Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant

Abstract

Pittosporum eriocarpum Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. In vitro regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine in vitro raised plantlets with mother plant. DNA fingerprints of in vitro regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of P. eriocarpum. The protocol would be useful for the conservation and large scale production of P. eriocarpum to meet the demand for medicinal formulations and also for the re-introduction of in vitro grown plants in the suitable natural habitats to restore the populations.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant

“…Even though we have carried amplification and produced monomorphic bands with RAPD primers, as we cannot ensure the reproducibility of results with RAPD primers, hence in our study along with RAPD primers we have also used other advanced primers, i.e. , ISSR and SCoT, which are reliable and reproducible type in assessing the genetic stability of regenerants compared to RAPD [48]. All amplification reactions with RAPD, ISSR, and SCoT primers were repeated three times to check the reproducibility and results were, i.e.…”

“…SCoT markers can be used effectively for both genetic diversity and similarity studies [51, 52]. SCoT markers were already reported for assessing the genetic stability of several plant species such as Cleome gynandra [53], Pittosporum eriocarpum [48] , Albizia julibrissin [54], and Simmondsia chinensis [55]. Similarly, ISSR primer based assessment of regenerans was reported in Tylophora indica [56], Simmondsia chinensis [57], Rauwolfia tetraphylla L. [58], Spilanthes calva [59], Cucumis melo L. [60], Cornus alba [61], and Zea mays [62].…”

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis

“…Besides flow cytometry, several DNA markers such as Random Amplification of Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP) and microsatellite-based ISSR are frequently used to evaluate somaclonal variation within the regenerated plants [ 40 ], [ 28 ], [ 41 ]. Of these, ISSR marker analysis has been used most frequently because of its simplicity as well as high degree of sensitivity, increased stringency, higher reproducibility and cost-effectiveness [ 42 ].…”

Genetic Structure and Connectivity of the Red Mangrove at Different Geographic Scales through a Complex Transverse Hydrological System from Freshwater to Marine Ecosystems

“…Several molecular techniques are available, and those that have been used to study the genetic population structure of mangroves include isozymes [27], microsatellites [14,17,22,28,29], chloroplast DNA [22,33,34], AFLP [35], RAPD [36], and ISSR [33,[36][37][38][39]. Inter-simple sequence repeat (ISSR) is a dominant molecular marker that allows the amplification of a large number of highly polymorphic loci [40], and have been used to evaluate genetic diversity and structure in many plant species [41][42][43], particularly mangrove species (see above for references).…”