In vitro Propagation of Crassocephalum crepidioides—An Endangered African Traditional Leaf Vegetable and Molecular Analysis of Micropropagated Plants

Abstract

ABSTRACTCrassocephalum crepidioides (Benth) S. Moore is an important African traditional leaf vegetable, but its production is very low and its existence threatened. An efficient and reproducible micropropagation protocol of C. crepidioides was developed from nodal stem segments of pot-grown plants. Shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with benzineamino purine (BAP) alone and in combination with zeatin and naphthalene acetic acid (NAA). Elongated shoots successfully rooted on zeatin alone and in combination with indole butyric acid (IBA). Molecular stability of micropropagated plants was evaluated using random amplified polymorphic DNA (RAPD) markers. Survival of nodal segments in micropropagation medium was greater than 70% in all treatments. The presence of plant growth regulators (PGRs) in MS medium influenced shoot formation, rooting, and number of shoots and roots per explant. The best shoot formation frequency (88.7%), number of shoots per explant (3.7), and shoot length (30 mm) were from a medium containing MS supplemented with 10 mg·L−1 BAP, 15 mg·L−1 NAA, and 1.5 mg·L−1 zeatin. Frequency of root formation and survival of regenerated plants were greater on medium containing half-strength MS salt than medium fortified with full-strength MS salt. The best frequency of root formation (average 87.4%) and survival of regenerated plants (average 95.4%) were from media containing 6.0 mg·L−1 zeatin and 1.0–1.5 mg·L−1 IBA and half-strength MS salt. Micropropagated shoots with well-developed roots were acclimatized under greenhouse conditions in pots containing rich sandy loam soil (pH = 7.2). The RAPD amplification products were monomorphic in micropropagated plants and similar to those of the mother plant. No polymorphism was detected, indicating genetic uniformity of micropropagated plants. This is the first report of micropropagation of C. crepidioides, which will facilitate in vitro mass propagation, conservation, and germplasm exchange of this endangered African vegetable.