1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Response of Ascocenda orchid to growth regulator and micronutrients(Department of Floriculture and Landscaping, College of Horticulture, Vellanikkara, 2019) Jesabel George; Shobhana, AA study entitled ‘Response of Ascocenda orchid to growth regulator and micronutrients’ was carried out at Department of Floriculture and Landscaping, College of Horticulture Vellanikkara, from May 2018 to May 2019. Ascocenda is a monopodial, epiphytic, bigeneric hybrid, which is mainly grown as pot plant in hanging baskets using bricks, charcoal, coconut husk pieces etc. as growing media. The objective of the study was to evaluate the influence of foliar application of different micronutrient treatments on growth and yield of Ascocenda orchid. The experiment was conducted with eleven treatments viz., 0.01% zinc + 150 ppm benzyl adenine + PoP (T1), 0.025% zinc + 150 ppm benzyl adenine + PoP (T2), 0.01% manganese + 150 ppm benzyl adenine + PoP (T3), 0.025% manganese + 150 ppm benzyl adenine + PoP (T4), 0.01% boron + 150 ppm benzyl adenine + PoP (T5), 0.025% boron + 150 ppm benzyl adenine + PoP (T6), 0.01% iron + 150 ppm benzyl adenine + PoP (T7), 0.025% iron + 150 ppm benzyl adenine + PoP (T8), 0.01% molybdenum + 150 ppm benzyl adenine + PoP (T9), 0.025% molybdenum + 150 ppm benzyl adenine + PoP (T10), 150 ppm benzyl adenine + PoP (T11 – control). Three month old tissue cultured plants of Ascocenda var. Big Suksamran were used for the study. The micronutrients were applied at fortnightly intervals and benzyl adenine was applied at monthly intervals. Application of NPK (3:1:1) weekly twice @ 0.2% and cow dung slurry (1:5) at monthly intervals was given to all treatments as per PoP recommendation of KAU. Observations were taken at monthly intervals. The results indicated that foliar application of 0.025% manganese along with 150 ppm BA and recommended dose of NPK (T4) was best for improving plant height. The maximum plant height obtained at 12MAP was 8.86 cm. This was followed by T5 (8.81 cm) and T3 (8.63 cm) which were statistically on par with T4. The maximum shoot diameter was observed in T5 (10.20 mm) at 12 MAP which was on par with T4 and T3 (9.96 mm and 9.84 mm respectively). The treatment T3 was superior in terms of leaf characters like leaf length and leaf area up to 7 MAP and thereafter these parameters were highest in treatment T5. However, there was no significant difference between T5 and T3 in terms of leaf length at 12 MAP (16.70 cm and 16.48 cm respectively). The highest leaf area at 12 MAP was observed in T5 (23.17 cm2) followed by T3 (22.73 cm2). Number of leaves and leaf breadth were found highest with the application of 0.01% boron along with 150 ppm BA and recommended dose of NPK. A maximum of 13.69 leaves were observed in T5 at 12MAP. The maximum leaf breadth observed in T5 after 12 months of planting was 1.52 cm, which was closely followed by T3 and T4 (1.51 cm each), and no significant difference between these three treatments could be noticed. Regarding interval of leaf production, only 4 treatments (T3, T4, T5, and T11) could produce the highest number of eight leaves, within a period of 386 days. Among these, T4 took the shortest period of 337.45 days to produce the 8th leaf. T10 produced only five leaves within a period of 386 days. Among the root parameters, highest root length was observed in T3 (0.01% manganese + POP + 150 ppm BA) at 12 MAP (26.59 cm) whereas the treatment T4 (0.025% Mn + PoP + 150 ppm BA) was superior in terms of number of roots and root diameter. The best treatment with respect to number of roots varied during initial months, even though, from 6 MAP onwards, highest number of roots was observed in T4 with a value of 10.28 at 12 MAP. In the case of root diameter, a highest of 2.91 mm was recorded in T4 at 12 MAP, which was on par with T8 (2.86 mm), T3 (2.84 mm) and T5 (2.82 mm). Among the eleven treatments, T3 (Mn 0.01% + PoP + 150ppm BA), T4 (Mn 0.025% + PoP + 150ppm BA), and T5 (B 0.01% + PoP + 150ppm BA)were found to be best for improving the vegetative characters of Ascocenda orchid, while application of Mo @ 0.025% (T10) at fortnightly intervals was inhibitory to the plants in terms of all the vegetative characters studied. The objective of studying the floral and postharvest characters could not be achieved since the plant did not bloom within the period