala Agricultural University is the 15th in the series of the SAUs. In accordance with the provisions of KAU Act of 1971, the Agricultural College and Research Institute at Vellayani, and the College of Veterinary and Animal Sciences, Mannuthy, were brought under the Kerala Agricultural University. In addition, twenty one agricultural and animal husbandry research stations were also transferred to the KAU for taking up research and extension programmes on various crops, animals, birds, etc. During 2011, Kerala Agricultural University was trifurcated into Kerala Veterinary and Animal Sciences University (KVASU), Kerala University of Fisheries and Ocean Studies (KUFOS) and Kerala Agricultural University (KAU). Now the University has seven colleges (four Agriculture, one Agricultural Engineering, one Forestry, one Co-operation Banking & Management), six RARSs, seven KVKs, 15 Research Stations and 16 Research and Extension Units under the faculties of Agriculture, Agricultural Engineering and Forestry. In addition, one Academy on Climate Change Adaptation and one Institute of Agricultural Technology offering M.Sc. (Integrated) Climate Change Adaptation and Diploma in Agricultural Sciences respectively are also functioning in Kerala Agricultural University.

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Open Access
In vitro micropropagation protocol for Vanda hybrids with clonal fidelity analysis
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Rosemol, Baby; KAU; Valsala, P A
Vanda orchids are one of the most sought after orchids in the international as well as domestic flower markets both as cut flower and potted plants. It is a monopodial orchid with vividly coloured, loosely arranged large beautiful flowers which has a long shelf life. Presently, many Vanda hybrids are becoming prominent even in the home gardens. However the present scenario of importing these hybrids from Thailand, Singapore and Malaysia to meet the Indian demands throws light on the need for developing an efficient propagation method for Vanda orchids. One of the major limiting factors for its spread and large scale cultivation in India is the non-availability of good quality and true to type planting material at a reasonable price. As the demand is more for the true to type plants, micropropagation is mostly recommended for orchid propagation. Hence this study was undertaken to develop an efficient micropropagation protocol for two Vanda hybrids namely Dr.Anek and Sansai Blue and to check the variability between the parents and regenerated plantlets. The different explants tested to initiate the cultures were leaf, root, stem and inflorescence segments. Initially the surface sterilization procedure was standardized for the explants. The results of the experiment showed that treating the explants with 0.1 per cent carbendazim for 20 minutes, followed by 70 per cent ethanol for 5 minutes and 0.1 per cent mercuric chloride for 5 min effectively reduced the microbial contamination with highest percentage of explant survival.Trial was made to initiate cultures using eight reported media compositions. The study showed positive results for inflorescence segments inoculated on to 1/2 MS + 10 mg l-1 BA+ 2 mg l-1 TDZ+30 g l-1 sucrose+7.5 g l-1agar + 250 mg l-1 cefotaxime as observed as direct shooting of the dormant buds. About 80 per cent and 60 per cent culture establishment was brought about in Dr. Anek and Sansai Blue respectively in 9 weeks. The established cultures successfully produced multiple shoots on MS+4.5 ml l-1BA +30 g l-1 sucrose + 7.5 g l-1 agar + 250 mg l-1 cefotaxime both when inoculated with and without the stalk in about 100 days of inoculation of explant. The micro-shoots from cultures without stalk were further transferred to hormone free basal MS media for elongation. Elongated shoots of about 4 cm were transferred to rooting media with a composition of MS + 0.5 mg l-1 NAA + 1 mg l-1 IAA +30 g l-1 sucrose +7.5 g l-1agar + 250 mg l-1 cefotaxime for better rooting of the regenerants. The percentage of rooting was observed to be 72.41 per cent for Dr. Anek and 70.37 per cent in Sansai Blue. The rooted plantlets with ample number of healthy roots were planted outm in small earthen pots with charcoal, coconut husk and brick pieces. These were successfully hardened in net house of 50 per cent shade and showed a hundred percent plantlet survival.Good quality DNA isolated from the mother plants and their respective clones using Rogers and Bendich procedure were analyzed for the clonal fidelity. ISSR analysis was done using 5 UBC (University of British Columbia) primers such as UBC 808, UBC 811, UBC 826, UBC 835 and UBC 841. An average of 8 to 9 bands was obtained from all primers in Dr. Anek and Sansai Blue. Out of 5 primers, UBC 808 and UBC 835 generated polymorphic bands in two clones of Dr. Anek. For Sansai Blue, all five primers generated monomorphic bands for all the mother plants and their respective clones analyzed. The per cent polymorphism in Dr. Anek was calculated to be 1.11 per cent whereas for Sansai Blue, there was no polymorphism detected revealing the true to type nature of the clones. The results showed that the identified protocol for in vitro regeneration of selected Vanda hybrids is a viable protocol since there were no changes in the banding pattern observed in tissue culture plants as compared with that of mother plant. Hence it can be concluded that the developed micropropagation protocol can be used for commercial production of Vanda hybrids without much risk of genetic instability. ISSR markers were effective to evaluate the genetic stability of the clones regenerated from the mother plants by the identified protocol.
