4401 University Drive, Department of Biological Sciences, University of Lethbridge, Lethbridge T1K 3M4, Alberta, Canada.
N Biotechnol. 2011 Dec 15;29(1):144-55. doi: 10.1016/j.nbt.2011.06.014. Epub 2011 Jun 21.
Brassica napus (canola) is the second largest oilseed crop in the world. It is among the first crops to be genetically transformed, and genetically modified cultivars are in commercial production at very significant levels. Despite the early lead with respect to transgenesis, there remain cultivars that are recalcitrant to transformation. To address this, we have conducted an elaborate investigation of the conditions for regenerating shoots from hypocotyl explants from four genetic lines: Invigor 5020, Westar and Topas as well as a microspore culture derived line of Topas (Line 4079). We analyzed the effect of hormonal combinations in regeneration medium, donor plant age and explant type on the regeneration capacity of these plants. The analysis showed that hypocotyls of eight-day-old seedlings grown on media supplemented with 1mg/L dinitrophenylhydrazine (2,4-D) produced the most shoots. Globular somatic embryos emerged following two weeks of 2,4-D treatment. When transferred to the medium containing 5mg/L benzyladenine (BA), approximately 82% of embryos produced shoots within six weeks. Invigor plants were shown to regenerate more efficiently than Topas; the number of plantlets regenerated from Invigor was approximately 40-50% more as compared to Topas or Line 4079. When hypocotyl explants were co-cultivated with the Agrobacterium strain GV3101 harboring a binary vector carrying a firefly luciferase reporter gene (LUC), significant numbers of plantlets were LUC-positive in a luciferase assay. Frequency of such plants were: Invigor 5020 (54.2 ± 2.5%), Westar (53.7 ± 5.3), Topas (16.0 ± 0.24) and Line 4079 (13.4 ± 4).
油菜（芥花）是世界上第二大油料作物。它是最早被基因转化的作物之一，并且已经有许多转基因品种投入商业化生产。尽管在转基因方面起步较早，但仍有一些品种难以进行转化。为了解决这个问题，我们对四个遗传品系（Invigor 5020、Westar、Topas 以及 Topas 的一个小孢子培养衍生系（Line 4079））的下胚轴外植体再生芽的条件进行了详细的研究。我们分析了再生培养基中激素组合、供体植物年龄和外植体类型对这些植物再生能力的影响。分析表明，在补充有 1mg/L 二硝基苯肼（2,4-D）的培养基上生长的 8 天大的幼苗的下胚轴产生的芽最多。在 2,4-D 处理两周后出现球形体细胞胚。当转移到含有 5mg/L 苄基腺嘌呤（BA）的培养基中时，大约 82%的胚在 6 周内产生芽。Invigor 植物比 Topas 更有效地再生；与 Topas 或 Line 4079 相比，从 Invigor 再生的植物苗数量增加了约 40-50%。当将下胚轴外植体与携带萤火虫荧光素酶报告基因（LUC）的二元载体的农杆菌菌株 GV3101 共培养时，在荧光素酶测定中，许多植物苗呈 LUC 阳性。具有这种特性的植物的频率为：Invigor 5020（54.2±2.5%）、Westar（53.7±5.3%）、Topas（16.0±0.24%）和 Line 4079（13.4±4.0%）。
