Adventitious shoot regeneration of oriental lily (Lilium orientalis) and genetic stability evaluation based on ISSR marker variation

Abstract

A regeneration system was developed for oriental lily (Lilium orientalis) based on both leaf and bulb scale. Adventitious shoots were regenerated from leaves of in vitro cultures on Murashige and Skoog medium containing thidiazuron (TDZ) or 6-benzylaminopurine (BA) and naphthaleneacetic acid (NAA). The highest percent regeneration from leaf explants was 74.2%, being observed on medium containing 10.8 μM TDZ and 0.54 μM NAA. The highest mean number of shoots generated was 4.4 and was obtained from bulb scale explants on medium containing 0.54 μM TDZ and 0.54 μM NAA. Adventitious shoots were successfully rooted at rates ranging from 79.2% to 100%. The rooted plantlets survived after acclimatization in the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated, a value of 100 mg l−1 being suggested as a lethal dose for lily transformation. Eighteen ISSR markers were employed to determine the genetic stability of the regenerated shoots in comparison to their mother plant. Eleven primers in total produced 70 clear and reproducible bands. Genetic similarity indicators among the clonal derivatives and the mother plant ranged from 0.92 to 1.0. All 15 micropropagated progenies and the mother plant could be grouped together in one major cluster with a similarity level of 92%. The somaclonal variation rate across the plantlets was estimated as 4.2%, indicating that direct shoot formation from explant regeneration is a safe method for multiplication of “true-to-type” plants.