Development of an Efficient Micropropagation System of Dragon Fruit (Hylocereus Undatus) and Assessment of Genetic Fidelity of in- Vitro Derived Plants Using Molecular Markers

Abstract

The present investigation on the “Development of an efficient micropropagation system of Dragon fruit (Hylocereus undatus) and assessment of genetic fidelity of in- vitro derived plants using molecular markers” was carried out in the Department of Horticulture. Hylocereus undatus is an important fruit crop belongs to the family Cactaceae. It grows best in dry, tropical, or subtropical climates where annual rainfall ranges from 20–50 cm per year. The fruit is red-skinned covered with large and long scales, red and green at the tips with white flesh and black seeds. The multiplication of H. undatus is easily by cutting off the stems and by sowing of seeds. But propagation by cutting is too slow to meet the demand for large scale plantation due to a shortage of young nodes and plants take 3 years to grow from seed, which are not genetically true-to-type. Hence, to meet the growing demand of dragon fruit, the in vitro multiplication of dragon fruits from younger shoots of mature plants is a best alternate and effective method. An efficient in vitro regeneration protocol has been established using apical meristem, areol and stem segment explant. Explants were sterilized using mercuric chloride (0.1%) for 30 seconds followed 1 minute by 70% ethanol. Explants were cultured on MS medium containing different concentrations and combinations of cytokines (BAP, GA and NAA). Among all type of explants taken under investigation the apical meristem takes minimum time (28.33±0.85) taken for shoot initiation, maximum number of shoot (4.73±0.47) were observed under BAP 2 mg/L + GA 1.5 mg/L + NAA 0.2 mg/L and the 100 % explants shows shoot initiation as well as longest shoot (4.58±0.71cm) among all the treatment was observed with BAP 2 mg/L + GA 2mg/L + NAA 0.05 mg/L. In vitro regenerated shoots when inoculated in MS medium supplemented with NAA 2 mg/L + BAP 0.1 mg/L shows 100 % roots in just 9.23±0.35 days and maximum number of roots 5.73±0.47 was obtained from this treatment. In vitro raised plantlets was transferred to hardening media containing different combinations of substrates. Among all the combinations, Soil + Sand + Vermicompost + Cocopeat (1:1:1:1) gives the highest survival rate of 91.33±1.15%. A large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, the genetic fidelity of micropropagated plantlets was evaluated by using inter specific sequence repeat (ISSR) analysis. During the study a total of 16 primers were screened, out of which 14 ISSR primers produced clear, distinct and reproducible amplicons. Fragments were scored for the presence of band as 1 (band present) and absence of band as 0 (band absent) and thus a matrix was obtained and further analysed with NTSYS-pc software by using Jaccard’s coefficient to calculate the similarity matrix for mother and in vitro raised plants. Dendrogram was plotted using UPGMA between mother plant and in vitro plants. The in vitro regeneration protocol provide large number of genetically similar plants with in short period of time