We developed a micropropagation protocol for Cleome gynandra, a C4 model plant with medicinal importance. Surface-sterilized nodal segments obtained from 1 to 2-month-old field grown plant were used as explants for culture establishment and plant regeneration. Multiple shoots differentiated through bud breaking on Murashige and Skoog (MS) medium with different concentrations of benzyladenine (BA) and kinetin (Kin). The optimum shoot differentiation occurred on medium with 1.5 mg l-1 BA. Out of various concentrations and combinations of cytokinins and auxins, MS medium containing 0.5 mg l-1 BA and 0.1 mg l-1 IAA (indole-3-acetic acid) was found best for shoot multiplication. However, the differentiated shoots exhibited hyperhydration, leaf curling and early leaf fall during subculturing. To overcome these problems, regenerated shoots were transferred to the modified MS medium with reduced nitrates (825 mg l-1 NH4NO3 and 950 mg l-1 KNO3) and 100 mg l-1 (NH4)2SO4. The micropropagated shoots were rooted (i) in vitro on one-fourth strength of MS salts with 0.25 mg l-1 each of IBA (indole-3 butyric acid) and NOA (2-naphthoxyacetic acid) ? 100 mg l-1 activated charcoal, and (ii) ex vitro, by treating the shoot base(s) with 200 mg l-1 of IBA for 3 min and transferred to soilrite moistened with one-fourth strength of MS macro salts in culture bottles. The plants were hardened in the greenhouse with 85 % survival rate. Micromorphological studies of the plants were conducted during hardening with reference to development and changes in vein spacing, glandular trichome and stomata. In comparison to leaves under in vitro condition, higher density of veins and glandular trichomes was observed in the leaves of hardened plants. In addition, stomata became functional during hardening which were non-functional under in vitro condition.
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