枸杞原生质体培养及高效成株体系的建立

Establishment of High Efficient Regeneration System from Protoplast of Lycium barbarum L.

由枸杞髓部组织诱导出胚性愈伤组织 ,并由此愈伤组织建立起稳定的细胞悬浮系。从悬浮细胞游离的原生质体在改良KM培养基 ( 1 .5mg/L 6_BA ,0 .5mg/LNAA和 0 .5mg/L 2 ,4_D)中进行液体浅层培养 ,3～ 4d后出现第一次分裂 ,第 7d统计分裂频率为 5 0 .3% ,1 5d左右可形成细胞团 ,3～ 4周后形成肉眼可见的愈伤组织 ,愈伤组织植板率为 1 .2 5 %。将细胞团转移到液体分化培养基 (MS + 6_BA 1 .5mg/L+ 2 ,4_D 0 .2mg/L) 8～ 1 0d可形成大量胚状体 ,及时将胚性愈伤组织块转移到固体分化培养基上 (MS +6_BA 0 .2mg/L) ,可形成大量绿芽 ,分化率 5 4 .1 7%。绿芽在生根培养基 (MS +NAA 0 .2mg/L)可形成完整植株 。

A stable cell suspension culture was established with callus that was induced from the ripe pith of Lycium barbarum L. The protoplasts were cultured in KM liquid medium supplemented with 1.5 mg/L 6_BA, 0.5 mg/L NAA and 0.5 mg/L 2,4_D. The protoplasts started to divide after 3～4 days. The frequency determined at 7 days was 50.3%, and cell colonies were formed after about 15 days. Microcalli could be observed in 3～4 weeks. The plating efficiency was 1.25%. Eight to seven days after the microcalli were transferred into the liquid proliferation medium (MS+1.5 mg/L 6_B+0.2 mg/L 2,4_D), a large amount of embryoids appeared on the calli. The embryonic calli were then moved on to a solid medium (MS+ 6_BA 0.2 mg/L) and green buds appeared. The frequency of shoot formation was 54.17%. The shoot rooted in the rooting medium (MS+NAA 0.2 mg/L) and the whole plants were transplanted into pots and grew well.