Analysis of genetic stability through intersimple sequence repeats molecular markers in micropropagated plantlets of Anoectochilus formosanus Hayata, a medicinal plant.

Anoectochilus formosanus HAYATA, commonly known as "Jewel Orchids," which has been used as Chinese folk medicines, is being subjected to a huge crisis, whose wild resources have gradually become more and more scarce. Hence, micropropagation protocol by axillary branching established for A. formosanus was employed for large-scale commercial production. In this study, due to the somaclonal variation and on the basis of some virtue of intersimple sequence repeats (ISSR) analysis, we firstly utilized the ISSR primers to investigate the genetic stability of A. formosanus propagated in vitro for a period of more than 5 years. Among the total 100 bands amplified by 17 ISSR markers, 77 bands were distributed in size from 500 bp to 1.5 kbp, while only 5 bands were beyond 1.8 kbp in size. Meanwhile, according to the cluster analysis, genetic similarity was more than 94% and the polymorphism rate was only 2.76% among the total 1810 scorable bands. All results demonstrate A. formosanus, multiplied by axillary branching, maintained high genetic fidelity even after a period of more than 5 years under in vitro propagation with only a low risk of genetic instability. The results from this study provide an important basis for giving evidence of genetic stability of A. formosanus before micropropagation for large-scale commercial production.