IN VITRO PRODUCTION OF SEEDS BY ENCAPSULATION OF ASEXUAL EMBRYOS

Asexual embryos of exalbuminous seed species might be transformed into artificial seed if they could be encapsulated with a synthetic seed coat that would maintain viability and permit germination with normal seedling development. The objective of this study was to investigate the feasibility of producing artificial seed by coating asexual embryos of carrot (Daucus carota L.). An assay using citrus embryos (coats removed from true seed) was developed to test synthetic coating candidates. Preliminary experiments using citrus embryos indicated the suitability of Polyox WSR-N 750 (Union Carbide) as a synthetic coating compound. Cell cultures of carrot were maintained in basal medium containing 0.1 mg/liter 2,4-D and 0.2 mg/liter kinetin. Embryos were initiated and developed by inoculating cell suspension into hormone-free medium and reculturing to fresh medium every 3 days during a 2-week induction-growth cycle. Artificial seed coats were applied to asexual embryos of carrot by mixing embryo suspension (size 0.15 - 1.0 mm) with Polyox (5% w/v) and dispensing 0.2 ml drops on sterilized teflon sheets in a laminar flow hood. Dried drops formed detachable wafers. Wafers were redissolved in fresh medium and the seed coat material was removed by washing. The rehydrated embryo suspension was plated on filter paper supports in Petri dishes and grown for 2-3 weeks. When dried to constant weight, a low percentage ((LESSTHEQ)4%) of asexual carrot embryos coated with 2.5% Polyox survived encapsulation, but survival of uncoated embryos was essentially nil. Hardening pretreatments applied to cultures of asexual embryos 10(\u27-6) M abscisic acid (ABA), a high inoculum density of 0.8 g/25ml, 12% sucrose, or chilling at 4(DEGREES)C for the last 3 days of an induction-growth phase all increased survival of encapsulated embryos. The pretreatment combinations tested (ABA + high inoculum density, ABA + high sucrose, ABA + chilling) resulted in lower embryo survival than would be expected if the effects were additive. Polyox-coated embryos pretreated with ABA plus chilling and stored in darkness survived up to 16 days at 4(DEGREES)C vs. 4 days at 26(DEGREES)C. Encapsulated asexual embryos surviving rehydration developed into normal-appearing plantlets