Dendrobium flower color : histology and genetic manipulation

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003.Includes bibliographical references (leaves 148-153).Mode of access: World Wide Web.Also available by subscription via World Wide Webxix, 153 leaves, bound ill. 29 cmDendrobium is the most important cut flower orchid in the world. Understanding the chemical, histological and molecular aspects of flower color is crucial for the development of breeding strategies for novel colors. The objectives of this research were to examine the histology of flower color, cloning and characterization of flavonoid biosynthetic genes, and metabolic engineering of Dendrobium flavonoid pathway to obtain new colors. In Dendrobium, anthocyanins can be confined to a single layer of cells (epidermal or suepidermal) in pale flowers. More intensely colored flowers had anthocyanin in several cell layers. Striped patterns on the perianth were due to the restriction of pigment to cells surrounding the vascular bundles. Color perception is markedly influenced by the presence or absence of carotenoids. Four types of epidermal cells were found in Dendrobium: flat, dome, elongated dome, and papillate. Epidermal cell shape and cell packing in the mesophyll affected the visual texture. Perianth parts with flat cells and a tightly packed mesophyll had a glossy texture, whereas dome cells and loosely packed mesophyll contributed a velvety texture. The labella in the majority of flowers examined had a complex epidermis with more than one epidermal cell shape, predominantly papillate epidermal cells. We were able to isolate a full clone of Dendrobium dihydroflavonol 4-reductase (dfr), and partial clones of chalcone synthase (chs), flavonoid 3'-hydroxylase (f3'h) and flavonoid 3', 5'-hydroxylase (f3'5'h), from Dendrobium Jaquelyn Thomas 'Uniwai Prince' (UH503). Expression data indicated that dfr and chs were expressed to the greatest degree in unopened buds. Amount of f3'h and f3'5'h mRNA was too small to detect. Southern analysis has shown that f3'h and f3'5'h is represented by 2 copies each in UH503. These clones will be extremely useful in future for flower color manipulation. Two different color genes, dfr and f3'5'h from two non-orchid plants, under the constitutive promoter ubiquitin3, were inserted into Dendrobium Icy Pink 'Sakura' with the intention of creating orange-red and blue shades, which are absent in commercial Dendrobium. Presence of the transgene in two sets of transformants was confirmed by PCR. Expression of the transgene from a few plants was indicated by RT-PCR and northern analyses