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ThesisItem Open Access Agrobactrium tumefaciens mediated genetic transformation in dendrobium variety sonia 17 with 1- aminocyclopropane- 1 carboxylic acid (acc) synthase antisense gene(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Karthika, Karunakaran; KAU; Rajmohan, KA study on “Agrobacterium tumefaciens mediated genetic transformation in Dendrobium sonia 17 using ACC synthase antisense gene.” was conducted at the Department of Plant Biotechnology, College of Agriculture, and Vellayani during 2004-2006. Orchid is an important cut flower crop. Dendrobium sonia 17 is the most popular commercial orchid grown in Kerala. It has sufficiently higher vase life. However, increasing vase life can reduce the per day cost of flower. The present study was undertaken with the main objective of evolving a protocol for Agrobacterium tumefaciens mediated genetic transformation in Dendrobium sonia 17 using ACC synthase antisense gene. PLBs were initiated from the meristematic shoot tip on half strength MS medium supplemented with growth regulators. MS medium supplemented with BA 0.2 mg l-1 was proved to be the best in terms of induction of PLBs (92.5 %). The Agrobacterium tumefaciens strain GV3101, harbouring the plasmid pA4A2AB was used for genetic transformation. As the plasmid harbour nptII and ACS2 genes, the sensitivity of Agrobacterium strain and PLBs to different concentrations of kanamycin was evaluated. The lethal doses of kanamycin to Agrobacterium and PLBs were 300 and 150 mg l-1, respectively. The effective dose of cefotaxime for the elimination of bacterial strain GV3101 was 50 mg l-1 and the lethal dose of cefotaxime to PLBs was 300 mg l-1. Genetic transformation was achieved by co-cultivating PLBs with bacterial suspension. Conditions like infection and co-cultivation time, selection agent were optimized. The most effective infection time was 20 min, followed by a co-cultivation period of four days. The survival of tissues transformed on the selection media was 76.47 per cent. The transformation efficiency was increased when acetosyringone 200 µM was added to infection and co-cultivation media. Transformation was confirmed by PCR and southern hybridisation of putative transformants. This study provides a protocol for genetic transformation in Dendrobium sonia 17 using ACC synthase antisense gene.ThesisItem Open Access Parasitism of Hirsutella thompsonii Fischer var. synnematosa Samson, McCoy & O`Donnell on coconut eriophtid mite Aceria guerreronis (Keifer)(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2002) Shabnaz Padiyath; KAU; Beena, SAn investigation on "Parasitism of Hirsutella thompsonii Fischer var. synnematosa Samson, McCoy & O'DonneU on coconut eriophyid mite Aceria guerreronis (Keifer)" was carried out at the Department of Plant Pathology, College of Horticulture, Vellanikkara. The main objectives of the study were to explore the cultural, physiological, toxicological characters of the fungus, compatibility of the fungus with pesticides and botanicals and mass multiplication characteristics of the fungus, which could be used as an efficient biocontrol agent on coconut mite. The results of this investigation revealed the following conclusions. From the mite infested young nuts collected from the coconut garden of College of Horticulture, Vellanikkara, the acaropathogen H. thompsonii var. synnematosa was isolated and its pathogenicity on mite was proved. Sabouraud's maltose agar + yeast was selected as the most suitable solid medium for the growth and sporulation of the fungus. Maximum number of fruiting bodies called synnemata were produced in Richard's medium. Correlation study revealed a significant positive correlation between mycelial growth and sporulation. Ninety six per cent germination of spores of the fungus grown in solid medium was recorded after eight hours of incubation. In the different liquid media tried, Sabouraud's maltose+yeast was selected as the best for maximum dry mycelial weight and sporulation. Only twenty five per cent germination was recorded after eight hours of incubation of spores of the fungus grown in liquid medium. Maximum sporulation and germination percentage of spores of the fungus were recorded in solid media compared to liquid media. Microscopic observations showed that in all the solid media the hyphae were hyaline, septate, smooth and branched producing conical to flask shaped phialides with narrow neck. Phialides were branched often once. Spores were spherical, verrucose and hyaline. Hyphal width, phialide width and spore diameter , ranged between 3.33 urn to 3.76 urn in different solid media. Maximum number of phial ides in a unit length of hypha was in Sabouraud's maltose agar + yeast. The fungus preferred an alkaline pH of 9. But it gave satisfactory growth on a wide range of pH from 5 to 9. Growth of the fungus was recorded only in temperatures of 25 and 30oe. No significant difference was recorded in the fungal growth at humidity levels of 50 to 90 per cent. Both exotoxin and endotoxin were extracted from the liquid culture of the fungus. In closed perianth condition there was no significant difference in the per cent mortality of mites due to spraying with both the toxins, but it was superior than spraying with water (control). In open perianth condition there was no significant difference in the percentage mortality of mites on spraying with both the toxins and water. The decreasing order of the inhibition of fungal growth by insecticides/acaricides was dicofol, carbaryl, triazophos, wettable sulphur and endosulfan. The fungicides bordeaux mixture and copper oxychloride showed cent per cent inhibition of fungal growth whereas potassium phosphonate (akomin) recorded minimum inhibition over control. Among the botanicals, maximum inhibition of fungal growth was with garlic + neem oil and azadirachtin showed least inhibition. In the two phase mass production study, wheat was identified as the most promising raw substrate for sporulation and germination percentage of spores of the fungus. In the powdered substrates tried, growth was present only in loose substrates like rice bran and tea waste.ThesisItem Open Access Identification, bionomics and control of infective larvae of common nematodes of domestic ruminants(Department of Parasitology, College of Veterinary and Animal Sciences, Mannuthy, 1995) Jeyathilakan, N; KAU; Sathianesan, VAn investigation by coprological examination and copro – culture into the occurrence of common nematodes of cattle buffaloes and goats, specific identity and bionomics of their infective larvae and assessment of ovicidal and larvicidal properties of common fertilizers like urea, ammonium sulphate, quick lime and ash for their control were carried out. The prevalence was highest in goats with strongyle having highest percentage and Haemonchus being the commonest nematode involved. As per the specific identity Haemonchus contortus, Oesophagostomum radiatum, Cooperia punctate, Trichostrongylus axei, T. colubriformis, Bunostomum phlebotomum and Strongyloides papillosus, H. contortus, O. radiatum, T. colubriformis and S. papillosus and H. contortus, O. columbianum, O. asperum, T. colubriformis, T. axei, B. trigonocephalum and S. papillosus were the species of nematodes encountered in cattle, buffaloes and goats respectively. Regarding bionomics none of the larvae showed phototropism and the highest and shortest viability was for Oesophagostomum species and Strongyloides species respectively. Urea and ammonium sulphate (1%) were found to have the highest ovicidal property (100%) with ash having the lowest, when mixed with faeces. The larvicidal property was 100% for quicklime (1%) with no efficacy for other fertilizers, when treated with larval suspension. Scanning electron microscopic study of some of the infective larvae was also carried out.