Controlling vitrification in Larixdecidua via culture media manipulation

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.