of study.Item Evaluation of selected underutilized flowers of Kerala for commercial exploitation(Department of Pomology and Floriculture, College of Horticulture, Vellanikkara, 2018) Sameera Sharief; Sudhadevi, P KFloriculture industry is unique among agricultural industries where novelty is an important attribute. World floriculture is expanding rapidly and new innovations and introductions are in great demand to feed the ever hungry market needs. It is in this interest that neglected or underutilized flower crop species (NUS) comes to the picture from which we could identify and develop diversified uses of floriculture. Even the present day top charactered crops of the industry are nothing but just derived and developed only from wild germplasm resourses, the most prominent among them being rose, chrysanthemum, carnation, gerbera and what not, the orchids and anthurium. Thirteen underutilized plants of Kerala were evaluated for use as cut flowers, dry flower, for essential oil extraction and pigment extraction and identification of components in their essential oils and pigments using GC-MS by conducting both field studies as well as postharvest studies. Out of the 10 plants selected for studying their use as cut flower, none were found suitable. Five plants were selected for their suitability for dry flower production. Among them, Cassia fistula got the highest cumulative score followed byAntigonon leptopus,Calicopteris floribunda and Barleria obtusa. Least cumulative score was obtained for Clerodendrum paniculatum. In Antigonon leptopus and Clerodendrum paniculatum best method of drying was microwave oven drying. Press drying was selected as the best method for Barleria obtusa and Calicopteris floribunda. Embedded drying was found the most suitable method of drying in Cassia fistula. The fragrant flowers selected for extraction of essential oils were Gardenia jasminoides, Plumeria spp and Quisqualis indica.Maximum essential oil yield was observed in Gardenia jasminoides (0.61%). The components in the essential oils were identified by gas chromatography and mass spectrometry. In Gardenia jasminoides the components identified were Ascalbin (0.7%), Nonadecane (0.80%), Dendaralasine (0.96%), Alpha famesene (1.12%), Linalool (1.38%), Beta famesene (2.75%), Famesene (3.04%), Henecosane (5.26 %), n-Tricosane (6.91%), n-octacosane (10.43%), Pentacosane (13.19%), Monoethylhexyl phthalate (44.74 %). The volatile components identified in Plumeria were α-Farnesene (1.08%), Benzoic acid, [(E,E)-3,7,11-trimethyl- 2,6,10-dodecatrien-1-yl] ester (1.08%), 1,6,10-Dodecatriene, 7, 11- dimethyl- 3- methylene-E(1.17%), Cyclohexasiloxane, dodeca methyl (1.92%). Phenyl ethyl alcohol (2.20%), 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-(Z,E)(2.93%), Oxalic acid, decyl 2-phenyl ethyl ester (4.3%), Triphenyl phosphare (4.3%), Heptacosane (6.32%), Nonacosane(6.40%) and Z-14-Nonacosane (6.40%) and Z-14- Nonacosane(11.65%). The components responsible for fragrance in Quisqualis indica were 2 H- Pyran-3-ol, 6-ethenyl tetra hydro-2,2,6-trimethyl(1.24%), Heptacosane (1.44%), Cyclotetra siloxane, octamethyl (1.95), Triphenyl phosphate (2.30%), Nonacosane(2.89%) and 5-Isoquinoline carbonitr (12.5%). The flowers selected for extraction of pigments were Caesalpinia pulcherrima, Cassia fistula, Clerodendrum paniculatum and Delonix regia. Two methods of extraction selected were solvent extraction after fermentation and solvent extraction after pretreatment with NaOH. In the entire species pigment yield was higher for solvent extraction after pretreatment. Highest oleoresin yield was observed in Clerodendrum paniculatum (0.60g), which was followed by Delonix regia (0.5g) and Cassia fistula(0.39). The lowest yield was observed in Caesalpinia pulcherrima (0.38g). After fermentation, Clerodendrum paniculatum gave highest oleoresin yield (0.43g). This was followed by Delonix regia(0.4g), Cassia fistula(0.3g) and Caesalpinia pulcherrima(0.28g). Highest carotenoid yield was observed in Cassia fistula (70.04mg/ 100 g) and highest anthocyanin yield was in Clerodendrum paniculatum (574.76mg/100g)). In Caesalpinia pulcherima carotenoid content was recorded as 15.35mg/100g and anthocyanin 488.75mg/100g. Anthocyanin yield of Cassia fistula was 0.35mg. Clerodendrum paniculatum recorded 2.98mg of carotenoid. Delonix regia recorded carotenoid and anthocyanin yield of 60.2 mg and 