Mass production of regenerated plants by artificial seed system

对毛状根不定胚高效生产系统、不定胚自动选择系统和毛状根人工种子系统进行了广泛的研究。在再生条件下，将芹菜愈伤组织包裹在固定化海藻酸钙凝胶珠培养物中，芹菜胚和植株被选择性释放。用5 mM CaCl_2反复分批培养，可长期（154d以上）产生胚和植株，且无凝胶珠破坏。固定化细胞培养的植株产量是悬浮培养的2 ~ 2倍。研究了一种利用辣根毛状根进行人工种子的高效育苗方法。为了制作毛状根碎片，在根生长后使用搅拌器。断片时间为30 s时，植株形成效果最好。用萘乙酸（1.0 mg/l）处理毛状根可缩短培养时间，提高植株产量。破碎后用0.1 mg/l的动素处理，植株数量最多。利用根源农杆菌介导的共转化技术，在番茄核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基启动子（rbcS3B）调控下，导入GUS基因的牛毛根。经gus转化后的毛状根也能有效地再生成植株。在再生植株的叶片组织中检测到GUS活性。采用摇瓶式生物反应器对山葵毛状根的碎片状茎尖进行再生。再生植株的数量与转速有关，120 rpm时再生植株数量最多。结果表明，振荡容器式生物反应器适合于再生植株的批量生产。

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.