Micropropagation of Zingiber rubens and assessment of genetic stability through RAPD and ISSR markers

Abstract

Protocol was developed for high frequency in vitro multiplication of an endemic species, Zingiber rubens Roxb. The sprouted buds of the rhizomes were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 0.5-5.0 mg dm-3), indole-3-acetic acid (IAA; 0.5-2.0 mg dm-3), kinetin (KIN; 1.0-3.0 mg dm-3), naphthaleneacetic acid (NAA; 0.5-1.0 mg dm-3) and adenine sulphate (ADS; 80-100 mg dm-3). MS basal medium supplemented with 3 mg dm-3 BA and 0.5 mg dm-3 IAA was optimum for shoot elongation. The elongated shoots (1-2 cm) were transferred to multiplication medium containing 2 mg dm-3 BA, 1 mg dm-3 IAA and 100 mg dm-3 ADS. The multiplication rate remained unchanged in subsequent subcultures. Upon ex vitro transfer, 85 % of plants survived. Genetic stability of micropropagated clones were periodically evaluated at an interval of 6 months up to 30 months in culture using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis and genetic uniformity in all regenerants was confirmed.