Abcisic acid (ABA) and osmoticum play important roles in cotyledonary embryo development. ABA inhibits cleavage polyem-bryony and allows embryos to separate and develop further.9 However, ABA alone could not inhibit precocious germination of cotyle-donary embryos; increased osmolality of the medium was found to be necessary for embryos to mature. ESM cultures were settled in a measuring cylinder for 30 minutes. Supernatant was discarded and 1.0 ml settled ESM culture was transferred onto pads imbibed in liquid medium. Good quality cotyledonary embryos were produced by combining activated charcoal, ABA, GA4/7, and osmoticum.10 Polyethylene glycol (PEG), MW 4,000-8,000, proved to be the best osmoticant for embryo maturation.11 Fifty to one hundred cotyledonary embryos were produced per ml of settled ESM suspension culture on development medium (Fig. 29.1). The number and quality of cotyledonary embryos produced varied considerably among genotypes.
For germination, good quality cotyle-donary embryos were selected individually (as looking similar to zygotic embryos) by hand from development medium under the stereo microscope. Selected embryos were transferred onto semi-solid medium for germination. Culture plates were incubated the first 5-7 days in the dark, followed by transfer to light. Percentage germination varied considerably among genotypes. Germinated embryos bearing an epicotyl were then selected by hand and transplanted into 10 cubic inch Supercell pots containing a mixture of peat, vermiculite and perlite, and incubated in the greenhouse with frequent misting for acclimatization and growth. After establishment in soil in the greenhouse, somatic seedlings were grown in the nursery for one year before transplanting to forest regeneration sites.
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