510 mg respectively. In the present study none of flowers were found suitable for use as cut flower. Out of the 5 plants selected for studying for use as dry flower, Cassia fistulawas the most suitable one. All the species selected for essential oil extraction were suitable for the purpose .In pigment extraction, highest oleoresin yield was observed in Clerodendrum paniculatum (0.60g), which is followed by Delonix regia (0.5g) and Cassia fistula(0.39). Future line of work suggested in this aspect based on the light of results are evaluation of more underutilized ornamental flowers available in our locality with a view of their commercialisation for specific traits and further evaluation of extracted pigments for their use in food industry.Item Development of recombinant coat protien for immunodetection of cucumber mosaic virus infecting banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Alan C Antony; Vimi LouisBanana (Musa spp.) is infected by four well characterised plant viruses viz., Banana bunchy top virus, Cucumber mosaic virus (CMV), Banana bract mosaic virus and Banana streak virus. Among these, CMV causes devastating effect on tissue culture banana plants. The study entitled “Development of recombinant coat protein for immunodetection of Cucumber mosaic virus infecting banana” was carried out using existing facilities of Department of Biochemistry, Indian Institute of Science, Bangalore, Division of Plant Pathology, Banana Research Station, Kannara and Department of Plant Pathology, College of Horticulture, Vellanikkara, Thrissur during 2018- 2019. The present study was carried out to produce recombinant coat protein, which can be utilised later for producing high quality antiserum for the detection of CMV infecting banana. Cucumber mosaic virus infected samples were collected based on various characteristic symptoms and screened by direct antigen coating immunosorbent assay using commercially available CMV polyclonal antiserum. Isolate namely, KANC- 4, KANC- 2 and NDRNS- 4 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS- 4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEM- T/CMV- CP was confirmed through colony PCR using coat protein specific primer, restriction digestion of recombinant plasmids using EcoR1 enzyme followed by sequencing. The vectors viz. pRSET- C and pET28a were selected for the expression of CMV- CP gene in E. coli. Coat protein specific forward (5’GGG GCT AGC ATG GAC AAA TCT GAA TCA ACC3’) and reverse primers(5’CCC GGA TCC TTA CTC TCC ATG GCG TTT AG 3’) were designed along with recognition sites of restriction enzymes BamH1 and Nhe1.The annealing temperature of designed primer was standardised as 55°C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and high fidelity Pfu DNA polymerase enzyme. Expression vectors as well as amplicon were subjected to ligation and the recombination in expression plasmids (pRSET- C/ CMV- CP and pET28a/CMV- CP) were confirmed through PCR and sequencing. The plasmid with maximum homology i.e., pRSET-C/CMV- CP was selected for further studies. The recombinant plasmid was transformed into E. coli BL21(DE3)pLysS cells for the expression of CMV- CP gene and the expression of 25 kDa recombinant CMV coat protein was confirmed in 12 per cent sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS- PAGE). Tris- NaCl buffer of pH 8.0 was selected for solubilising the recombinant protein using ExPASy - protein translation tool. The recombinant protein was further purified through Nitrilotriacetic acid column purification, in which the 6X histidine tagged recombinant protein was bound with agarose coated nickel beads. Buffers containing imidazole were used for the elution of histidine tagged recombinant protein, since imidazole competes with histidine for the binding site in nickel beads. Each fraction viz., cell pellet, supernatant, flow through, wash and elution were collected and later detected for protein using SDS- PAGE. Absence of 25 kDa protein in cell pellet indicated that the recombinant coat protein completely soluble in Tris- NaCl buffer (pH 8.0). Confirmation of recombinant coat protein was carried out through DAC- ELISA and western blotting using commercially available polyclonal CMV antiserum (1: 2000; NRCB, Trichy). The recombinant coat protein developed through this study could be utilised for large scale production of antiserum for immunodetection of CMV.