Genetic fidelity assessment of wild and tissue cultured regenerants of a threatened orchid, Cymbidium aloifolium using molecular markers

Molecular markers play an effective role in estimating the genetic similarity, variation, diversity, and population structure of different plants. Various factors associated with in vitro culture conditions may cause genetic variation in tissue cultured regenerants. The main goal of the present study was to determine the genetic uniformity of plantlets regenerated through in vitro culture of protocorms, shoot tips, and sodium alginate coated artificial seeds of Cymbidium aloifolium (L.) Sw. and its non-tissue cultured source mother plant using molecular markers such as Random Amplified Polymorphic Deoxyribonucleic acid (RAPD) and Inter Simple Sequence Repeats (ISSR). Ten RAPD and five ISSR primers were used to amplify the genomic DNA isolated from the in vivo plant and randomly selected micropropagated plants. Nine out of ten RAPD primers amplified a total of 256 loci while five ISSR primers amplified a total of 99 loci. The combined data of RAPD and ISSR markers showed low polymorphism. Among the tested plants, dendrograms constructed through UPGMA analysis of RAPD and ISSR markers revealed high genetic similarity between the mother plant and in vitro cultured regenerants. Among the different in vitro regenerants, protocorm-derived plants showed 91% genetic homogeneity to that of the mother plant. Thus, both molecular markers proved to be equally efficient for genetic fidelity studies in C. aloifolium. Hence, this research successfully assessed the genetic fidelity of in vitro cultured plants which could be useful in reintroducing true-to-type plants through plant tissue culture techniques.