Somatic Embryogenesis of Phalaenopsis

Abstract

The growth and differentiation of a unique Phalaenopsis callus was characterized. The callus was determined to be friable and embryogenic producing plantlets which matured and flowered. In contrast to other orchid calli reported previously, the callus was unique in its extreme friability and undifferentiation with no trace of organization. The callus was maintained on a modified Vacin and Went medium containing sucrose 20.0 g*liter-1 supplemented with coconut water (CW) 150 ml-liter-1 without any morphological change for more than five years. Analyses of callus growth (fresh weight) showed that the callus had an extremely low growth rate, which was not affected by sucrose levels between 2.5 to 30.0 g-liter-1. Although CW stimulated callus growth, lAA (indole-3-acetic acid) or BA (benzyl adenine) was not effective. Somatic embryogenesis was induced by lowering sucrose level to 2.5 g*liter-1 or deletion of sucrose and supplementing with CW. Callus turned green and subsequently formed numerous embryoids at periphery of green callus. The development of embryoids into plantlets morphologically resembled germination of zygotic embryos of Phalaenopsis. Sucrose (20.0 g-liter-1) inhibited greening and somatic embryogenesis. Sugar inhibition of greening and somatic embryogenesis was not overcome by transfer of callus from auxin-enriched medium to auxin-free medium or increasing nitrogen level, but only by reduction of carbohydrates available in medium. Pretreatment with lAA or NAA (a-naphthaleneacetic acid), and supplement of CW, casein hydrolysate (CH), glutamic acid or glutamine with 2.5 g-liter-1 of sucrose were marginally effective in promoting embryogenesis. Readily utilizable monosaccharides, glucose, fructose, mannose, ribose, xylose and disaccharide, sucrose at 20.0 g-liter-1 were inhibitory, while other disaccharides, multisaccharides and sugar-alcohols supported greening and somatic embryogenesis. Amylose (20.0 g-liter-1) was the most effective in induction of embryogenesis among carbohydrate sources. Regeneration of plantlets through indirect somatic embryogenesis is shown to be a viable pathway for clonal propagation of recalcitrant Phalaenopsis orchids.