In Vitro Micropropagation of 'Yukiwariso' (Hepatica nobilis Schreber var. japonica f. magna) by Leaf Segment Culture

Abstract

This study was conducted to establish a system of in vitro micropropagation of Hepatica nobilis. Leaf segments, excised from commercial plants, formed calli at high frequency in MS medium that contained 0.1-10 mg·liter^-1 NAA and BA. The highest percentage of bud formation was observed to be about 20% in the medium supplemented with 0.5 or 1 mg·liter^-1 NAA and 5 mg·liter^-1 BA. Embryoid formation was stimulated on the medium containing 0.1 mg·liter^-1 NAA and 0.5 mg·liter^-1 BA, or 1 mg·liter^-1 NAA and BA. When 1 to 2 mm long adventitious buds were excised from leaf segments and recultured every 8 weeks on MS medium supplemented with BA, GA₃ or both for 24 weeks, they developed into shoots (referred to as the primary shoots in this text). The survival rates of the primary shoots were higher in the medium containing both BA and GA₃ than in the medium with BA alone. When primary shoots were cultured on MS medium containing 5 mg·liter BA and 10 mg·liter^-1 GAs, they formed the highest number of new shoots, referred to as secondary shoots in the text. When excised secondary shoots were cultured in MS medium containing 10 mg·liter^-1 NAA or IBA in the dark for a week and then transplanted into MS-medium without any hormones, they developed roots at a high frequency, whereas shoots transplanted to MS-medium with NAA or IBA formed only callus at the base. Following acclimatization, rooted plants that were transplanted into pots and grown in a glasshouse, flowered within 2 years.