A culture line of asparagus forming green bulbous structures consisting of numerous multiple bud clusters designated "bud clusters" was induced from a meristem culture of asparagus (Asparagus officinalis L.cv. Hiroshimagreen, 2n=30). Small cubic segments (2 mm3) cut from bud clusters were cryopreserved using three different cryogenic protocols. Only vitrification produced very high levels of shoot formation after cooling to -196°C. Segments were treated with a vitrification solution (PVS2) at 25°C for 45 min or at 0°C for 120 min prior to a direct plunge into liquid nitrogen. After rapid warming, the segments were expelled into Murashige and Skoog medium containing 1.2 M sucrose for 10 min and then plated on agar shoot outgrowth medium. The average rate of shoot formation of vitrified segments producing normal shoots was near 90% without any preculture and/or cold-acclimation treatment. Revived segments resumed growth within 3 days and developed about three shoots per segment. In vitro-cultured bud clusters appear promising as material for cryopreserving asparagus germplasm.
A micropropagation system for 'Yamatoimo' Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 laM benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2-3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2-3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 laM a-naphthaleneacetic acid (NAA), 0.89 p.M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3-7 mm diameter, were obtained.
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