Chloroplast Genetic Transformation Of Brassica Napus Mediated By Biolistic Bombardment

Rapeseed (Brassica napus L.) is one of the most important oil-producing crops in China. It is important to improve the production and quality of rapeseed by genetic engineering. However, the foreign genes in cultivars introduced by nuclear transformation technology could escape through pollen from transgenic rapeseed plants to other closely related species, such as B. rapa or B. juncea by cross-pollination in fields. Chloroplast transformation in plants has many advantages over nuclear transformation. Chloroplast genes are inherited in a strictly maternal fashion in most angiosperm plant species including rapeseed. This minimizes the possibility of out-crossing transgenes to related weeds or species and reduces the potential toxicity of transgenic pollen to non-target insects. It will have good applications in rapeseed genetic engineering.In this study, we first established a new tissue culture system from cotyledons and leaves of elite rapeseed cultivars, which was subsequently used for chloroplast transformation by bombardment through spectinomycin selection conferred by aadA gene. Then we demonstrated for the first time that the foreign genes were integrated into the rapeseed chloroplast genome by molecular hybridization, and the foreign genes could be inherited into the next generation. All of the results laid a solid foundation for further research of chloroplast transformation in rapeseed. The main results were as follows:1. Establishment and optimization a new tissue culture system for rapeseed chloroplast transformation. Six media for callus induction and three media for shoot regeneration were compared, and results showed that calli could be retained good conditions in medium contained 0.6 mg/L 2,4-D and 0.2 mg/L KT for a long time. The calli were displayed a light green, healthy appearance, and had good potential to regenerate. The most suitable regeneration medium contained 1 mg/L 6-BA,1 mg/L KT and 0.5 mg/L NAA. Although a high concentration of 6-BA and NAA could made explants to quicly produce calli, all the calli differentiated into roots rapidly, which is not suitable for selecting chloroplast transformants.2. Screening the rapeseed genotypes suitable for chloroplast transformation. We studied the differences of callus induction and shoot regeneration abilities for 48 cultivars, and found that there was a significant difference in shoot regeneration frequency among these genotypes. The highest frequency could reach 85.42%, and the lowest was 18.75%. Cultivars FY-4, FY-15, FY-26, T13 and T15 were suitable for chloroplast transformation by their high regeneration frequency.3. Construction of rapeseed chloroplast transformation vectors. The vectors contained different lengths of trnI-trnA region from the FY-4 rapeseed chloroplast genome for integration via homologous recombination. Vectors pCL318-5 and pCL1013-8 contained 1.5 kb trnI and trnA, respectively, whereas pCL1031-F4 contained 2.5 kb trnl and trnA, respectively. The HSA gene and aadA gene expression cassette contains a 16S rRNA promoter linked to the 5'untranslated region (UTR) of the bacteriophage T7 gene 10L and a tobacco psbA terminator.4. Transformation and regeneration of transplastomic rapeseed plants from cotyledonary explants. Chloroplast transformation of FY-4 rapeseed with the vector pCL318-5 was mediated by biolistic bombardment. After selection of the explants under low selection pressure (10-20 mg/L Spec), we obtained 73 green resistant plants with high green plant percentage 0.35%. But only 19 plants obtained the foreign gene, with PCR-positive rate being 26.02%. It was obviously that it had high percentage of non-transgene under the low selection pressure. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable.5. Transformation and regeneration of transplastomic rapeseed plants from leaf explants. Chloroplast transformation of T15 rapeseed leaves with the vectors pCL1013-8 was mediated by biolistic bombardmend. After selection of the explants under high selection pressure (100-200 mg/L Spec), we obtained 7 green resistant plants with green plant percentage being 0.14%. After identified by PCR,6 plants were positive, with a PCR-positive rate being 85.71%.