Rapid micropropagation of monopodial orchid hybrid (Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.) through direct induction of protocorm-like bodies from leaf segments

Abstract

A competent protocol for accelerated plant regeneration system via direct induction of protocorm-like bodies (PLBs) from leaf of orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl. was developed for the first time to establish a basis for mass production. Using tissue culture technique, the conditions for PLB induction from leaf explants and conversion of PLBs into plantlets were investigated. Leaves were transferred to MS medium containing different types and concentrations of cytokinins (namely, N6-benzyladenine, 6-furfurylaminopurine, N-phenyl-N′-(1,2,3-thidiazol-5-yl)urea/TDZ and zeatin) for PLB induction. By means of exploring the effects of cytokinins, it was determined that the optimum PLB induction occurred on MS media supplemented with 1.5 mg l−1 TDZ; whereby accordingly, PLB induction (with a frequency of 94.8 %) was observed as early as 8 days of culture and an average of 25 PLBs was obtained from 1 cm2 leaf segment after 40 days of culture. Variable pressure scanning electron microscopy indicated the different developmental stages of PLBs in detail. Light/stereo microscopic observation showed the maturation of PLBs and gradual formation of shoot and leaf primordia. More than 96 % conversion (with well-developed shoots and roots) was achieved within the next 30 days of culture, when well developed PLBs were transferred in MS medium supplemented with 1 mg l−1 BA, 0.5 mg l−1 IBA plus 60 mg l−1 adenine sulphate. After 60 days of transfer in plastic pots filled with sand and perlite (2:1; v/v) and with charcoal and coconut fibre (1:1; v/v), subsequently, 90 % well-acclimatized plantlets were recovered.