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.
    Plant Cell Tissue and Organ Culture 07/2015; 123(1). DOI:10.1007/s11240-015-0826-z · 2.13 Impact Factor
    • "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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    ABSTRACT: An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l(-1)) and produced (4.56 +/- A 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0-2.0 mg l(-1)) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05-0.4 mg l(-1)). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l(-1)) and IAA (0.1 mg l(-1)) formed the maximum number of shoots (68.40 +/- A 2.74 per culture bottle) with an average height (6.59 +/- A 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 +/- A 0.64 roots per shoot and an average length of 2.76 +/- A 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.
    Acta Physiologiae Plantarum 05/2014; 36:1115-1122. DOI:10.1007/s11738-014-1486-z · 1.58 Impact Factor
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    ABSTRACT: An efficient in vitro protocol for large-scale multiplication of Nepenthes khasiana, a threatened insectivorous plant of India, has been developed from nodal stem segments. The highest shoot proliferation of 19.16 ± 0.23 shoots/explant was recorded in half-strength Murashige and Skoog (MS) medium supplemented with 2.5 mg/l kinetin, 2.0 mg/l 6-benzyl aminopurine, 3 % sucrose and 0.8 % agar. The best rooting was achieved in half-strength MS medium supplemented with 2.0 mg/l α-naphthalene acetic acid with an average of 9.04 ± 0.46 roots/shoot. The plantlets were successfully transferred to the greenhouse with survival rate of 92 %, exhibiting normal development. Cytological and random amplified polymorphic DNA (RAPD) analyses were carried out to assess the genetic integrity of the regenerated plantlets. Cytological analysis revealed no change in chromosome number with cells studied showing 2n = 80. Of the 80 primers screened for RAPD analysis, 14 primers resulted in clear and scorable bands. A total of 72 amplification products were obtained out of which only 4.1 % bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient ranging from 0.98 to 1.0, thus suggesting genetic stability in the micropropagated plants of N. khasiana.
    Acta Physiologiae Plantarum 09/2013; 35(9). DOI:10.1007/s11738-013-1314-x · 1.58 Impact Factor
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