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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

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 -Ben-zyladenine (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 combina-tion 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 dendro-gram 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. Keywords Cytokinin . Fidelity . ISSR . Micropropagation . RAPD . Spilanthes calva DC. Abbreviations 2iP N 6 -(2-isopentenyl) adenine BA N 6 -benzyladenine CTAB Cetyl trimethyl ammonium bromide DMRT Duncan's multiple range test IAA Indole-3-acetic acid IBA Indole-3-butyric acid ISSR Inter simple sequence repeat Kn 6-furfuryl aminopurine MS Murashige and Skoog NAA α-naphthalene acetic acid NTSYS Numerical taxonomy and multivariate analysis system RAPD Random amplified polymorphic DNA SAHN Sequential agglomerative hierarchical and nested clustering method program in NTSYS SIMQUAL Similarity for qualitative data program in NTSYS SPSS Statistical package for social sciences UPGMA Unweighted pair group method with arithmetic mean Introduction

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