1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Improvement of Anthurium andreanum Lind. in vitro(Department of Pomology and Floriculture, College of Horticulture, Vellanikkara, 1998) Mini Balachandran; Ramachandran Nair, SItem Establishment of in vitro regeneration systems from callus and protoplast in capsicum frutescens L.(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Jancy J Sathyaraj; Deepa S NairThe present study entitled “Establishment of in vitro regeneration systems from callus and protoplast in Capsicum frutescens L. was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2015-2017. The objective of the study was to establish callus culture from different explants in C. frutescens, to establish protocol for protoplast isolation from callus/leaf mesophyll and to culture protoplast. The study was carried out in two phases viz., establishment of callus culture and organogenesis; and standardization of protocol for protoplast culture. Callus was induced from leaves and internodal segments from in vitro raised seedlings. Among the 44 treatments in MS medium with different combinations of auxins (NAA, IAA, IBA, 2,4-D and picloram) and cytokinins (BA and Kn), 100 per cent callus induction was obtained in MS media supplemented with picloram 0.50, 1.00, 1.50 and 2.00 mg L-1 (C41, C42, C43, and C44), NAA 1.50 mg L-1, NAA 2.00 mg L-1(C3, C4) and BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29). Among the 78 treatments tried for organogenesis, calli obtained from C29 treatment showed organogenesis in MS + BA 3.00 mg L-1 + IBA 1.00 mg L-1 (R37) and (MS + BA 5.00 mg L-1 + IAA 2.00 mg L-1 (R61) in 41 and 90 days, respectively. The microshoots obtained recorded 83.33 per cent rooting in MS medium supplemented with IAA 1.50 mg L-1(Rt7) in 12.83 days. Leaves excised from in vitro seedlings, and calli produced in MS + BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29), were used as explants for protoplast isolation. Leaf bits incubated in cell protoplast washing (CPW) solution containing cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M (maintained at pH 5.8) for 6 h (DM28) in dark at 27oC, yielded (124 x 105) protoplast per g, with a viability of 95.16 per cent. The callus yielded maximum protoplast (36 x 105 protoplasts per g) in an enzyme combination of cellulase 2.00 per cent + macerozyme 0.50 per cent + mannitol 0.60 M (maintained at pH 5.8) after 4 h 91 (DM47) of incubation under same conditions. In protoplast purification, floatation medium with 21 per cent sucrose recorded maximum protoplast yield (30 x 105 protoplasts per g tissue and 10 x 105 protoplasts per g callus) and maximum viability (90.91 per cent and 100 per cent), from leaf derived and callus derived protoplast, respectively. The purified protoplasts with 10 x105 plating density initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1 (PCM5) in 45 days. Further development to visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1, in 60 days from callus derived protoplast and in 70 days from leaf derived protoplast. In the study, maximum callusing response was obtained in MS medium with picloram 1.50 mg L-1. Organogenesis was obtained from the calli derived in MS medium with BA 3.00 mg L-1 and NAA 1.00 mg L-1. The shoot initiated from the calli in MS medium with BA 3.00 mg L-1 and IBA 1.00 mg L-1. The rooting of microshoots could be obtained in MS medium with IAA 1.50 mg L-1. In protoplast isolation, leaf gave higher protoplast yield and viability in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M, incubated in dark for 6 h and callus, in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.60 M, incubated in dark for 4 h. The protoplasts purified in 21 per cent sucrose supplemented floatation medium and adjusted to a plating density of 10 x 105, initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1. The visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1. In this study, a callus mediated in vitro regeneration system has been established in C. frutescens. A protocol has also been developed for protoplast isolation from leaf and calli, and its culture resulting in microcalli formation.Item Somatic embryogenesis in black pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Afnamol, O P; Soni, K BItem Elicitation mediated carotenoid prodouction and capsanthin capsorubin synthase gene expression in byadagi chilli (capsicum annuum L.)(Centre of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2018) Pooja, S L; Shylaja, M RByadagi chilli is well-known for its deep red colour and zero pungency. The fruits of Byadagi chilli are brown to deep red at full maturity. The red colour of chilli fruits are due to several carotenoid pigments. They are good source of natural colourants used in food, feed, textile and cosmetic industries. The chilli type is best suited for oleoresin extraction and exported as a substitute for paprika oleoresin. The present study ‘Elicitation mediated carotenoid production and Capsanthin capsorubin synthase gene expression in Byadagi chilli (Capsicum annuum L.)’ was undertaken at Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during 2016-2018. The study was carried out using two genetically distinct chilli genotypes based on their colour at fully ripe fruit stage namely Byadagi Dabbi and variety Anugraha. Callus cultures were produced from tender leaves of both the genotypes using MS media augmented with different plant