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Subject: Biotechnology and Molecular Biology × Author: KAU × End date: 2018 × Start date: 2016 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    In vitro micropropagation protocol for Vanda hybrids with clonal fidelity analysis
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Rosemol, Baby; KAU; Valsala, P A
    Vanda orchids are one of the most sought after orchids in the international as well as domestic flower markets both as cut flower and potted plants. It is a monopodial orchid with vividly coloured, loosely arranged large beautiful flowers which has a long shelf life. Presently, many Vanda hybrids are becoming prominent even in the home gardens. However the present scenario of importing these hybrids from Thailand, Singapore and Malaysia to meet the Indian demands throws light on the need for developing an efficient propagation method for Vanda orchids. One of the major limiting factors for its spread and large scale cultivation in India is the non-availability of good quality and true to type planting material at a reasonable price. As the demand is more for the true to type plants, micropropagation is mostly recommended for orchid propagation. Hence this study was undertaken to develop an efficient micropropagation protocol for two Vanda hybrids namely Dr.Anek and Sansai Blue and to check the variability between the parents and regenerated plantlets. The different explants tested to initiate the cultures were leaf, root, stem and inflorescence segments. Initially the surface sterilization procedure was standardized for the explants. The results of the experiment showed that treating the explants with 0.1 per cent carbendazim for 20 minutes, followed by 70 per cent ethanol for 5 minutes and 0.1 per cent mercuric chloride for 5 min effectively reduced the microbial contamination with highest percentage of explant survival.Trial was made to initiate cultures using eight reported media compositions. The study showed positive results for inflorescence segments inoculated on to 1/2 MS + 10 mg l-1 BA+ 2 mg l-1 TDZ+30 g l-1 sucrose+7.5 g l-1agar + 250 mg l-1 cefotaxime as observed as direct shooting of the dormant buds. About 80 per cent and 60 per cent culture establishment was brought about in Dr. Anek and Sansai Blue respectively in 9 weeks. The established cultures successfully produced multiple shoots on MS+4.5 ml l-1BA +30 g l-1 sucrose + 7.5 g l-1 agar + 250 mg l-1 cefotaxime both when inoculated with and without the stalk in about 100 days of inoculation of explant. The micro-shoots from cultures without stalk were further transferred to hormone free basal MS media for elongation. Elongated shoots of about 4 cm were transferred to rooting media with a composition of MS + 0.5 mg l-1 NAA + 1 mg l-1 IAA +30 g l-1 sucrose +7.5 g l-1agar + 250 mg l-1 cefotaxime for better rooting of the regenerants. The percentage of rooting was observed to be 72.41 per cent for Dr. Anek and 70.37 per cent in Sansai Blue. The rooted plantlets with ample number of healthy roots were planted outm in small earthen pots with charcoal, coconut husk and brick pieces. These were successfully hardened in net house of 50 per cent shade and showed a hundred percent plantlet survival.Good quality DNA isolated from the mother plants and their respective clones using Rogers and Bendich procedure were analyzed for the clonal fidelity. ISSR analysis was done using 5 UBC (University of British Columbia) primers such as UBC 808, UBC 811, UBC 826, UBC 835 and UBC 841. An average of 8 to 9 bands was obtained from all primers in Dr. Anek and Sansai Blue. Out of 5 primers, UBC 808 and UBC 835 generated polymorphic bands in two clones of Dr. Anek. For Sansai Blue, all five primers generated monomorphic bands for all the mother plants and their respective clones analyzed. The per cent polymorphism in Dr. Anek was calculated to be 1.11 per cent whereas for Sansai Blue, there was no polymorphism detected revealing the true to type nature of the clones. The results showed that the identified protocol for in vitro regeneration of selected Vanda hybrids is a viable protocol since there were no changes in the banding pattern observed in tissue culture plants as compared with that of mother plant. Hence it can be concluded that the developed micropropagation protocol can be used for commercial production of Vanda hybrids without much risk of genetic instability. ISSR markers were effective to evaluate the genetic stability of the clones regenerated from the mother plants by the identified protocol.
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    ThesisItemOpen Access
    Cloning and characterization of fusarium wilt resistance gene analogs in banana (Musa spp.)
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Ramesh; KAU; Valsala, P A
    Banana is one of the important fruit crops of India. It is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production are Fusarium wilt or Panama wilt caused by Fusariumoxysporumf. sp. cubense (Foc). Control of the pathogen is difficult and mainly involves the use of disease free suckers. Although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult due to its triploid nature and sterility factors of banana. The study entitled "Cloning and characterization of Fusarium wilt resistance gene analogs in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2013- 2015 with an objective to PCR amplify the genomic DNA from Fusarium wilt resistant banana genotype with primers specific to ‘R’ genes of TIR-NBS-LRR class for cloning and characterization of resistant gene analogs. 105 Palayankodanresistant (Mysore Poovan AAB) and Poovan susceptible(Rasthali AAB)varieties of banana were used for the present study.Fusarium culture was isolated from roots of infected banana plant and cultured inPDA (Potato Dextrose Agar) media. Fungal spore was suspended in sterile water and filled in small polythene bag. Artificial inoculation – root feeding of inoculum of water suspension was done. The symptoms of Fusarium wilt was observed two months after infection. Results confirmed resistance of Palayankodan and susceptibility of Poovan The DNA was isolated from Palayankodan (resistant) and Poovan (susceptible)genotypesand isolated DNA was subjected to RNase treatment. Quality checking was done using 0.8% agarose gel electrophoresis and quantity analysis was done using nanodrop ND-1000 spectrophotometer. Thirty five ng of DNA was used as template DNA for PCR amplification using reported degenerate primers. Twenty five primer combinations were made using five pair of degenerate primers. Among those combinations only one primer combination F9(F)+F6(R) showed polymorphic band of 700bp which was eluted, purified and cloned in pGEMT easy vector system.The presence of insert was confirmed by colony PCR and the eluted product was sequenced by outsourcing. The sequence obtained was subjected Blastn, Blastx and Blastp analysis and was compared with NCBI database. The sequence showed similarity with NBS-LLR resistant gene of Musa spp. Open reading frames (ORFs) were also identified using ORF finder software and four ORFs were identified For further validation, new primers were designed from 675 bp region of NBS-LLR class of resistant gene using primer3 plus software with an expected band size of 430bp. The DNA from both infected and healthy samples were amplified with designed primers and the expected bands were obtained in DNA samples of healthy 106 (resistant) plants where as it was absent in DNA samples of infected (susceptible) plants. The degenerate primer F9(F)+F6(R) and designed primer FTGAGCAGCATCGCCTA. R- GCCTGACACCAGTGAAGC can be used for Fusarium wilt disease diagnostics. Sequence information with respect to above primers amplicons can be used for synthesis of gene construct for genetic engineering.