MSc

The study entitled “Refinement of micropropagation protocol and RAPD assay for sex determination in Kodampuli (Garcinia gummi-gutta var. gummigutta)” was carried out in the laboratory of Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Kerala Agricultural University, Vellanikkara to attempt and refine the micropropagation protocol in Kodampuli and to determine the sex of this polygamodioecious tree taxon. The relevance of the study is known by the fact that this polygamodioecious perennial tree spice poses hindrance to large scale multiplication of propagules due to seed dormancy, lack of orthrotropic shoots for grafting, poor germination percentage etc. The diabetes and cardiovascular diseases are the two serious lifestyle diseases, which spread at alarming rate throughout the world. In India, Kerala holds first position in these two obesity related diseases. The fleshy fruit rinds of Garcinia spp. are the richest natural source for anti-obesity metabolite, (-)Hydroxy Citric Acid (-HCA).

The major objectives of the study were to 1) Refinement of standardized micropropagation protocol (nodal segments) and somatic embryogenesis (immature endosperm) for large scale multiplication to cope up the anticipated extensive cultivation. 2) Standardization of DNA isolation techniques from male and female trees in vitro seedling progenies and polyembryonic seedlings in Kodampuli. 3) RAPD assay using selected oligoneucleotide decamer primers to ascertain the sex at juvenile phase in this polygamodiocious species so as to substantiate the findings of isozyme analysis of esterase using SDS- PAGE .

The nodal segements (immture parrot green shoots) collected in fungicide solution and then with HgCl2 for 1-3 minutes under laminar flow chamber. These explants inoculated in half MS and full strength WPM medium fortified with various concentrations of plant growth regulators and activated charcoal. Best results obtained in ½ MS + 5.0mgl-1 IBA + 1.0mgl-1 BA + 0.5% AC. They showed 25% survival in the above media with proliferation of multiple shoots. All cultures transferred to the shoot elongation media were lost due to fungal contamination.

Immature kodampuli fruits (30-45 day old) were collected from the field grown trees of the Department of Olericulture and pretreated with any one of the systemic fungicides like Fytolan/Companion/bavistin (0.1%) for 30 minutes. Seeds from these immature fruits were extracted under the aseptic condition of laminar air flow chamber and thereafter sterilized in 0.1per cent HgCl2 for a period of 1-3 minutes. The two ends of seeds were cut and only the endosperm part was inoculated into jam bottles containing half strength MS medium fortified with different concentrations of plant growth regulators .These were kept for dark incubation in the culture room for a period of 2 weeks to reduce phenolic exudation and later transferred to light condition. Temperature of the culture room was maintained at 26+ 2oC. The media composition ½ MS + 2.0mgl-1 BAP showed good response with 78 per cent of the inoculated immature endosperm survived at 15DAI.

The excised ends of the immature endosperm containing embryo were inoculated into half strength MS basal .These inoculated embryo with a bit of endosperm were initially kept in dark for two week and later transferred to light in the culture room. The in vitro seeds on germination were transferred to MS basal medium with different hormonal compositions. These immature seeds took 21 days for germination. These in vitro germinated seeds transferred to rooting media and they were initiated roots 60 DAI. These cultures were shown 100% fungal contamination and thus they were discarded.

DNA isolation was standardized by using CTAB method with an extended incubation at – 20oC for a period of 1½ hours. These isolated DNAs from the established male and female trees in the field as well as in vitro germination seedlings were subjected to sex determination studies using RAPD analysis.

The RAPD analysis for sex determination was carried out by using 35 decamer primers for screening. Among the OPE (six), OPF (nine) and Kit-C (twenty) primers were tried, and the Kit-C-1 olegonucleotide decamer primer were explicited distinct monomorphic and polymorphic banding pattern. So, this primer can be used for further sex determination studies of Kodampuli in future.