Culture initiation, medium composit

Culture initiation, medium composition and tissue culture conditions

Merwilla plumbea seeds were germinated in vitro on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962). Seeds were obtained from the University of KwaZulu-Natal Botanical Garden (Pietermaritzburg, South Africa) and decontaminated as described previously (Baskaran et al., 2012). Excised leaf explants of approximately 1 cm by 1 cm were inoculated on 10 mL of onetenth strength MS medium supplemented with 30 g L−1 sucrose and dispensed in culture tubes (100 mm×25 mm, 40 mL volume). Apart from control medium which was devoid of any CK, 1 M of five different CK treatments (2iP, BA, mT, mTR, MemTTHP) in acompletely randomized experiment were tested. The control and the CK-supplemented media were adjusted to pH 5.8 with either 0.1 M KOH or HCl. After the addition of 8 g L−1 agar for solidification, the media were autoclaved at 103 kPa and 121 ◦C for 20 min.Cultures were incubated in a growth room at 25±2 ◦C with 16 hlight/8 h dark conditions and photosynthetic photon flux density (PPFD) of 40–45 mol m−2 s−1 provided by cool white fluorescent tubes (Osram® L58W/640, Germany). After 12 weeks culture,plantlets from each treatment including the control were divided into two groups; the first group was harvested and separated into aerial (shoots and leaves) and underground (bulbs and roots) sections while the second group was acclimatized ex vitro.