Abstract
An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cuphea procumbens Orteg. using cotyledonary node explants excised from 15 days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at a concentration of 2.5 mu M was effective in inducing multiple shoots. The highest number of multiple shoots (9.33 +/- 0.60) and maximum average shoot length (4.16 +/- 0.44 cm) was standardized on MS medium supplemented with 2.5 mu M BA alongwith 0.5 mu M NAA. Addition of 200 mg/l Casein hydrolysate (CH) to the shoot induction medium enhanced the growth of regenerants. Rooting of in vitro regenerated shoots was best achieved on 1/2 strength MS medium. The in vitro raised plantlets with well developed shoots and roots were hardened, successfully established in earthen pots containing garden soil and maintained in greenhouse with 80% survival rate. Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate the genetic stability among in vitro regenerated progenies. All RAPD profiles from the micropropagated plants were monomorphic and similar to control plant. These results suggests that the culture conditions used for the axillary bud proliferation are appropriate for clonal propagation of this medicinally important plant as they do not appear to interfere with genetic integrity of in vitro regenerated plants. The described method can be successfully employed for large-scale multiplication and in vitro conservation of C. procumbens.