Morphological Hormonal and Genetical Analyses of Early in Vitro Flowering in Dendrobium Chao Praya Smile

Abstract

Dendrobium Chao Praya Smile was induced to flower in two-layer medium within 6 months from seed germination using BA. Besides, the functionality of the in vitro-developed flowers was verified through sporad analysis and pollen grains germination tests. More importantly, the in vitro-developed flowers were able to form seedpods and produce viable seeds upon self-pollination. With successful seed production in culture, the plantlets had completed a life cycle entirely in vitro in a time span as short as 11 months, approximately one-third of the time in field-grown plants. Histological analysis revealed that floral transition, as indicated by bolting, in D. Chao Praya Smile took place at 54 days after being grown in BA-containing liquid medium. While floral buds were developed in plantlets after being transferred to two-layer medium. During floral transition, the expression of DCPSknox, a gene involved in maintaining the indeterminacy of shoot apical meristem, was found to decrease. On the other hand, segregation of colors was observed in in vitro-developed flowers in which 4 types of flowers with different intensity of pink coloration were produced. It was suggested that color segregation was naturally occurring as BA treatment has not affected the expression of DCPSCHS, a key gene involved in anthocyanins biosynthesis, in the plantlets. Besides, one-third of the flowers produced in vitro was found to be incomplete with missing or defective floral organs. Using HPLC-ESI-MS/MS, changes in cytokinins and IAA contents were analyzed in flowering-induced D. Chao Praya Smile at different growth stages as well as in different tissues during floral transition. It was found that iPR was significantly increased in the plantlet and shoot apex at floral transition. Furthermore, higher cytokinins/IAA ratios were also exhibited in the plantlet and shoot apex at this vegetative-to-flowering transitional stage. Hence, it was proposed that the cytokinin/IAA ratio, and not the absolute amount of cytokinins, determined flowering in D. Chao Praya Smile. The inductive and inhibitory effects of iPR and IAA, respectively, on the flowering in D. Chao Praya Smile have also been verified. DCPSCKX, a gene involved in cytokinin homeostasis, was cloned and its expression was found to be strongly stimulated by BA treatment. Finally, a model of mechanisms underlying the BA-induction of flowering in D. Chao Praya Smile was proposed.