Abstract
BackgroundBrassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus.ResultsBased on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets.ConclusionA rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.
Highlights
Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production
High efficiency of Agrobacterium‐mediated hypocotyl transformation in rapeseed For Agrobacterium-mediated hypocotyl transformation in rapeseed, the over-expression vector pCMABIA-1303 and RNAi vector p35S-1390 were used for transformation (Fig. 1)
Hypocotyls from eight different rapeseed varieties were used for Agrobacterium mediated transformation
Summary
Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. In B. napus, various technologies, including PEG-mediated DNA uptake [16, 17], electroporation [18], particle bombardment [19], Agrobacterium-mediated transformation and microspore transfection [20, 21], have been used to obtain genetically modified plants Among these technologies, Agrobacterium-mediated transformation is the most general, reliable and effective method [22,23,24]. Maheshwari et al (2011) investigated the effect of hormonal combinations, donor plant age and explant types on the transgenic frequency and regeneration capacity of plants in four different rapeseed lines (nvigor 5020, Westar, Topas and its microspore derivative-Line 4079) [25] They found that the transformation frequency was 54.2 and 53.7% in cultivars of Invigor 5020 and Westar, but 16.0 and 13.4% for Topas and Line 4079, respectively. Due to the diverse genetic transformation ability caused by distinct genetic backgrounds within the cultivars [5], recalcitrance continued to persist in several cultivars which were not capable of being genetically transformed, especially in commercial varieties
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