Open Access
Cloning and characterization of fusarium wilt resistance gene analogs in banana (Musa spp.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Ramesh; KAU; Valsala, P A
Banana is one of the important fruit crops of India. It is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production are Fusarium wilt or Panama wilt caused by Fusariumoxysporumf. sp. cubense (Foc). 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 "Cloning and characterization of Fusarium wilt resistance gene analogs in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2013- 2015 with an objective to PCR amplify the genomic DNA from Fusarium wilt resistant banana genotype with primers specific to ‘R’ genes of TIR-NBS-LRR class for cloning and characterization of resistant gene analogs. 105 Palayankodanresistant (Mysore Poovan AAB) and Poovan susceptible(Rasthali AAB)varieties of banana were used for the present study.Fusarium culture was isolated from roots of infected banana plant and cultured inPDA (Potato Dextrose Agar) media. Fungal spore was suspended in sterile water and filled in small polythene bag. Artificial inoculation – root feeding of inoculum of water suspension was done. The symptoms of Fusarium wilt was observed two months after infection. Results confirmed resistance of Palayankodan and susceptibility of Poovan The DNA was isolated from Palayankodan (resistant) and Poovan (susceptible)genotypesand isolated DNA was subjected to RNase treatment. Quality checking was done using 0.8% agarose gel electrophoresis and quantity analysis was done using nanodrop ND-1000 spectrophotometer. Thirty five ng of DNA was used as template DNA for PCR amplification using reported degenerate primers. Twenty five primer combinations were made using five pair of degenerate primers. Among those combinations only one primer combination F9(F)+F6(R) showed polymorphic band of 700bp which was eluted, purified and cloned in pGEMT easy vector system.The presence of insert was confirmed by colony PCR and the eluted product was sequenced by outsourcing. The sequence obtained was subjected Blastn, Blastx and Blastp analysis and was compared with NCBI database. The sequence showed similarity with NBS-LLR resistant gene of Musa spp. Open reading frames (ORFs) were also identified using ORF finder software and four ORFs were identified For further validation, new primers were designed from 675 bp region of NBS-LLR class of resistant gene using primer3 plus software with an expected band size of 430bp. The DNA from both infected and healthy samples were amplified with designed primers and the expected bands were obtained in DNA samples of healthy 106 (resistant) plants where as it was absent in DNA samples of infected (susceptible) plants. The degenerate primer F9(F)+F6(R) and designed primer FTGAGCAGCATCGCCTA. R- GCCTGACACCAGTGAAGC can be used for Fusarium wilt disease diagnostics. Sequence information with respect to above primers amplicons can be used for synthesis of gene construct for genetic engineering.
