Mass production of regenerated plants by artificial seed system

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（1.0mg/l）处理毛状根，可缩短培养时间，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。

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.