甘蓝CMS系的原生质体培养与植株再生

PLANT REGENERATION FROM PROTOPLASTS OF A CMS LINE OF CABBAGE(BRASSICA OLERACEA L.VAR CAPITATA)

对雄性不育结球甘蓝“CMS 黑叶大平头BC_5”的叶及下胚轴原生质体的分离、培养与植株再生等的研究表明,以2%纤维素酶(OnozukaR-10)+0.5%离析酶(R-10)的酶液游离CMS 的试管苗叶、下胚轴及原生质体产量高,去壁效果好。适合于CMS 叶原生质体及下胚轴原生质体纯化的漂浮液蔗糖浓度分别为16%和17%。原生质体的清洗和收集均以500r/min 的离心效果为佳。在修改后的MS 培养基(MS_1+NAA0.2mg/l+BA2mg/l)上获得了较快的细胞壁再生和分裂启动、最高的分裂频率和植板率。叶原生质体第一次分裂在培养3～4天后,下胚轴原生质体在48小时左右发生分裂。培养20～30天即形成肉眼可见的微愈伤颗粒,40天左右即可达1mm 大小。MB_2培养基(MB+甘氨酸2mg/l+NAA0.2mg/l+BA2mg/l)对微愈伤组织增殖的效果最佳.在MS_2(MS+NAA0.2mg/l+BA3mg/l)和MS_4(MS+NAA0.4mg/l+BA3mg/l)培养基上,芽分化效果最好。在不加任何激素的MS 培养基上诱导生根,2周后即可获得完整的再生植株。

Protoplasts of the cytoplasmic male sterile line of cabbage,“Heida BC_5”(CMS),wereisolated from leaves and hypocotyls of plantlets grown in vitro,in enzyme mixture con-taining 2% cellulase(Onozuka R-10)and 0.5% macerozyme R 10.Good results ofprotoplasts collection were obtained with sucrose concentration of the floating solution be-ing 16% and 17% for leaf hypocotyl protoplasts,resp.and with centrifugation at the rateof 500 rpm.All the collected protoplasts were plated in 5 different media for liquid culture.The best results of culture were observed on MSI medium for both leaf and hypocotylprotoplasts with the highest cell division rate and plantig effieiency.Cultured for about 2weeks,numcrous cell colonies and a few embryo-like structures were obserced.The cellcolonies developed into visible microcalli in 20-30 days and grew up to 1 mm or so in di-ameter about 40 days after culture.For further growth,the calli were transferred to 7 dif-ferent agar media,from which one suitable medium,MB_2,was selected.Cultured for 40-50days,the calli grew up and were then transferred to 4 solid media for organ differentiation.About 2 weeks after rooting on the MS medium without any auxin,complete plants wereregenerated.