菜心下胚轴原生质体培养和植株再生

PROTOPLAST CULTURE AND PLANTLET REGENERATION FROM HYPOCOTYLS OF BRASSICA CAMPESTRIS VAR.PARACHINESIS

以萌发３—４ 天（长约４ ｃｍ ）的菜心（Ｂｒａｓｓｉｃａ ｃａｍｐｅｓｔｒｉｓ ｖａｒ．ｐａｒａｃｈｉｎｅｓｉｓ）无菌苗苍白下胚轴为材料，酶解分离原生质体。经纯化的原生质体，在含０．５ ｍ ｇ／ＬＺＴ、０．５ ｍ ｇ／Ｌ２，４－Ｄ、１．０ ｍ ｇ／ＬＮＡＡ 和０．４ ｍ ｏｌ／Ｌ葡萄糖的Ｋ８ｐ 培养基中，进行微滴培养。在起始培养１４—１８小时，原生质体再生新的细胞壁。３６ 小时再生细胞开始第一次分裂。第三天分裂细胞频率可达３５％ 。培养第８—９ 天，可见含８—１６个细胞的小细胞团，植板率为１５％ —１８％ 。３ 周后将发育成直径为２ ｍ ｍ 的白色小愈伤组织，转到含０．３ ｍ ｇ／Ｌ ２，４－Ｄ并用ｇｅｌｒｉｔｅ半固化的培养基上，增殖成４—５ ｍ ｍ 直径的愈伤组织。在ＭＳ＋ ３．２（或１．６） ｍ ｇ／Ｌ ＢＡ＋ １．６（或０．８） ｍ ｇ／ＬＺＴ＋ ０．０１ ｍ ｇ／Ｌ ＮＡＡ＋ ０．１ ｍ ｇ／ＬＧＡ３ 和０．２％ 蔗糖的分化培养基上，获得芽的分化。切下约２ ｃｍ 长的芽苗，转移到含０．２ ｍ ｇ／ＬＩＡＡ 和２％ 蔗糖的培养基上。

Protoplasts isolated from 3—4 day old (ca 4 cm in length) etiolated hypocotyls of Brassica campestris var.parachinesis (Baily) Tsen et Lee and purified with 20% sucrose were cultured on K8p medium suplemented with 0 5 mg/L ZT, 0 5 mg/L 2,4 D, 1 0 mg/L NAA and 0 4 mol/L glucose.When initially cultured for 14—18 hours the protoplasts formed new walls and by first division after 36 hours.The divided protoplasts reached 35% after being cultured for three days.When cultured under optimum conditions for 8—9 days, the protoplasts formed 8—16 cell colonies with a plate effeciency as high as 15%—18%.Rapidly growing and dividing calli of 2 mm in diameter were transferred onto semisold gelrite media with 0 3 mg/L 2,4 D enabling them to proliferate further towards the size of 4—5 mm in diameter.Shoot differentiation was carried out in MS medium with 3 2 (or 1 6) mg/L BA, 1 6 (or 0 8) mg/L ZT, 0 01 mg/L NAA, 0 1 mg/L GA 3 and 0 2% sucrose.Shoots were cut down and rooted on medium with 0 2 mg/L IAA and 2% sucrose where whole plants were evatually developed.