Development of Genetic Engineering in sugarbeet (Beta vulgaris L.)

甜菜是一个因不愿体外操作而闻名的物种，这一特征是该作物常规遗传修饰的主要限制。我们最近开发了一种可靠的方法，从甜菜小叶培养中高频再生植株。种子在固化的MS培养基上无菌萌发，然后将幼苗（子叶和下胚轴的上部）培养在含有0.25mg/l BA（苄基腺嘌呤）和0.1mg/l IBA（吲哚丁酸）的MS培养基上。当幼苗4周龄时，我们从新展开的叶片分离叶片外植体（5* 5 mm片段）并将它们置于相同的培养基上。叶片外植体在培养1至2周后形成多个芽，芽生长迅速。在附加1.0mg/l IBA的MS培养基上，大量的新生芽容易生根，并能转移到温室中继续正常生长。下一步的目标是利用叶片培养策略建立农杆菌介导的转化体系。使用携带HPT-II（潮霉素磷酸转移酶）和GUS（β-葡糖醛酸糖苷酶）基因的二元载体。接种培养的小叶外植体，导致转化植物的产生，通过（1）引入的潮霉素抗性，（2）引入的GUS活性，和（3）PCR和/或Southern印迹分析以显示外源DNA的整合来确定。

Sugarbeet is a species which has acquired a reputation for recalcitrance to manipulation in vitro, a characteristic which is a major limitation to the routine genetic modification of the crop. We have recently developed a reliable method for high frequency regeneration of plants from leaflet culture of sugarbeet. Seeds were aseptically germinated on solidified MS medium and seedlings (cotyledons with the upper part of hypocotyls) were then cultured on MS medium containing 0.25mg/l BA (Benzyladenine) and 0.1mg/l IBA (Indole butyric acid) . When the seedlings were 4 week old, we isolated leaf explants (5*5mm segments) from newly expanded leaves and placed them on the same medium. Leaf explants formed multiple shoots after 1 to 2 weeks of incubation, with rapid growth of shoots. A large number of newly formed shoots readily rooted on MS medium supplemented with 1.0mg/l IBA and 'could be transferred to the glasshouse where they continued normal growth. The next aim was to develop the Agrobacterium-mediated transformation system using leaflet culture strategy. Binary vectors were used, carrying both HPT-II (hygromycin phosphotransferase) and GUS (beta-glucuronidase) genes. Inoculation of cultured leaflet explants, resulted in the production of transformrd plants, determined by (1) introduced resistance to hygromycin, (2) introduced GUS activity, and (3) PCR and/or Southern blot analysis to show the integration of foreign DNA.