Evaluation of physiochemical factors affecting high-frequency plant regeneration of Blyttia spiralis (synonym: Pentatropis spiralis ), a threatened climber of medicinal value

Blyttia spiralis (Forssk.) D.V.Field & J.R.I.Wood, a threatened and medicinally important plant, belongs to the family Apocynaceae. Widespread habitat destruction has adversely affected natural populations of this species. This paper reports the first efficient plant propagation system for B. spiralis using nodal segments as explants. The greatest number of shoots was obtained on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP; 4.0 mg L−1) through bud breaking. Shoots were further multiplied through repetitive transfer of original explants and by subculturing of in vitro-raised shoots. Key physiochemical factors affecting various stages of micropropagation were systematically tested. The maximum number of shoots (35.45 ± 2.64), with a mean length of 6.69 ± 1.03 cm, was achieved on 0.8% agar-gelled shoot proliferation medium (SPM: MS + 0.5 mg L−1 each of BAP and kinetin [Kin] + 0.1 mg L−1 α-naphthaleneacetic acid [NAA]), with a combination of 50 mg L−1 ascorbic acid; 25 mg L−1 each of citric acid, l-arginine, and adenine sulfate; and 150 mg L−1 ammonium sulfate and 100 mg L−1 activated charcoal (AC) in culture bottles. In tests of rooting conditions, 61.5% of the shoots rooted in vitro on ¼-strength MS medium containing 2.0 mg L−1 indole-3-butyric acid (IBA) and 200 mg L−1 AC; 89.4% of the shoots rooted ex vitro in Soilrite®, if the shoot bases were pretreated with 250 mg L−1 IBA for 5 min. Plantlets rooted by both procedures were gradually acclimatized in a greenhouse. About 85% of ex vitro-rooted and 55% of in vitro-rooted plantlets were successfully established in soil. The described method can be employed for the large-scale propagation and conservation of Blyttia spiralis.