growth regulators viz., Indole-3- acetic acid (IAA), Kinetin (Kin) and Benzyl adenine (BA) at different concentrations as reported by Kintzios et al. (1996), Kehie et al. (2012), Kabby et al. (2015) and Santos et al. (2017). Though, callogeneses were obtained in all the media tried, early callusing and higher callus index were achieved in MS medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 Kin. Elicitation of two month old calli was done with two different chemical elicitors viz Salicylic acid and Methyl jasmonate at different concentrations like 20, 40 and 60 mgL-1 for 72h. The carotenoids were extracted and β-carotene was quantified using HPLC. Elicitor treatment with salicylic acid increased β-carotene content significantly. The highest β-carotene was recorded in Byadagi calli elicited with salicylic acid 20 mgL-1 for 72 hours. In this treatment Byadagi Dabbi recorded 6.75 times higher β-carotene (42.85 μgg-1 fresh weight) than the control (6.34 μgg-1 fresh weight). In methyl jasmonate elicitation, both the genotypes were found on par with respect to β-carotene production in the different concentrations studied. Expression of Capsanthin capsorubin synthase (Ccs) gene was studied in the highest β-carotene yielding treatment viz elicitation with salicylic acid 20 mgL-1 for 72 hours using real time PCR. The total RNA extracted from elicited calli were converted to cDNA. The Capsicum annuum L. β-tubulin gene was used as the endogenous control. Dissociation curves were obtained as a single dominant peak denoting that there was specific gene amplification for both endogenous control and Ccs gene in different treatments. Relative quantification of the Ccs gene expression was done using the Comparative CT method reported by Livak and Schmittgen, (2001). The Ccs gene was found up-regulated 2.35 fold in Byadagi Dabbi elicited calli with salicylic acid 20 mgL-1 for 72 hours. The expression of Ccs gene in the variety Anugraha was down-regulated (0.906 fold) when elicited with salicylic acid 20 mgL-1 for 72 hours. The major outcome of the present investigations are the suitability of leaf and calli induced from leaves for carotenoid production, scaling up of carotenoid production through salicylic acid elicitation and upregulation of Ccsgene expression in highest carotenoid yielding elicited calli. Another significant finding is the response of calli to salicylic acid elicitation which is a cheaper elictor as compared to Methyl jasmonate and the highest content of β-carotene at lower concentration of elicitor which are plus points as far as commercial exploitation of carotenoids is concerned. However, much more elaborate studies are required on explant stage, culture systems, age of cultures, culture conditions, duration and concentration of elicitors and activity of regulatory enzymes and elicitor mediated expression of genes involved in carotenoid metabolic pathway for commercial exploitation of the system for carotenoid production. A more in-depth understanding of the underlying biological mechanisms will enable to fully harness the potential of cell cultures and to enhance carotenoid production on an industrial scale.Item 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; 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.Item Response of immature inflorescence for in vitro regeneration on coconut (cocos nucifera L)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2006) Siny, C V; Nazeem, P AInvestigations on 'Response of immature inflorescence for in vitro regeneration in coconut (Cocos nucifera L.)' were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2005 to 2006. Y3 medium was found to be the best basal medium for in vitro culture of immature inflorescence of coconut. Inflorescence of length 40 to 50 cm was found to posses male flowers at pollen mother cell stage of microsporogenesis. Wiping the spathe with 70 per cent ethyl alcohol before excising the immature inflorescence parts could effectively control contamination with 100 per cent culture establishment. The young inflorescence parts could survive up to 12 minutes treatment with 0.1 per cent HgCl2. Among the different explants tried, anthers at premeiotic stage and immature rachillae were found to be the best for callus induction and embryo formation. When the explants were inoculated the exudation of polyphenols from the explants adversely affected their survival. Polyphenol exudation was checked by incorporating PVP 0.1 per cent and activated charcoal 0.2 per cent in the medium and by incubation under dark condition. 2,4-D at 15 to 30 mgl-1 was found to be the most effective auxin for callus induction and proliferation. Y3 basal medium with growth regulator combinations of 15 mg l-1 2,4-D, 0.5 mg l-1 picloram, 1mg l-1 NAA and 0.1 mg l-1 TDZ was identified as the best medium for callus induction and embryogenesis of immature anther. Sucrose at 5 per cent concentration was identified as the optimum concentration for callus induction. Pretreatment of inflorescence at 4°C for 24 hrs or 30 hrs doubled the callus induction and reduced the browning of explant. Callus induction was observed from rachillae tissue in Y3 medium containing15 mg l-1 2,4-D, 1 mg l-1 picloram, 1 mg l-1 IAA and 0.1 mg l-1 TDZ.Item Induction of variability in anthurium (Anthurium andreanum Lind.) through in vitro mutagenesis(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Yashawant Kumar Srivastava; Nazeem, P A