In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker

Abstract

An efficient in vitro protocol has been established for clonal propagation of elite plant of Spilanthes calva DC., an important source of spilanthol, an antimalarial larvicidal compound. Nodal explants excised from field grown plant of S. calva DC. when reared on Murashige and Skoog's medium augmented with different cytokinins, viz. N 6 -Benzyladenine (BA), N 6 -(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn), differentiated multiple shoots from the axils. BA at 10 μM proved optimum for elicitation of multiple shoots in 91.6 % cultures with an average of 7.12 shoots per explant within 6 weeks. The excised shoots rooted on half strength Murashige and Skoog's medium supplemented with 0.1 μM IBA. Micropropagated plants were hardened and transferred to field for acclimatization, where 95 % plants survived and were phenotypically similar to the donor plant. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic fidelity amongst the regenerants. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 71 scorable bands, ranging in size from 100 bp to 1,100 bp were generated by a combination of the two markers in the aforesaid plants. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant. The similarity values amongst the aforesaid plants varied from 0.967 to 1.000. The dendrogram generated through UPGMA (Unweighted Pair Group Method with arithmetic mean) analysis revealed 98 % similarity amongst them, thus confirming the genetic fidelity of the in vitro clones.

Male adventitious roots of Rumex thyrsiflorus Fingerh. as a source of genetically stable micropropagated plantlets

“…That explanation needs future studies and if confirmed could open up a new way for obtaining genetically stable material for biotechnological purposes. Recently, RAPD analysis was used as an efficient tool to evaluate the clonal fidelity of micropropagated plants in many systems and indicated that the pattern of monomeric bands that are observed in Ajuga bracteosa (Kaul et al 2013), Spilanthes calva (Razaq et al 2013) and Rhinacanthus nasutus (Cheruvathur and Thomas 2014) are in agreement with our observations. The plantlets regenerated from roots during our experiments exhibited normal morphological characters and no detectable variation was recorded in their morphology.…”

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

“…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].…”

Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC

“…Significance of BA in inducing multiple shoots has been already reported in Simmondsia chinensis (Link) Schneider (Agrawal et al 2002) and Spilanthes spp. (Razaq et al 2013). For rhizogenesis, IBA yielded the optimum response in terms of percentage root induction and average root number per shoot.…”

“…The banding profiles of micropropagated plants vis-a-vis that of mother plants shown by RAPD marker has clearly revealed almost complete uniformity and monomorphism thereby, indicating a high level of genetic similarity amongst the regenerants and the elite donor plant of C. angustifolia. The low level of genetic variation in DNA may be attributed to naturally occurring variation or due to the accumulation of mutation by various factors such as in vitro process and its duration, auxin to cytokinin ratio (hormonal balance), in vitro stress induced by added biochemicals, or other nutritional conditions (Razaq et al 2013;Singh et al 2013). The genetic variation induced by such genetic and epigenetic mechanism can be reflected in the banding profile developed by different marker system (Phillips et al 1994).…”