Abstract
Vitrification rates obtained from our inverted embryo system were significantly decreased by lowering the cytokinin concentrations in Brown and Lawrence medium containing 10 mM L-glutamine from 10 to 1 mg/L, or by replacing 10 mM L-glutamine with equimolar concentrations of Ca(NO3)2 or by adding 1 g/L Gelrite to the normal 10 g/L Difco–Bacto agar in the culture media. In all treatments, decreased vitrification was accompanied with decreased adventitious bud production. With reduced N6-benzylaminopurine, vitrification decreased from 77 to 29%, but bud production decreased from 61 to 17 buds per explant and mortality increased from 3 to 33%. Incorporation of Ca(NO3)2 into the media decreased vitrification from 87 to 21%, but the number of adventitious buds per embryo decreased from 75 to 42. Vitreous shoots were reverted to normal development with an 81% reversion frequency and a 6% mortality rate by culturing these shoots on Gresshof and Doy medium with one-half total nitrogen. Elongation of previously vitreous shoots was best when these shoots were cultured on Gresshof and Doy medium + 0.5 mg/L N6-benzylaminopurine for 2 weeks, followed by subculture on Gresshof of and Doy medium + 10 g/L charcoal.
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