Protoplast isolation and plant regeneration from leaves of Rhodiola sachalinensis / 中草药

ABSTRACT

Objective To isolate protoplasts from tube plant leaves of Rhodiola sachalinensis and regenerate plantlets after protoplast culture.Methods Preculture treatment and size of explants,enzyme concentration,mannitol concentration in enzyme mixture related with protoplasts isolation were studied to determine the superior optimized conditions.Results Explants could be used to isolate protoplast without dark preculture,and leaf length should be longer than 1.5 cm.The best incubating enzyme solution contains 1.0% cellulase Onzuka R-10,0.5% Macerozyme R-10,10 mmol/L CaCl2?2H2O,0.1% MES,0.7 mmol/L KH2PO4,and 0.5 mol/L mannitol.The enzyme and explants mixture were shaken for 4 h at 25 ℃.The protoplasts yield and viability were 39.43?106/g fresh weight and 78.6%,respectively.Purified protoplasts were cultured in medium 1/2 MS+1 mg/L 2,4-D+0.5 mg/L ZT+0.5 mol/L mannitol +500 mg/L hydrolysis of casein initially with shallow liquid layers,and calli formed within 40 d.After calli were transfered to MS+1 mg/L 6-BA+0.1 mg/L NAA,adventitious buds were induced from calli.Shoots longer than 2 cm rooted within 30 d when they were transfered to 1/2 MS medium.Conclusion The study provides the scientific base for protoplast fusion in polyploidy breeding of R.sachalinensis.