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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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20191
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Subject: Plant Biotechnology × Author: Jadhav Pritam, Ramesh × Author: KAU × Start date: 2008 ×
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    ThesisItemOpen Access
    Development of resistance against banana bract mosaic virus in musa spp. var. grand naine using small interfering RNA (siRNA)
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Jadhav Pritam, Ramesh; KAU; Soni, K B
    The study entitled “Development of resistance against Banana bract mosaic virus in Musa spp. var. Grand Naine using small interfering RNA (siRNA)” was carried out during 2015-2019 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to develop resistance against Banana bract mosaic virus in banana var. ‘Grand Naine’ using siRNA mediated technology. Embryogenic calli of banana var. Grand Naine were used for agrobacterium mediated transformation of ihpRNA cassette as the embryogenic cells have single-cell origin and do not produce chimeric plants. The rapid and efficient protocol standardised for somatic embryogenesis in Nendran developed in the Department of Plant Biotechnology, College of Agriculture, Vellayani was followed for inducing somatic embryogenesis in banana var. Grand Naine. Immature male flowers from position 0 to 11 were used as explants. Pale white embryogenic callus was initiated within 45 days on Murashige and Skoog (MS) medium supplemented with 6-Benzyladenine (BA) (8 mgL-1) and Thidiazuron (TDZ) (0.6 mgL-1) under dark conditions. The percentage of embryogenic calli obtained was 13.88. For regeneration of somatic embryo, the embryogenic calli were transferred to MS medium supplemented with BA (2 mgL-1) and incubated under light (16 hr), which resulted in 100 per cent germination and plantlet regeneration. The plantlets were transferred to coir pith compost and were hardened for one month in mist chamber. Plants were then transferred to polybags with soil and cow dung (1:1) mixture and kept in shade net house for secondary hardening. An intron hairpin RNA (ihpRNA) vector was constructed to produce small interfering RNA (siRNA) against the coat protein gene of BBrMV. The construct was designed using the partial coat protein gene sequence of BBrMV isolated from the infected banana var. Grand Naine. The coat protein gene was amplified from the cDNA prepared from the total RNA isolated from the infected plants by RT-PCR. For this coat protein gene specific primers were designed using the whole genome sequence of BBrMV retrieved from NCBI GenBank. The partially amplified coat protein gene fragment of 745bp was sequenced and analysed using BLASTn tool for similarity with the sequences of BBrMV deposited in NCBI genome database. Sequence was closely related to BBrMV infecting cardamom with 97.83 percent similarity. The sequence was subjected to miRNA target prediction for selection of the target site to be used in preparation of ihpRNA construct. The sequence that frequently occurred in probable dicer substrate sites was selected. It was then checked for the presence of restriction sites of AscI, PacI, KpnI and SpeI as these sites were the cloning sites of the primary vector, pSTARLING. Based on this information the primers with anchored restriction sites were designed to amplify a fragment of 326bp towards the 5’ end. The sense fragment of coat protein gene (326bp) was amplified with the primers anchored with AscI and PacI sites and the antisense fragment (326bp) was amplified with the primers having KpnI and SpeI sites. The respective restriction sites were anchored at 5’ end of forward primer and 3’ end of reverse primer. The use of two restriction sites helped in proper orientation of sense and antisense strand in ihpRNA construct. The amplified sense and antisense fragments were eluted from agarose gel and cloned in pJET1.2 cloning vector. The cloned fragments were released with sticky ends from pJET1.2 using the corresponding restriction enzymes and integrated in pSTARLING vector flanking the cre intron to favor the formation of the hairpin structure. Presence of the inserts was confirmed by restriction digestion and PCR. The ihpRNA casette in pSTARLING consisted of ubiquitin promoter, ubiquitin intron, sense coat protein strand, cre intron, antisense coat protein strand and termination sequence in the order within the NotI restriction sites. For agrobacterium mediated transformation, the complete cassette was released using NotI and ligated at NotI site within the lacZ gene of the binary vector pART27 containing antibiotic resistance markers nptII and Spec. Integration of cassette within lacZ gene facilitated the selection of the binary vector with the ihpRNA cassette by blue-white screening. The white positive colonies were confirmed with PCR. The binary vector with the insert was transferred to Agrobacterium tumefaciens strain GV 3103 by freeze-thaw method. Transformed colonies were picked and the presence of the vector and the ihpRNA insert was confirmed by PCR and restriction digestion. Embryogenic calli were transformed with A. tumefaciens strain GV 3103 carrying the ihpRNA construct and the transformed embryos were selected under antibiotic pressure (kanamycin 200 mg L-1). Transformed calli were transferred on MS medium containing 2 mg L-1 BA which gave a maximum regeneration of 12 percent. The regenerants were confirmed for the presence of ihpRNA construct using PCR with the primers for insert, npt II and cre intron. The formation of siRNA in the transformed plant was analysed using Northern hybridisation. The small RNAs were isolated from the leaves of transformed plants and separated on 15 percent acrylamide gel containing 7M urea and electroblotted on the positively charged nylon membrane. It was then subjected to hybridization with biotin labelled probes designed against the target site. The small RNAs were detected after DAB staining. The study was successful in developing ihpRNA construct for resistance against BBrMV in Musa spp. var. Grand Naine. This is the first report on development of ihpRNA construct for siRNA mediated resistance against BBrMV. The transgenics developed in this study need to be evaluated for virus resistance by challenging with viruliferous aphids. The technology developed in this study can be applied in other banana varieties also for imparting virus resistance. Compared to other recombinant DNA techniques, RNAi offers specificity and efficacy in gene silencing. Since the small gene fragment used for construct preparation do not code for any protein, this technology does not arise much biosafety concerns. In banana, where traditional breeding for virus resistance is very difficult, this technology is a promising alternative.