草地早熟禾午夜Ⅱ号原生质体培养及植株再生

Protoplast Culture and Plant Regeneration of Midnight Ⅱ(Poa Pratensis L.)

对草地早熟禾(Poa Pratensis L.)品种-午夜Ⅱ号进行了原生质体培养及其植株再生的研究。以午夜Ⅱ号成熟种子诱导的松软胚性愈伤组织为材料,利用酶解法分离出大量有活力的原生质体。原生质体经培养,持续分裂形成愈伤组织,并分化出再生绿苗和白化苗。研究了不同酶液组成和酶解时间对原生质体游离的影响。结果表明:采用继代培养8-10 d的胚性愈伤组织在1.0%纤维素酶+1.0%离析酶+0.5%果胶酶+0.3%崩溃酶条件下,酶解14-16 h可得到产量和活力较高的原生质体。原生质体以2.0×105-5.0×105个/mL的植板密度,采用液体浅层法在附加1.0 mg/L 2,4-D0、.3 mg/L 6-BA、100 mg/L水解酪蛋白1、00 mg/L水解乳蛋白、1.0%蔗糖、0.4 mol/L甘露醇的KM8P培养基中培养,可形成较小的愈伤组织。愈伤组织经过增殖在分化培养基上(MS+0.5mg/LNAA+2.0 mg/L 6-BA)可诱导芽、根的形成,再生出完整植株。

The friable calli induced from mature seeds of Poa pratensis L.cv.Midnight II were used for protoplast preparation and the protoplasts were isolated using enzyme digestion.Calli appeared after sustained protoplast divisions in culture medium and the green shoot and albino plantlets were obtained from the protocalli on differentiated medium.The effects of different enzyme composition and enzyme digestion time on protoplast isolation were discussed in this paper.The results show that it was available to harvest high yield and viabilities of protoplasts from embryogenic calli with treatment of digestion in 1.0% Cellulase Onozuka R-10,1.0% Macerozyme R-10,0.3% Driselase,and 0.5% Pectolase Y-23 in 8 to 10 d.It was better to digest for 14-16 h in enzyme digestion solution mentioned above.Calli formed on KM8P medium supplemented with 1.0 mg/L 2,4-D,0.3 mg/L 6-BA,100 mg/L casein hydrolysate,100 mg/L lactablumin hydrolysate,1.0%（W/V） sucrose,and 0.4 mol/L mannitol at plating density of 2.0×105-5.0×105 mL-1.The protoplast-derived calli developed shoots and roots on differentiation media（MS＋0.5 mg/L NAA＋2.0 mg/L 6-BA） after further subculture,and then complete plants were obtained.