《植物生理学报》 2009, 45(4): 367-371

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

张改娜^1,2, 贾敬芬^2,*

1 河南科技大学农学院, 河南洛阳471000; 2 西北大学生命科学学院, 西安710069

通信作者：贾敬芬；E-mail: zgnluck1@163.com；Tel: 0379-64282340

摘　要：

从发根农杆菌A4转化的荒漠植物——骆驼刺毛状根愈伤组织中分离的原生质体培养的结果表明, 酶解新转代7~10 d 的淡黄色松软愈伤组织, 可获得大量有活力的原生质体。原生质体在附加有 1.5 mg•L-1 2,4-D、0.2 mg•L-1 6-BA、0.3 mol•L-1 甘露醇、2% (W/V)蔗糖和 500 mg•L-1 水解酪蛋白的 DPD 培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为 (450±3) mOsm•kg-1, 原生质体的最适植板密度为4×105 个•mL-1。制备原生质体的愈伤组织以低温(4 ℃)预处理后, 原生质体 的产率和分裂频率均提高, 分裂频率最高可达 50%。原生质体分裂形成的愈伤组织转移在附加 1~2 mg•L-1 6-BA (或 KT)和0.2 mg•L-1 NAA 的 MS 培养基上培养后, 可以分化并获得再生植株。纸电泳检测表明, 原生质体再生的愈伤组织和分化植 株仍然含有毛状根转化系的特异产物——冠瘿碱。

关键词：骆驼刺; 发根农杆菌A4 转化系; 原生质体培养

收稿：2009-01-09   修定：2009-03-25

资助：国家自然科学基金(30671082)。

Protoplast Culture and Plantlet Regeneration from Cell Line of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi Desv

ZHANG Gai-Na^1,2, JIA Jing-Fen^2,*

¹College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471000, China; ²School of Life Science, Northwest University, Xi’an 710069, China

Corresponding author: JIA Jing-Fen; E-mail: zgnluck1@163.com; Tel: 0379-64282340

Abstract:

The protoplasts were isolated from calli which were induced from hairy root segments of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi. After cultured in the DPD medium supplemented with 1.5 mg·L^-1 2,4-D, 0.2 mg·L^-1 6-BA, 0.3 mol·L^-1 mannitol, 500 mg·L^-1 casein hydrolysate (CH) and 2% (W/V) sucrose, the protoplasts underwent sustained divisions and formed calli. The protoplast density of 4×10⁵ mL^-1 and (450±3)mOsm·kg^-1 osmotic pressure in culture medium were proved to be appropriate for obtaining higher division frequency of protoplasts. A lot of protoplasts could be obtained by the enzymatic hydrolysis of yellowish subcultured calli after cultured on MS medium supplemented with 1.5 mg·L^-1 NAA, 1.0 mg·L^-1 6-BA, 500 mg·L^-1CH and 2% (W/V) sucrose for 7–10 d. Lower temperature (4 ℃) pretreatment of subcultured calli enhanced ratios of protoplast isolation and subsequent divisions. The division frequency of protoplasts was about 50%. After transferred on the MS medium added with 1–2 mg·L^-1 6-BA (or KT) and 0.2 mg·L^-1 NAA, the protoplastderived calli differentiated and formed the regenerated plantlets. Paper electrophoresis analysis indicated that the protoplast-derived calli and regenerated plantlets still contained special product––opine in transgenic root hairs.

Key words: Alhagi pseudalhagi; Agrobacterium rhizogenes A4-transformed cell line; protoplast culture