Regeneration System And Agrobacterium-Mediated Transformation In Brassica Napus L.

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 AgNO₃ 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 MShormone-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 (3⁶mg/L) was a prerequisite forefficient 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.