Open Access
DNA barcoding of spider mites (Prostigmata: Tetranychidae) in vegetable crops
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2015) Shruti, Bennur; KAU; Abida, P S
DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short DNA sequences from a standardized region of the genome. It helps in precise identification of species that can be applicable as a diagnostic tool for quarantine and other pest management activities. DNA barcoding is based on the variation on the sequences identified in genomic regions, which can distinguish individuals of a species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e. the ‘barcode’ from a standard region of the genome, a specific gene region either from mitochondria or nuclear genome. The barcode sequence from the unknown specimen could be compared with a library of reference barcode sequences derived from individuals of known identity. The family Tetranychidae or spider mites include the most injurious plant-feeding mites. Some infest a wide range of host plant whereas some others are highly specific. The family includes about 12,000 species of phytophagous mites which can rapidly disperse to exploit new feeding sites, damage agricultural and horticultural crops causing severe economic losses. Precise identification up to species level is difficult since all species look similar. So molecular methods are increasingly applied in tetranychid mite taxonomy to establish species identity and DNA barcoding is the best option for this. The study entitled “DNA barcoding of spider mites (Prostigmata : Tetranychidae) in vegetables” was done at Centre for Plant Biotechnology and Molecular Biology and Department of Agricultural Entomology, College of Horticulture, Vellanikkara. The objective of this study was to generate DNA barcodes for different species of spider mites in vegetable crops and to study the intra and inter species genetic relationship. For this, spider mites were collected from different locations viz; Anthikad, Alathore, Elenad, KVK, Thrissur and Vellanikkara and from different vegetable crops viz; Amaranthus, brinjal, cowpea, cucumber, dolichos bean, okra and ridge gourd. After collection, spider mites were reared in the laboratory to get an isoline from which few males and females were used for microscopic slide preparation. Acarologists used slides to identify the spider mite species based on morphological taxonomic keys. Total genomic DNA isolated using modified CTAB method (Rogers and Benedict, 1994) was subjected to PCR assay using markers for two different loci ITS2 (second internal transcribed spacer) and COI (mitochondrial cytochrome c oxidase subunit I) which yielded bands of 620 and 868bp respectively. The obtained bands were eluted and subjected for sequencing. Nucleotide divergence among the sequences was calculated using Barcode of Life Data (BOLD) tool ‘Distance summary’. It is desirable for barcodes to show very low sequence divergence within species. Multiple sequence alignment using clustalW of MEGA6 software was performed for all the sequences and phylogenetic analysis has discriminated three different species of Tetranychidae family: Tetranychus truncatus, T.macferlanei and T.okinawanus. Barcoding gap, a position in the sequence at which unique nucleotide is present in all the members of a Tetranychus species was also assessed. Morphological and molecular analysis results were correlated with each other and results matched. Tetranychus okinawanus found on cucumber during the study is first time getting reported from India. The study had shown that both ITS2 (99.25%) and COI (98.45%) sequences efficiently classified the spider mite species. However, ITS2 was found to be an efficient tool which gave species level resolution in spider mites. This can be used as supporting marker for COI to barcode spider mites species. In future, these primers can be used to barcode other Tetranychus species also and sequence analysis can also be done using ITS1 locus and its efficiency can be measured for DNA barcoding of Tetranychus species.
Open Access
DNA fingerprinting of selected chilli (Capsicum spp.) varieties
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Manibala Kumari; KAU; Kesavachandran, R
Chilli (Capsicum spp.) is considered as one of the most important commercial spice crops and is a widely used universal spice, named as the wonder spice. It is raised over an area of 18 lakh ha. in the world, with a production of 29 lakh t. India is not only the largest producer but also the largest consumer of chilli in the world. The study entitled “DNA fingerprinting of selected chilli (Capsicum spp.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2012-2013. The objectives of the study were to characterize the released chilli 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. Six chilli varieties, namely Ujwala, Anugraha, Jwalamukhi, Jwalasakhi, Vellayani Athulya and Vellayani Samrudhi collected from CoH, Vellanikkara and CoA, Vellayani and maintained at CPBMB, CoH were used for the study. Morphological parameters of six chilli varieties were taken such as stem colour, branching habit, leaf size, leaf colour, fruit colour, fruit shape and fruit surface. DNA extraction was done by CTAB (Rogers and Bendich, 1994) method. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A 260 /A ) 1.80 - 1.91 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assays. Thirty RAPD, 30 ISSR and 30 SSR primers were screened with bulked DNA of Ujwala, Anugraha and Jwalamukhi variety for amplification and those which gave reliable distinct banding patterns were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analyses were separated on two per cent agarose gel and the amplification patterns were recorded. Genomic DNA from each variety was amplified with ten selected primers of RAPD, ISSR and SSR primer pairs. The amplification patterns were scored and depicted to develop DNA fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 8.33 (S 12) to 12.9 (OPAH 06) for RAPD primers and 8.66 (SPS 03) to 14.33 (ISSR 07) for ISSR primers, indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.80 (S 12) to 0.86 (OPAH 06) for RAPD primers and it was 0.82 (SPS 03) to 0.88 (UBC 840) for ISSR primers. Distinct bands were used to develop DNA fingerprint of chilli varieties (Ujwala, Anugraha, Jwalamukhi, Jwalasakhi, Vellayani Athulya and Vellayani Samrudhi) through RAPD, ISSR and SSR analyses. 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 for each variety with RAPD, ISSR and SSR data was also developed. The amplification patterns observed in RAPD, ISSR and SSR analyses were scored and analyzed for quantifying the variability among the varieties. The computer software NTSYS-Pc was used for cluster analysis (Rohlf, 2005). Maximum variability observed was 41 per cent for the variety Vellayani Samrudhi. The varieties Ujwala and Anugraha indicated 91 per cent similarity. The fingerprint developed was sufficient to provide varietal identity and the analysis could reveal variability/ relatedness among the six varieties.
