Analysis of Genetic Stability through Intersimple Sequence Repeats Molecular Markers in Micropropagated Plantlets of Anoectochilus formosanus HAYATA, a Medicinal Plant

Abstract

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.

Evaluation of genetic variability in in vitro sodium azide-induced Dendrobium ‘Earsakul’ mutants

“…Similarly, mutants or somaclonal variants have been successfully identified using ISSR analysis in several plants including B. rapa subspecies yellow sarson and A. formosanus (Zhang et al, 2010;Kumar et al, 2011).…”

“…Mutations resulting from deletions/duplications or single nucleotide changes at the primer binding sites can be identified. In orchids, ISSRs have been utilized to assess the level of genetic diversity in cultivated spring orchid (Cymbidium goeringii), to estimate genetic stability of micropropagated jewel orchid (Anoectochilus formosanus), and to determine the phylogenetic relationship of Dendrobium species (Wang et al, 2009;Zhang et al, 2010). In this study, we evaluated the effectiveness of ISSRs in detecting and fingerprinting Dendrobium 'Earsakul' mutants induced by in vitro mutagenesis of protocorm-like bodies (PLBs) using different concentrations of NaN 3 , and we assessed the genetic variability of these mutants.…”

Promoting role of an endophyte on the growth and contents of kinsenosides and flavonoids of Anoectochilus formosanus Hayata, a rare and threatened medicinal Orchidaceae plant

“…In a previous study, we used a micro-propagation technique for the large-scale production of A. formosanus to protect the wild population of this rare and threatened orchid species. The results demonstrate that A. formosanus maintains high genetic fidelity even after a 5-year propagation in vitro (Zhang et al, 2010).…”

“…After surface sterilization, aboveground parts of A. formosanus samples collected from Fujian Province, China, were cut into small pieces of stems with an axillary bud about 1 cm, and cultured in sterile micropropagation medium (MM) containing MS (Murashige and Skoog, 1962) basic medium and supplemented with 0.5 mg/L naphthaleneacetic acid (NAA), 1 mg/L 6-benzyl adenine (6BA), 1 g/L activated charcoal, and 30 g/L sucrose. The medium was solidified with 10 g/L agar and then incubated at (252) °C under a 12-h photoperiod and a photosynthetic photon flux density of 50 μmol/(m 2 ·s) (Zhang et al, 2010). After four months of micropropagation in vitro, the aseptic in vitro micropropagated plants of A. formosanus were transferred to fresh MM without NAA and 6BA and cultured for another two weeks.…”

“…Its wild sources are gradually decreasing (Zhang et al, 2010). Consequently, the prices of its fresh and dry states have increased to approximately CNY 320 and 3 200 per kilogram, respectively (Shiau et al, 2002), thereby limiting its clinical application.…”