摘要
十字花科(Cruifrae)芸薹属(Brassica genus)包含了一些重要的油料作物和蔬菜作物,其中有6个物种被广泛种植。甘蓝型油菜(Brassica.napus L.)是全球继大豆和棕榈油之后第三大油料作物。随着人类需求的增加和新科技手段的不断涌现,油菜品种改良正呈多元化发展,植物转基因技术在油菜遗传改良中广泛应用,推动了油菜遗传育种工程技术进行一次新的革命。转基因技术已经成为向油菜导入新型外源基因的重要手段,转基因抗除草剂油菜、抗虫油菜、高油脂含量油菜、油菜新型不育系等已大规模投入商业化生产,抗病、抗环境逆境胁迫(干旱、盐碱、高温和低温)等新型油菜育种材料的创制已成为重要研究课题。
本研究主要目标是,立足西北地区油菜品种,筛选最佳转化受体,建立农杆菌介导的油菜高效转化体系,获得CMO和BADH抗盐耐旱基因的油菜转化体,为利用基因工程改良油菜奠定基础。
研究了甘蓝型油菜花茎外植体高效诱导不定芽的再生体系,结果表明:以含有2,4-D(0.5～1.0mg/L)的培养基对外植体进行预培养,以及在分化培养基中加入AgNO_3(2～6mg/L),可显著降低外植体褐化坏死的频率,提高了不定芽的发生频率及其再生能力;6BA(2.5mg/L)与NAA(0.1mg/L)配合使用有利于提高不定芽发生频率;再生的不定芽90%可长根。
研究了油菜未成熟胚胎离体培养条件下的几种再生方式。结果表明,油菜幼胚离体培养以三种方式实现再生:①幼胚早熟萌发,在茎顶端分生组织区依次产生多个额外子叶,表明幼胚外植体仍保持着胚性发育特点;②子叶近轴端和下胚轴表面直接产生类胚结构,继代后部分类胚结构可发育成体细胞胚,其它则由于无茎顶端分生组织,最终不能形成完整植株;③外植体细胞脱分化形成愈伤组织,继代后胚性愈伤组织产生单生的体细胞胚,最终可发育成完整的小植株。2,4—D是影响外植体再生方式的重要因素,它抑制了幼胚早熟萌发。在无2,4—D的培养基上,幼胚早熟萌发,主要以第一种方式实现再生;在2,4—D诱导下,外植体主要以第二或第三种方式再生。不同基因型间在外植体再生方式和体胚诱导率上存在差异。
以30个甘蓝型油菜品种(系)的子叶和下胚轴为外植体,研究了子叶和下胚轴的再生能力和再生方式,以及在抗菌素(羧苄青霉素和头孢霉素)和筛选剂(卡那霉素)胁迫下,各基因型子叶和下胚轴外植体的再生能力。结果表明,油菜子叶和下胚轴外植体主
The genus Brassica includes many economically important vegetable, condiment and oilseed crops. Within this genus, Brassica napus L. is an important oil crop, ranking third only to soybean and palm oil in global production. Canola oil is widely used as a cooking oil, salad oil, and for the production of margarine. There are several studies on Canola transformation with respect to the introduction of various new traits such as modified oil composition, herbicide tolerance, altered protein composition and insect resistance. The key objective of this work was to develop an efficient Agrobacrerium-mediated transformation system for Canola to facilitate crop improvement through genetic engineering. Transformation has been carried out using various explants, such as stem internodes, stem segments, cotyledonary petioles and hypocotyl segments. The efficiency of A. tumefticiens mediated transformation technique in oilseed rape is influenced by cultivar, donor plant age and explant type. An increase in the transformation efficiencies is desirable in order to decrease the amount of resources needed to produce transgenic plants, and to potentially provide a higher baseline for subsequent transformation of other canola varieties.Excised peduncle segments of three Brassica napus L. cultivars were used to optimize conditions for high-frequency shoot regeneration. The presence of 2,4-D(0.5-1.0 mg/L) in pre-cultured medium markedly enhanced the subsequent frequency of responding explants and the number of shoots/responding explant. The use of AgNO_3 was a prerequisite for efficient shoot regeneration due to it strongly increased morphogenesis and decrease the frequency of explants death. BA (2.5mg/L) in combination with a low concentration of NAA(0.1mg/L) gave optimum shoot bud differentiation. The regenerated shoots could be rooted at a frequency of 95%.The well-established tissue culture system to regenerate plants from a single transformed cell is one of the main prerequisites for crop plant genetic transformation. Somatic embryos provide a tool for efficient transformation because of their excellent ability to regenerate into full plants. This paper describes the different patterns of plant regeneration from excised immature embryo of Brassica napus L.. When immature embryos were placed on MS
hormone-free medium with 1% sucrose, they precociously germinated and produced 2-5 extra cotyledons. This indicated that immature embryos explant contain some cells that retain embryogenic competence. While immature embryos were cultured on MS media supplemented with various concentration of 2,4-D and 3% sucrose, somatic embryos and embryo-like structures were obtained by directly or indirectly. Upon transfer to MS hormone-free medium, most of the somatic embryos developed into plantlets, while the most of embryo-like structures could not due to lacking defined shoot meristems. However, the response to tissue culture is highly genotype dependent.Although genetic transformation protocols are now available for most of the major crop species, the protocols are applicable within each species to only a few genotypes/varieties that regenerate in vitro at high frequency. Cotyledons and hypocotyls of 30 different varieties of Brassica napus from