沙葱叶基愈伤组织原生质体再生体系的建立

Establisment of Plant Regeneration System from Protoplasts of Leaf Base-Derived Calli in Allium mongolicum Regel

沙葱是一种具有抗旱抗寒、抗病性和适应性强等生理特性的荒漠植物。为开发利用其固有的遗传资源,本研究利用细胞工程技术建立了沙葱（Allium mongolicum Regel）叶基愈伤组织原生质体的分离、培养和植株再生实验体系。研究结果表明,酶法分离原生质体的产率和分裂频率明显取决于用于制备原生质体的愈伤组织的状态。转代培养7-10d的松软愈伤组织可分离出大量有活性的。在附加2.0mg/L2,4-D、0.2mg/L激动素、500mg/L水解乳蛋白、0.4mol/L甘露醇和2%蔗糖的MS培养基中进行液体浅层培养,4~5d后出现第一次原生质体分裂;7~10d出现第二次分裂。结果显示原生质体的分裂频率大约为5%;4周后,可见到小愈伤组织。当将原生质体分裂形成的愈伤组织转移到附加2.0mg/L6-苄氨基嘌呤（或激动素）和0.4mg/L萘乙酸（NAA）的MS固体培养基上,并在低光照条件下培养后,从愈伤组织上分化出了不定芽,进而发展成小植株,并移栽成活。本研究对沙葱抗逆遗传品质用于经济植物遗传改良的研究奠定了可行的实验基础。

Allium mo ngolicum Regel is a kind of desert plant which contains physiolgical characteritics of drought-resistence, cold-resistence, disease stress-resistence and strong adaptability. In order to develop and utilize these inherent genetic resources, in this research, we established an experimental system for leafbase-derived calli of A llium mongolicum Regel from the isolation of protoplasts, cultivation and plant regeneration by cell engineering techniques. The result showed that the rate of production and their frequency of protoplasts division were strongly dependent on the state of the calli which were isolated by enzymatic method. A large amount of activated protoplasts could be isolated from 7 to 10 day-old friable calli after transferring to the fresh subculture medium. And first divisions of protoplasts appeared after 4 to 5 days of cultivation in liquid MS medium supplemented with 2.0 mg/L 2,4-D, 0.2 mg/L kinetin, 500 mg/L lactalbumin hydrolysate, 0.4 mol/L mannitol and 2% sucrose. The second divisions were followed after 7 to 10 days in cultivation. The result still displayed that frequency of protopIast division was about 5%. Four weeks later, we could observe some small calli. When the protoplast-derived calli were transferred into the MS solid medium added with 2.0 mg/L benzylamino-purine （or kinetin） and 0.4 mg/L naphthaleneacetic acid and cultivated in the codition of low illumination, after which the adventitious shoots were induced, then we could obtain numerous plantlets and could transplant them survival to pots in the greenhouse. This research would establish a feasible experiment foundation for studying the stress resistance heredity characters of A llium mongolicum Regel utilizing in genetic improvement of economic plants.