Open Access
Identification of molecular marker for self-incompatibility in selected germplasm accessions of cocoa (Theobroma cacao L.)
(College of Horticulture, Vellanikkara, 2015) Goradevaishali, Shivaji; KAU; Abida, P S
Cocoa (Theobroma cacao L.) is a diploid species (2n = 2X = 20) with a small genome size of 380Mbp. It belongs to the family of Malvaceae (alternatively Sterculianceae). Theobroma cacao L. is a highly cross pollinated crop due to its peculiar flower structure and existence of self-incompatibility. Self-incompatibility is exploited in hybridization process of breeding programmes and also in selecting clones for polyclonal garden. Traditional method to access self- incompatibility is by hand pollinating 100 flowers per tree. This is very tedious and time consuming method. This necessitated the identification of a marker associated with self-incompatibility. The cocoa germplasm accessions are maintained at Cocoa Research Centre, KAU, Vellanikkara. The centre has identified and maintained Selfincompatible and self-compatible cocoa accessions. Self-incompatibility was manually assessed by selfing 100 flowers per accessions. The cocoa accession setting fruits on selfing classified as self-compatible and which do not set fruit as self-incompatible. Five self-incompatible [IMC 20, PNG 299, IMC 105, ICS 5 and R 10 (MEX)] and five self-compatible cocoa accessions (NA 149, EET 397, SIC 5, POUND 18, GV 13.5) were selected for the present study. Isolation of good genomic DNA from all self-incompatible and self-compatible accessions of cocoa was carried out from the young leaves; using Doyle and Doyle (1987) method was used for analysis of RAPD, ISSR and SSR markers. Random Amplified Polymorphic DNA (RAPD) fragments were generated in the bulked DNA samples in order to identify markers that were polymorphic between self-incompatible and self-compatible plants. Among the 76 RAPD primers screened, 15 primers were selected based on amplification. A 550 basepairs (bp) DNA fragments were generated with RN-6 primers in three selfcompatible specific genotypes. The polymorphic band was cloned and sequenced at SciGenom Pvt. Ltd., Cochin. The sequence analysis gave no information to develop SCAR marker. In Inter Simple Sequence Repeats (ISSR) assay, among the 42 primers screened initially, 20 primers were selected for the study. There was no polymorphism between self-compatible and self-incompatible genotypes. Additionally Simple Sequence Repeats (SSR) assay was also performed with genome specific primers. Total genomic DNA of the self-incompatible and self-compatible accessions was amplified with 11 SSR primers and out of them seven primers were selected. The primer mTcCIR 33 yielded distinctly polymorphic band of 350-400bp size in self-incompatible genotypes. mTcCIR 33 SSR primer also produced polymorphic band obtained on PAGE. The PCR product was directly sequenced at SciGenom Pvt. Ltd., Cochin. The sequence analysis showed 86 per cent identity to Theobromo cacao. More number of genus specific primers has to be screened to develop better markers to distinguish self-comptability and self-incompatability in cocoa or an attempt to biochemical markers can also be resorted.

Kerala Agricultural University, Thrissur

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