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

DOI: 10.1007/s12298-012-0152-4


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. N6-Benzyladenine (BA), N6-(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. © 2012 Prof. H.S. Srivastava Foundation for Science and Society.

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    • "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. "
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    ABSTRACT: Rumex thyrsiflorus Fingerh. is one of the few dioecious plant species that have sex chromosomes. The chromosome constitution of females is 2n = 12A + XX and 2n = 12A + XY1Y2 of males. It is a medicinally important plant species and has also been the object of studies on the structure and function of sex chromosomes and sex ratio. An efficient plant regeneration protocol was developed from karyologically stable male roots that had been derived from a long-term liquid culture. The root segments were grown on MS medium supplemented with the following plant growth regulators: 2.4-D, NAA, kinetin, BAP and TDZ. The highest frequency (81.73 %) of adventitious shoot formation (16.27 shoots/explant) was obtained on MS + 0.5 mg/l TDZ. Regenerated shoots were successfully rooted on ½ MS + 2 % sucrose + 0.5 mg/l IBA and acclimated to in vivo conditions. Histological analysis revealed indirect (via callus) adventitious shoot formation. The cells of the morphogenetic callus were surrounded by a fibrillar structure that was similar to the extracellular matrix. Molecular analysis based on genetic sex markers confirmed that all of the root explants were male. The genetic stability of the regenerated plantlets was confirmed using random amplified polymorphic DNA analysis. This is the first report concerning the micropropagation protocol for R. thyrsiflorus Fingerh. from male roots derived from a long-term liquid culture, which offers a unique opportunity to obtain true-to-type plants of the same sex.
    Full-text · Article · Jul 2015 · Plant Cell Tissue and Organ Culture
    • "Acta Physiol Plant (2014) 36:1115–1122 1119 and both RAPD and ISSR markers are broadly being suggested by many researchers in different plants (Rawat et al. 2013; Razaq et al. 2013; Nayak et al. 2013; Singh et al. 2013). "
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