Isolation Of Protoplasts And Anther Culture In Chinese Jujube And Wild Jujube

Chinese Jujube originated in China belongs to Ziziphus genus, Rhamnaceae family, and is an important resource in our country. This study revealed effects of optimal stage, pretreatment, concentrations of sucrose and plant growth regulator on anther culture in wild jujube and'Zanhuangdazao','Pingguozao','Junzao', and obtained plant regeneration of anther. Callus were induced from leaves, stem and anther of wild jujube, and obtained the best combination of plant growth regulator; The research isolated protoplast from pollen and suspension cell lines of wild jujube, and found the best concentration of enzyme, enzemolysis time and concentration of mannital. The study was the foundation to early times of ploidy breeding.The main results were as followed:1. Obtained plant regeneration on anther(1)Microspore development had correlativity with the morphological characteristics of fiower organ, studied by both of cytological and morphological methods. Late-uninucleate was the optimal stage of anther culture, this stage flower bud was yellow-green, anther was light yellow.(2)Low tempreture pretreatment of anther significantly influnced wild jujube,'Pingguozao'and'Zanhuangdaza'(P<0.05). Induction rate and differentiation rate of wild jujube were the biggest by 7 d low tempreture, 3 d was helpful to differentiation of callus; Induction rate of'Pingguozao'was the biggest by 3 d low tempreture, 5 d and 7 d were helpful to differentiation and regeneration of callus; Induction rate and differentiation rate of'Zanhuangdazao'were the biggest by 3 d low tempreture. Low tempreture pretreatment of'Junzao'anther didn't have effect.(3)Mannital pretreatment of anther significantly influnced'Pingguozao','Zanhuangdaza'and wild jujube(P<0.05). Induction rate of'Pingguozao'and wild jujube was the biggest by mannital pretreatment of 3 d; Induction rate of'Zanhuangdaza'was the biggest by mannital pretreatment of 4 d. Mannital pretreatment of'Junzao'anther didn't have effect.(4)When concentrations of sucrose was 6%, which may help induce anther callus on'Zanhuangdaza'and wild jujube; When concentrations of sucrose was 9%, which may help induce anther callus on'Junzao'. (5)Callus could be efficiently induced from anther in the medium MS+TDZ+NAA+6 g/L agar+30 g/L maltose, TDZ 0.5 mg/L and NAA 0.5 mg/L were suitable for anther culture of'Junzao'and'Zanhuangdaza', TDZ 0.5 mg/L and NAA 1.0 mg/L were suitable for anther culture of wild jujube, TDZ 1.5 mg/L and NAA 0.5 mg/L were suitable for anther culture of'Pingguozao'. This study obtained plant regeneration of anther, wild jujube was 25 tissue culture seedlings,'Zanhuangdaza'was 5 tissue culture seedlings,'Pingguozao'was 2 tissue culture seedlings,'Junzao'was 2 tissue culture seedlings.2. Induction and regulation on callusCallus could be efficiently induced from anther of wild jujube. Callus could be efficiently induced from leaves of wild jujube in the medium MS+6-BA 1.0 mg/L+NAA 1.0 mg/L+6 g/L agar+30 g/L maltose. Medium of callus subculture was MS+TDZ 0.2 mg/L+NAA 0.1 mg/L+CH(600 mg/L)+Gln(300 mg/L)+ME(500 mg/L)+6 g/L agar+30 g/L maltose, Medium of suspension cell lines was not added to agar.3. Isolated protoplast(1)The optimal enzyme solution for isolation of pollen protoplasts from tetrad of wild jujube was: snailase (1.0%) +cellulase (0.5%) +mannital 0.3 mol/L+0.1%MES, enzymolysis time was 4 h.(2)The optimal enzyme solution for isolation of protoplasts from suspension cells of wild jujube was: cellulose 10 g/L+macerozyme 5 g/L+mannital 0.7 mol/L+0.1%MES, enzymolysis time was 16 h.