IFAS, CREC, University of Florida, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA.
Biochemistry Department, College of Agriculture, Zagazig University, Zagazig, 44511, Egypt.
Transgenic Res. 2018 Apr;27(2):179-191. doi: 10.1007/s11248-018-0065-2. Epub 2018 Feb 14.
Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.
利用遗传工程方法对柑橘属“W. 默科特”蜜橘（由‘默科特’和未知花粉亲本杂交育成）进行品种改良是目前全世界许多地区最具商业价值的品种之一。利用原生质体转化系统，通过直接 DNA 摄取实现了对‘W. 默科特’蜜橘的转化。该 DNA 构建体（pAO3）编码了绿色荧光蛋白（GFP）和来自水稻的 Xa21 基因的 cDNA，用于转化‘W. 默科特’蜜橘的原生质体。继柑橘原生质体培养和再生之后，通过 GFP 表达对转化的小愈伤组织进行了显微镜下鉴定，将其与未转化组织物理分离，并在体细胞胚发生诱导培养基上进行培养。共回收了超过 150 个转基因胚，从中再生了 10 个转基因系，并在生根培养基上进行了茎伸长培养。对转基因芽进行微嫁接并在温室中建立，每个系有 3-5 个重复。通过 PCR 和 Southern blot 分析证实了 Xa21 和 GFP 的插入。通过荧光显微镜验证了 GFP 的表达，通过 Western blot 分析显示尽管在不同的转基因系中表达水平有所不同，但 Xa21 得到了表达，这一点通过 RT-qPCR 得到了证实。通过注射器接种柑橘溃疡病菌对转基因植株进行了挑战，与非转基因对照植株相比，转基因植株的病斑数量和病斑内细菌数量均有所减少。通过从胚性愈伤组织进行原生质体转化，将水稻中的 Xa21 基因导入‘W. 默科特’蜜橘，获得了溃疡病抗性增强的转基因系，目前正在田间条件下进行评估，以验证其抗性水平。
