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ThesisItem Open Access Development of small interfering RNA (siRNA) mediated resistance in banana against banana bract mosaic virus(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Lekshmi, R S; KAU; Soni, K BThe present study entitled “Development of small interfering RNA (siRNA) mediated resistance in banana against Banana bract mosaic virus (BBrMV)” was carried out during 2012-2016 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The study was carried out with an objective to develop siRNA mediated technology for the development of banana plants resistant to Banana Bract Mosaic Virus (BBrMV). The study was conducted in banana cv. Nendran. A protocol for somatic embryogenesis in banana cv. Nendran was standardized by using immature male flowers as explants. Pale white friable callus with rich cytoplasm was initiated in Murashige and Skooge (MS) medium supplemented with BA (0.1 – 0.5 mgL-1) and picloram (0.5 – 2 mgL-1) incubated in dark with a maximum explant response of 30 per cent. For embryogenesis, the developed embryogenic calli were transferred to semisolid MS medium supplemented with BA 2 mgL-1 and IAA 0.5 mgL-1 which resulted in a maximum of 10 per cent embryogenesis. The glassy elongated monocot embryos were germinated in half strength semisolid MS medium (0.3 per cent Gelrite) supplemented with BA 2 mg L-1 and IAA 0.5 mg L-1 and incubated in dark. A maximum germination rate of 80 per cent was obtained in this medium. The germinated embryos were transferred to MS medium with BA 2 mg L-1 and NAA 1 mg L-1 resulted in 100 per cent Plant regeneration. The plantlets were transferred to coirpith compost in pot trays in mist chamber for one month for hardening and then transferred to polybags with soil and cowdung (1:1) mixture. To develop siRNA technology to silence the replicase gene of BBrMV, an intron hairpin RNA (ihpRNA) construct was developed. For this a partial mRNA sequence of replicase gene was isolated from BBrMV banana plants. Gene specific primers designed based on the whole genome sequence information retrieved from the GenBank, NCBI. Total RNA from infected banana leaves was isolated and cDNA was prepared using RT-PCR. The partial gene fragment isolated was sequenced and analysed using the bioinformatics tool BLAST. The sequence was subjected to miRNA target prediction and restriction mapping to select suitable region for the construct and further processing. Based on this information a fragment of 400 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with BamHI and PacI sites were used for the amplification of sense strand and primers anchored with KpnI and SpeI sites were used for antisense strand amplification. The sense and antisense fragments amplified were cloned to pTZ57R/T cloning vector. In the next step the inserts were released from pTZ57R/T using the corresponding restriction enzymes and were integrated in pSTARLING (primary vector), on either side of the cre intron which facilitated the formation of the hairpin (ihpRNA) construct. Presence of the inserts was confirmed by restriction digestion and electrophoresis. The ihpRNA construct in pSTARLING now contained ubiquitin promoter, ubiquitin intron, sense strand of replicase gene, cre intron, antisense strand of replicase and termination sequence in the order with the NotI restriction sites. This construct was released from pSTARLING and ligated to the digested NotI site in the lacZ gene of the binary vector pART27 containing antibiotic resistance marker nptII and spec. The binary vector was confirmed for the insert by transferring to DH5α and colony selection by blue-white screening. The binary vector with the insert isolated from the white colony, was transferred to Agrobacterium tumefaciens strain LBA 4404 via freeze-thaw method. Transformed colonies were picked up and confirmed the presence of the vector and the ihpRNA insert by PCR. Somatic embryos were transformed with LBA 4404 carrying the ihpRNA construct the ihpRNA construct and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). Transformed embryos were subjected to regeneration. A maximum regeneration of 25 per cent was obtained after transformation. The regenerants were confirmed for the presence of ihpRNA construct using PCR with specific primers for sense-intron-antisense fragment, npt II and cre intron. The study was successful in developing a siRNA construct for resistance against BBrMV and obtaining transformed Nendran banana plantlets.ThesisItem Open Access Agrobacterium tumefaciens mediated transfer of exogenous hydroxy methyl glutaryl CoA (HMG CoA) reductase gene to Centella asiatica L(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2008) Lekshmi, R S; KAU; Soni, K BA study on “Agrobacterium tumefaciens mediated transfer of exogenous hydroxyl methyl glutaryl CoA (HMG CoA) reductase gene to Centella asiatica L.” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2006-2008. Centella asiatica is an important medicinal plant, which is used in many ayurvedic formulations. It contains a blend of compounds including triterpenes (asiatic acid, madecassic acid and asiaticoside), which are responsible for its medicinal properties. Among the various secondary metabolites in Centella asiatica, asiaticoside possesses remarkable pharmaceutical value due to its anti-inflammatory, antitumour, neuroprotective, skin care and toning effects. Since the asiaticoside content in the Indian ecotypes is less, the industrial demands are met by importing this plant from African countries. By improving asiaticoside content, utilization of Indian ecotypes could be improved and thereby the cost of medical preparations could be reduced. Metabolic engineering is becoming a popular approach for the modification of medicinal plants for altering the metabolite content. Medicinal plants with quantitatively and qualitatively improved pharmacological properties have been produced by metabolic pathway engineering. The present study was undertaken with an objective to enhance the asiaticoside content by introducing exogenous hmgr gene to Centella asiatica. This gene is responsible for coding hydroxy methyl glutaryl CoA reductase enzyme which acts at the upstream of the triterpene biosynthetic pathway and produce mevalonic acid. Callus was induced from leaf and node explants of Centella and MS medium supplemented with Kn 4 mg l-1 and NAA 2 mg l-1 was found to be the best for callus induction. Callus initiation was faster (23 days) from node compared to leaf (25 days), with 100 and 92.85 per cent induction respectively. Of the various media tried for regeneration, the highest regeneration (0.052%) was obtained on MS medium supplemented with Kn 4mg l-1 and NAA 2 mg l-1. Agrobacterium tumefaciens strain EHA105 harbouring the plasmid pBE2113 containing nptII and hmgr gene was used for transformation. The sensitivity of Agrobacterium strains and Centella callus to different concentrations of kanamycin was evaluated. The lethal doses of kanamycin to Agrobacterium and Centella callus were 350 and 125 mg l-1, respectively. The effective dose of cefotaxime for the elimination of bacteria was 50 mg l-1 and the lethal dose of cefotaxime to callus was 100 mg l-1. Genetic transformation was achieved by co-cultivating callus with bacterial suspension (OD600 of 0.1) containing 100 μM acetosyringone. An infection time of 20 min and co-cultivation period of four days were given. The transformed tissues were selected on MS medium containing 200, 250 and 300 mg l-1 of kanamycin. The survival of the tissues on these media after three weeks was 18.75, 19.35 and 13.63 per cent, respectively. Transformation was confirmed by PCR analysis with npt II gene specific primer. All the three samples gave appreciable quantity of the product of size 700 bp which was comparable to positive control. In the present study regeneration of the transformed tissues was not obtained in the media standardized. Even though an attempt was made to analyse asiaticoside content in the transformed callus using thin layer chromatography (TLC), there was no detectable quantity of asiaticoside. This could be due to the undifferentiated nature of the callus. As the accumulation of asiaticoside is mainly in the leaves, a better protocol for regeneration needs to be developed so that further studies on triterpenoid analysis could be carried out.