Mass production of regenerated plants by artificial seed system

利用人工种子系统大规模生产再生植物

• 批准号: 06403019
• 负责人: KOBAYASHI Takeshi
• 金额: $ 21.95万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06403019/
• 关键词: artificial seed; regeneration; hairy root; adventive embryo; mechanical cut; secondary metabolite; preservation; cultivation engineering

Efficient prodution system of adventive embryo, automatic selection system of adventive embryo and artificial seed system from hairy roots were studied extensively.Celery embryos and plantlets were found to be selectively released in a culture of immobilized Ca-alginate gel beads in which celery callus was entrapped under regeneration conditions. Repeated batch culture with 5 mM CaCl_2 provided long-term(more than 154d) embryo and plantlet production without gel beads disruption. Productivity of plantlets in the immobilized cell culture was 2,2-fold as high as that in the suspension culture.An efficient plantlet production from horseradish hairy roots was studied to apply the hairy roots for artificial seed. To make the hairy root fragments, a blender was applied after the root growth. A fragmentation time of 30 s gave the best result on the plantlet formation. Treatment of the hairy roots with naphthaleneacetic acid(1.0 mg/l) made the culture time shorter and increased plantlet productivity. Treatment with kinetin at 0.1 mg/l after fragmentation yielded the highest number of plantlets.Ajuga hairy root in which the beta-glucuronidase(GUS)gene was introduced under control of the tomato ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS3B) promoter was constructed by the Agrobacterium rhizogenes-mediated cotansformation. The GUS-transformed hairy root could also be efficiently regenerated into plantlets through this procedure. GUS activity was detected in leaf tissue of the regenerated plants.For plant regeneration from fragmented tips of horseradish hairy root, a shaking-vessel type bioreactor was used. Number of regenerated plantlet depended on a rotational spped, and the maximum number was obtained at 120 rpm. It was concluded that the shaking-vessel type bioreactor was suitable for mass-production of regenerated plantlet.

本研究对芹菜毛状根不定胚高效生产系统、不定胚自动选择系统和人工种子系统进行了深入研究，发现在固定化海藻酸钙凝胶珠包埋芹菜愈伤组织的培养条件下，芹菜胚和植株的再生有选择性。用5 mM CaCl_2重复分批培养可获得长期（154 d以上）的胚和植株生产，而凝胶珠不破裂。固定化细胞培养的成苗率是悬浮培养的2.2倍。为了制备毛状根片段，在根生长后应用搅拌器。破碎时间为30 s时，成苗效果最好。用萘乙酸（1.0 mg/l）处理毛状根可缩短培养时间并提高成苗率。以发根农杆菌介导的共转化法，将番茄核酮糖-1，5-二磷酸羧化酶/加氧酶小亚基（rbcS 3B）启动子控制下的β-葡萄糖醛酸酶（GUS）基因导入筋骨草毛状根中。GUS转化的毛状根也能高效地再生成植株。以辣根毛状根为外植体，采用摇瓶式生物反应器培养再生植株。再生植株的数量取决于转速，以120 rpm为最高。说明摇瓶式生物反应器适合于大规模生产再生植株。

项目成果

Nobuyuki Uozumi, Takeshi Kobayashi: "Measurement techniques plant cell culture" Intelligent Agriculture. 76-89 (1996)

Nobuyuki Uozumi、Takeshi Kobayashi：“植物细胞培养测量技术”智能农业。

中島田豊ら: "Efficient Culture method for production of plantlets from mechanically cut norseradish hairy roots" Journal of Fermentation and Bioengineering. 81. 87-89 (1996)

Yutaka Nakashimada 等人：“从机械切割的北欧萝卜毛状根中生产幼苗的有效培养方法”发酵和生物工程杂志 81. 87-89 (1996)。

Hiroyuki Honda, Toshiyuki Hattori, Nobuyuki Uozumi, Takeshi Kobayashi, Yoshihito Kato, Setsuro Hiraoka: "Production of regenerated plantlet using a shaking vessel-type bioreactor" Journal of Chemical Engineering of Japan. 30(1). (1997)

Hiroyuki Honda、Toshiyuki Hattori、Nobuyuki Uozumi、Takeshi Kobayashi、Yoshihito Kato、Setsuro Hiraoka：“使用振荡容器型生物反应器生产再生植株”日本化学工程杂志。

魚住信之ら: "Efficient regeneration from GUS-transformed Ajuga hairy root" Journal of Fermentation and Bioengineering. 81・5. 374-378 (1996)

Nobuyuki Uozumi 等人：“GUS 转化的 Ajuga 毛状根的有效再生”《发酵与生物工程杂志》81・5（1996）。

中島田,豊: "Production of plantlets available to artificial seed from horseradish hairy roots fragmented by a blender" Journal of Fermentation and Bioengineering. (in press). (1995)

Yutaka Nakashimada：“用搅拌机破碎的辣根毛状根生产可用于人工种子的植物”《发酵与生物工程杂志》（1995 年出版）。