埃塞俄比亚芥的原生质体培养及植株再生

PLANT REGENERATION FROM PROTOPLASTS OF BRASSICA CARINATA BRAUN.

以埃塞俄比亚芥“84A165”为材料,从3—5日龄无菌苗的子叶、下胚轴或温室生长的三叶期苗的第一真叶游离原生质体,悬浮于 P-B 液体培养基中,用0.15—0.3%低融点琼脂糖固化,薄层漂浮,暗培养。原生质体密度为5×10~3—1×10~4/ml。下胚轴原生质体在培养后48小时出现一次分裂,3天后分裂频率达21%,6天后达34%。子叶和真叶原生质体起始分裂稍晚(第5天),分裂频率也较低。培养1周后,加入稀释培养基 DPDK_3,并转至光下,以后每隔一周加液一次。一个月后获得肉眼可见的小愈伤组织。植板率为2—3%。小愈伤组织在固体增殖培养基上继代长大。子叶和真叶原生质体来源的愈伤组织在转至补加 NAA0.1、BA3mg/1的分化培养基上后获得芽分化,把这些芽切离,转至 IAA0.2mg/l 的生根培养基上,再生出完整植株。

Protoplasts of Brassica carinata Braun.(accession No.84A165) were enzymatically isolated from hypocotyls and cotyledons of 3-5 day-old test-tube seedlings or first true leaves taken from greenhouse grown plants at a three-leaf stage.The protoplasts were suspended in a P-B liquid medium solidified with 0.15-0.3% low melting agarose which formed a thin layer float- ing on the surface of the liquid medium.The optimum protoplast density was ranging from 5×10~3 to 1×10~4/ml.As for the hypocotyl protoplasts,the first division was observed after 48h in the culture.The division frequency reached 21% and 34% at day 3 and 6 respectively.The initiation of celi division in the case of cotyledon and mesophyll protoplast culture was late, usually at day 5,and the division frequency was also somewhat lower.One week after culture, the cultures were transferred to fluorescent light condition with an intensity of about 1,000 1x.A dilution medium DPDK_3 was then added and the dilution procedure was repeated at one week interval thereafter.One month after culture,microcalli with 300-500μm in size were formed,It was also found that in some cases globular embryoid structure protruded on the cal- lus surface.Totally,a 2-3% plating efficiency was achieved.Shoot regeneration occurred when cotyledon and mesophyll protoplast-derived calli were transferred onto a modified MS medium supplemented with NAA 0.1,BA 3mg/l.Individual shoots were rooted on a rooting medium supplemented with 0.2mg/l of IAA.Intact plants with normal morphology were ev- entually produced.