Plant regeneration from petiole callus of Amorphophallus albus and analysis of somaclonal variation of regenerated plants by RAPD and ISSR markers

摘要

A simple procedure has been outlined for plant regeneration of Amorphophallus albus Liu & Wei, a native medicinal plant in China, from petiole-derived callus. Calli were induced at a high frequency of 76.4±3.2% from petiole explants excised from two-month-old plants on Murashige and Skoog (MS) medium supplemented with 5.37 mu M a-naphthaleneacetic acid (NAA) and 4.44 uM 6-benzyladenine (BA). Of the different types of callus induced, type III callus was selected for morphogenesis induction. Culture of the callus on MS medium containing proper NAA and BA or KT combinations resulted in formation of corm-like structure (CLS) that produced shoots and roots during further culture. The optimal morphogenetic response was observed on the media with a cytokinin/auxin ratio of about 4:1, which resulted in more than 70% CLS formation and 6~8 CLSs per callus. Complete plantlets with well-developed root systems were obtained from these CLSs by subculturing them on the original media from which they had been derived without a separate rooting culture. Transfer of the plantlets with roots to soil resulted in a more than 90% survival rate. Analysis of 20 regenerated plants by two molecular markers, randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), revealed somaclonal variation in the regenerated plants. The percentage of polymorphic bands in RAPD and ISSR analysis were respectively 20.8% and 39.0% for the 20 plants. Cluster analysis indicated that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 21 plants (20 regenerated and one donor plant) were, respectively, 0.973 and 0.917, which allowed classification of the plants into distinct groups. A high-frequency somaclonal variation induced in A.albus tissue culture may help in the selection of useful variants that may be induced to improve this important corp.