Mousavi Mousa, Mousavi Amir, Habashi Ali Akbar, Dehsara Bahareh
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran,
Mol Biol Rep. 2014 Dec;41(12):8185-94. doi: 10.1007/s11033-014-3720-6. Epub 2014 Sep 9.
In this study, an efficient transformation system for gene delivery in date palm was established. The effects of different physical and biological parameters were optimized for transient transformation of uidA gene in somatic embryos of Estamaran cultivar. The tissues were bombarded with constructs harboring the uidA gene driven by CaMV 35S or rice Act1 promoter. Efficiency of expression was estimated by comparison of the number of blue spots resulted from GUS assay. Optimal transient expression was observed when explants were precultured on a media containing 0.4 M mannitol with air desiccation and bombarded at acceleration pressure of 1,350 psi, target distance of 6 cm with gold particles size of 0.6 µm which coated with 2.5 µg of DNA and at chamber vacuum pressure of 28 inHg. Significantly higher expression levels were obtained in tissues when the construct having the Act1 promoter was employed. After bombardment, somatic embryos were transferred to the regeneration media containing MS basal salts supplements with 3 mg/l 2ip, 40 mg/l adenine, 1 mg/l 2,4-D, 30 g/l sucrose and 3 g/l activated charcoal. Regenerated plantlets were checked by PCR using gene-specific primers. About 16 % of the plantlets were reported to be stably transformed. Southern analysis of genomic DNA from transformed plants showed that 1-2 gene (uidA) copies were integrated and GUS-negative plants did not contain any transgene. Achievement of these data considered as the first report of its kind is believed to facilitate transfer of desirable traits in date palm.
在本研究中,建立了一种用于枣椰树基因传递的高效转化系统。针对Estamaran品种体细胞胚中uidA基因的瞬时转化,对不同物理和生物学参数的影响进行了优化。用携带由CaMV 35S或水稻Act1启动子驱动的uidA基因的构建体对组织进行轰击。通过比较GUS检测产生的蓝色斑点数量来估计表达效率。当外植体在含有0.4 M甘露醇的培养基上预培养并进行空气干燥,以1350 psi的加速压力、6 cm的靶距离、0.6 µm的金颗粒大小(包被2.5 µg DNA)以及28 inHg的腔室真空压力进行轰击时,观察到最佳瞬时表达。当使用具有Act1启动子的构建体时,组织中获得了显著更高的表达水平。轰击后,将体细胞胚转移到含有MS基本盐补充物、3 mg/l 2ip、40 mg/l腺嘌呤、1 mg/l 2,4-D、30 g/l蔗糖和3 g/l活性炭的再生培养基中。使用基因特异性引物通过PCR对再生植株进行检测。据报道,约16%的植株被稳定转化。对转化植株的基因组DNA进行Southern分析表明,整合了1 - 2个基因(uidA)拷贝,而GUS阴性植株不含有任何转基因。这些数据的获得被认为是此类研究的首次报道,有望促进枣椰树优良性状的转移。