Establishment of high frequency plant regeneration system from leaf explants of Pinellia koreana via bulblets formation

Abstract

Pinellia koreana K-H Tae & J-H Kim is a recently discovered Korea endemic medicinal plant species whose natural habitat is rapidly destroyed by industrial development. Described in this paper are culture conditions for high frequency plant regeneration via bulblet formation from leaf explant cultures of P. koreana. Leaf explants formed white nodular structures and off-white calluses at a frequency of 91.2% when cultured on MS medium supplemented with 2 mg/L BA and 0.5 mg/L NAA. However, the frequency of white nodular structures and off-white calluses formation was slightly decreased with an increasing concentration of NAA up to 4 mg/L, where the frequency reached 31.7%. Most petiole explants did not form white nodular structures and off-white calluses except the combination treatment of 2 mg/L BA and 2 mg/L NAA. Upon transfer onto MS basal medium, over 90% of nodular structures gave rise to numerous bulblets and developed into plantlets. Plantlets regenerated from bulblets were transplanted to potting soil and grown to maturity at a survival rate of over 95% in a growth chamber. Therefore, the in vitro plant regeneration system of P. koreana obtained in this study will be useful for mass propagation and long-term preservation of genetic resources of P. koreana. *Corresponding author Tel 042-860-4646 Fax 042-860-4677 E-mail: kimsw@kribb.re.kr Introduction Pinellia koreana K-H Tae & J-H Kim is a new plant species discovered at Chrisan in Korea (Tae and Kim, 2005). This plant differs from other plants belonging to the same genus Pinellia by having 3-foliolate leaves with bilobed lateral leaflets at maturity. The genus Pinellia is herbaceous medicinal plants belonging to the Araceae. The chemical composition of Pinellia is not well established as there are only a few reports on the occurrence of small amounts of alkaloids ephedrine in the tuber. Also a glycoprotein fraction was reported to have notable antiemetic effects (Kurata et al. 1998). And root contains toxins (presence of calcium oxylate), that are destroyed by drying or cooking. Pinellia is mostly used for indigestion, nausea, vomiting, gastritis, and ulcer (Lee and Cho 1987; Bown 1995). The genus of Pinellia consists of a few species in eastern Asia (Kitamura et al. 1980). Pinellia is propagated by tubers and bulbils. However, the yield and quality of tubers are gradually reduced over cultivation because of viral diseases. Therefore, introduction of a mass propagation system using in vitro culture technology such as development of virus-free strains and genetic manipulation of tubers is necessary to enhance the productivity of tubers. Despite its medicinal importance, few scientific studies of P. koreana have been reported. Only several in vitro culture studies have reported on the related species P. ternata including protoplast culture and plant regeneration (He et al. 1996), formation of micro-tubers and plantlet regeneration by thidiazuron (Yoo and Lim 1997), and clonal propagation by bulbils (Chen et al. 1989; Tsay et al. 1989). Also natural habitats of P. koreana decreased gradually because of industrialization. Tissue culture techniques may provide an alternative mean for its mass multiplication and ex-situ conservation of genetic resources. This study describes culture conditions for high frequency plant regeneration via bulblet formation from leaf explants cultures of P. koreana. 식물생명공학회지 제36권 제2호 J Plant Biotechnol Vol. 36, No. 2, 193-196 (2009) 194 ‧ Journal of Plant Biotechnology Figure 1. Plant regeneration from leaf explants of P. koreana via bulblets formation. A: Leaf explants; B: Off-white callus formation; C: White nodular structure formation; D: Bulblets formation from leaf-derived callus; E: Leaf and root development from bulblets; F: Regenerated plantlets transferred to soil; G: Morphological variation in leaf shape of regenerated plantlets; H: Morphological variation of plants after 2 year cultivation; I: Flowering of mature plants. Scale bars represents 1 mm (A, B, C, D and E), 2 cm (F, H) and 5 cm (G, I), respectively Materials and methods