Development Of An In Situ Pistil Transformation Method In Brassica Species

Brassica crops include many important vegetables and oil crops. Conventionally genetic transformation method for these crops is Agrobacterium-mediated transformation method based on tissue culture and explants regeneration. The main disadvantages of this method are genotype dependency, low efficiency and hard to reproduce. It is urgent to develop a high throughput and genotype-independent gene transferring method to meet the genetic improvement and gene functioning when whole genome sequences are to be issued.This study describes a new high efficient method for producing transgenic Brassica plants without tissue culture and genotype dependency. The method involves placement of a liquid drop containing binary vector DNA on the cut surface of pollinated Brassica pistils. To improve the efficiency and application, pollen tube growth of different Brassica species is observed and factors of transformation are studied for optimization. Furthermore, the mechanism of this method is studied by tracing the expression of GUS gene and genetics of bar gene in different generations.The main results are as follows:1. Observation on pollen tube growth.The pollen tube growth of B. napus, B. rapa and B. oleracea is observed using aniline blue staining method to understand the interaction between pollen tube and pistil ready to be transformed. It is found that the pollen tube grows faster in B. rapa and reaches the ovary at 6h after pollination, while in B. napus and B. oleracea, tubes arrive in embryo sac after 12 to 18h.2. Set up an in situ pistil transformation procedure for B. napus cv Zhong Shuang 9.Healthy plants at the stage of 3-5 days after flowering are chosen for transformation. Keep the buds to be open in the next 1 to 3 days as receptors. Pollinate the buds. After 4 to 9h, half stigma is removed from the pistil and vector buffer is placed on the fresh surface. Then the pistils are allowed to set seed before selection. Using this method, 12 Basta resistant seedlings are selected.3. Main factors affecting transformation.1) Appropriate transformation time.The optimal timing for transformation is obtained using binary vector pGreen0229 drop on pollinated pistils at different time after pollination. The transformation rate (Basta-resistant seedlings per 100 pistils) is 1.8 % at 6 h, and then increased rapidly to 3.9% at 9h. Peaks occur at 12h and 18 h with the highest transformation rate of 4.1% and then decrease rapidly. No positive transgenic seedlings are obtained when transformation is carried later than 24h.On the contrary, seed setting rate generally increases with the delaying of transformation. Time of 15 to 18 h is shown to be the balance period of high yield and high transformation rate. The optimal time of high transformation efficiency agrees with that of pollen tube arriving in embryo sac.2) The optimal transformation buffer is tested using 0.1×SSC+10mMCaCl2, 0.1×SSC, 0.5×SSC and 1×SSC. Buffer 0.1×SSC is determined to be the best resulting to high efficiency of 5.4%.3) The optimal pistil excision for transformation is tested among three treatments, half stigma, half style and total style. It is found that removing the half stigma resulted in medium yield and high transformation rate as 3.8%.4. Molecular detection of transgene.PCR and Southern blot of Basta resistant plants confirmed bar gene transformation events. No hybridization signal is shown in non-transgenic control. One or two copies of bar gene are detected in the transformants tested through Southern blot and self pollination. .5. Inheritance and expression of transgenes.Inheritance study of seven T2 and T3 generations shows single loci of bar gene segregating in a 3:1 ratio for self pollinated progeny and a 1:1 ration for back cross progeny. The result agreed to Southern blot. Reciprocal hybrids indicated no maternal effect in 5 lines tested.Binary vector pGrGUS, conferringβ-glucuronidase gene, is used to transform B. oleracea and B. napus cv ZS9. GUS histochemical assay shows positive color in transgenic tissue. The results proved the validity of in situ pistil transformation and correct expression of foreign gene.The transformed immature seeds are analyzed using GUS assay. Three GUS patterns are found including blue embryo, blue endosperm and both blue. It suggested that gene delivery might be occurred around double fertilization, and both fertilized egg cell and polar nuclei are targets in the in situ pistil transformation method.6. Independency of species and genotypes.The method is valid on introducing different binary vectors into other six B. napus materials, two lines of B. rapa and three cultivars of B. oleracea. The transformation frequencies are ranged from 0.3% to 3.1% among B. napus and B. oleracea, while the efficiency of B. rapa is as high as 10～16%. There is no obvious genotype dependency among species.7. Mutant development.Mutants are discovered from transgenic lines and their T2 generations. The phenotype included male sterile, yellow seed color and higher oil content. One of the sterile lines 05-84 showed co-segregation of bar gene and sterility. It is suggested that the in situ pistil transformation method can be used as a mutagenesis approach.