Cypripedium formosanum Hayata is a rare slipper orchid species endemic to Taiwan that has a pinkish single flower above fan-shaped leaves at the stem apex (Cribb, 1997; Lin, 1987). C. formosanum is one of the popular potted and perennial gardening plants. Usually, the rhizomes with dormant flower buds are collected in the winter, and buds bloom after bud breaking from dormancy. However, conventionally propagation by vegetative division of C. formosanum is time-consuming, because the rhizome usually produces only one shoot bud yearly. Therefore, a rapid, reliable protocol for propagation is required. Nevertheless, attempts to culture the slipper orchids (Cypripedium Linnaeus, Paphiopedilum Pfitzer, Phragmipedium Rolfe, and Selenipedium Rchb. F.) in vitro are still challenging (Arditti and Ernst, 1993; Stewart and Button, 1975). Recently, plant regeneration through seed-derived explants has been reported in Paphiopedilum (Chen et al., 2004; Huang et al., 2001; Lin et al., 2000) and Cypripedium (Lee and Lee, 2003; Shimura and Koda, 2004; Yan et al., 2006). In C. montanum Douglas ex Lindl., multiple shoots were successfully obtained from the seedling node cultures on the medium with 8.88 μM N6-benzyladenine (BA) and 1.07 μM 1-naphthaleneacetic acid (Hoshi et al., 1994). However, those seed-derived explants are not true-to-type and heterozygous.
It has been reported that the initial success of an aseptic culture in the mature perennial plants is determined before the explant has been collected (Evers et al., 1988; Kartsonas and Papafotiou, 2007), suggesting that seasonal influence may affect the internal physiological state of explants. C. formosanum is an alpine plant, and the rhizome comprises a chain buried in soil forming new growths at the anterior end during April every year. It is not clear whether the seasonal influence of explant collection plays an important role in establishing the aseptic culture. Therefore, the objective of this study was to develop an efficient method for micropropagation of C. formosanum using explants from adult plants. The effects of timing for explant collection, BA and thidiazuron (TDZ) concentrations on shoot multiplication, and organic supplements on plantlet rooting were investigated.
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