the present varieties, used in the North-west of China, and plant breeding lines were tested for their regeneration response. The result indicated that regeneration in B. napus is highly variable and genotype specific. The frequency of regeneration among the varieties(or lines) are verity different , from 27.5% to 100% in Cotyledon, and from 33.4 to 100%. While under the stress of Cb(600mg/L) or Cef(500mg/L), which usually used as selector agent, the frequency of regeneration were decreased in different extent, indicating that both Cb and Cef can inhibit the regeneration of shoots. Two varieties were excluded as the acceptor of transformation because of the far low frequency of regeneration under the stress of the selective agent. Kanamycin was confirmed as an efficient selective agent for the transformation of Brassica napus, and the maximum transformation efficiency was very different among the different genotype of B. napus.Cotyledon and hypocotyl explants of 9 varieties (lines) of B. napus, from above test, were used to investigate the competence of regeneration of these varieties under the stress of Agrobacterium, for evaluating them, and establish an efficient Agrobacterium-based transformation experiments. The result indicated that the 9 varieties could be divided into three types according to the response to the infection with Agrobacterium: resistant type (R);supper susceptive type (SS) and middle type (M). The R type could not form regeneration shoot owing to hypersusceptibility response at the cut of the explant;the SS type could not also form regeneration shoot owing to propagate so far on the surfer of the explant;and only the M type could form regeneration shoot, but as low frequency. The frequency of regeneration shoot can be enhanced by optimizing the co-cultivate condition: using filter paper as co-cultivate support, result in not only avoiding vitrification of explant, but also prolong the time of co-cultivate, thus the cells (include transformed cells) on the explant could have enough time to regenerate. The use of AgNO3 (3~6mg/L) was a prerequisite for
efficient shoot regeneration under selective conditions, but the mount is dependented on genotype. The hypocotyl explant was more easy death infected by Agrobacterium than cotyledon explant.Glycinebetaine is an important quaternary ammonium compound that is produced in response to salt and other osmotic stresses in many organisms. In several higher plants, the synthesis of glycinebetaine requires the catalysis of choline monooxygenase (CMO) protein and betaine aldehyde dehydrogenase (BADH) protein. We transformed the BADH gene, cloned from Atriplex hortensis and controlled by two 35S promoters of the cauliflower mosaic virus, into two Brassica napus lines of breeding, PI 37 and 487, using Agrobacterium tumefaciens strain AGL1 carrying a binary vector pBin438, with cotyledon and hypocotyl regeneration system. Polymerase chain reaction analyses demonstrated that the BADH gene had integrated into the genome of B. napus. Transgenic B. napus plants showed the levels of activity of BADH were similar to or higher than wild-type plants leaves after exposure to salt stress. Observations on rooting development under the salt stress with concentrations up to 0.5% suggested that the transgenic plants exhibited tolerance to salt stress than wild-type plants. Several transformed lines resistance to Km, from CM9-transformation, were achieved from two lines of Brassica napus. The transgenic plants exhibited tolerance to salt stress than wild-type plants.In plant a transformation method to produce transgenic Brassica napus plants. The procedure included Agrobacterium-mediated inoculation of plants at various development stages along with a vacuum infiltration step. The flowering stage appeared to be the most receptive stage for transformation and production of transgenic plants. The floral-dip method for Agrobacterium-medisAed transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. Application of Agrobacterium to pollen recipient plants yielded transformants. Agrobacterium strains with T-DNA carrying BADH and CMO gene under the control of 35S. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.
引文
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