Tissue Culture And Cryopreservation Shoot-tips In Vitro Of Duchesneae Indicae By Vitrification

Shoot-tips of wild Duchesneae Indicae which came from Baotianman in Henan were used as explants in this research in order to establishing the system of tissue culture. In addition, Vitrification was used on cryopreservation of Duchesneae Indicae. This paper supply more scientific indication for researching Duchesneae Indicae. The major consequences are displayed as follows:(1) The optimal induction culture medium was MS + 2.0 mg·L^-1 6-BA, induction ratio reached to 100%, average differentiation index was 19.87±1.51; the optimum reproduction medium was MS + 2.0 mg·L^-1 6-BA+0.01 mg·L^-1 IBA, average reproduction coefficient was 9.47±1.51; when MS without any hormone as rooting medium, results were better, average root length and quantity can reach (4.15±0.92) cm and 6.73±1.58 separately.(2) The effect of various factors of vitrification, for example, cryoacclimation time, pre-culture time, pretreatment time, PVS2 treatment time, cryopreservation time in Liquid Nitrogen, was studied on survival rate of Duchesneae Indicae shoot tips after cryopreservation. The results showed that shoot tips 1² mm length was cut from Duchesneae Indicae after 30 d subculture and cryoacclimatized 3⁴ weeks at 4℃; cultured in solid pre-culture medium (SPCM: MS + 184.2 g·L^-1 glycerol + 136.9 g·L^-1 sucrose + 7 g·L^-1 agar) after 4 d at room temperature; put them into sterile frozen tube with liquid pretreatment medium (LPTM: MS + 184.2 g·L^-1 glycerol + 136.9 g·L^-1 sucrose) at 20℃for 20³0 min; replaced LPTM with Plant Vitrification Solution2 (PVS2: MS + 300 g·L^-1 glycerol + 150 g·L^-1 ethylene glycol + 150 g·L^-1 DMSO + 136.9 g·L^-1 sucrose) at 0℃for 50⁷0 min; changed fresh PVS2, preserved in Liquid Nitrogen for 1 h; threw rapidly into 40℃water for 1 min; washed the shoot tips with solution contained MS and 1.184.2 g·L^-1 sucrose 3 times, 10 min a time; planted tips to regeneration medium (RM: MS + 1.0 mg·L^-1 GA3 + 1.0 mg·L^-1 6-BA + 30 g·L^-1 sucrose + 7 g·L^-1 agar) in dark for 4 d; then normally cultured, After 7 d, counted the survival rate. The best survival rate was (43.00±7.584) %.