Micropropagation of Tolumnia Orchids through Induction of Protocorm-like Bodies from Leaf Segments

Abstract

A protocol for plant regeneration via direct induction of protocorm-like bodies (PLBs) from leaf segments of Tolumnia Snow Fairy was developed as a basis for mass production. Ten-month-old, in vitro–grown donor plantlets were obtained by inducing shoots from buds on the flower stalk. Leaf segments harvested from plantlets of different heights and from expanding leaves at different positions were compared, as were two BA concentrations with 0.5 mg·L−1 NAA. The greatest rate of PLB induction (16.7%) was observed when leaf segments taken from 1- to 2-cm height plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 2 mg·L−1 BA and 0.5 mg·L−1 NAA after 16 weeks of culture. When using leaf explants, only inner, expanding leaves cultured on MS basal medium supplemented with 4 mg·L−1 BA and 0.5 mg·L−1 NAA resulted in PLB induction, at an average rate of 25.5 PLBs per explant. After 16 weeks of culture, histological and scanning electron microscopy (SEM) observations revealed that PLBs originated from epidermal cells of leaf explants. PLBs of 1 to ≤2 mm in diameter continued to proliferate after 4 weeks of culture. These secondary PLBs could be produced from either whole PLBs or the upper side of PLBs. Finally, PLBs were regenerated into plantlets. After ≈14 months of culture, fully developed plants exhibiting well-developed roots and shoots were acclimatized. These plants grew well, with 1-year survival rates of nearly 73%, for plants originating as explants taken from 1- to 2-cm tall plants, and of 79%, for plants originating as explants taken from inner leaves. Some mature plants flowered 1 year after transplantation. This study presents a simple system that can provide a large number of PLBs for mass propagation in a short time that can be converted into plants and also used for the new cultivars of Tolumnia orchids.