Ali Shawkat, Mannan Abdul, El Oirdi Mohamed, Waheed Abdul, Mirza Bushra
Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan.
BMC Res Notes. 2012 Jun 12;5:285. doi: 10.1186/1756-0500-5-285.
Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants.
Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L(-1) along with 250 mg L(-1) each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L(-1) of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis.
Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.
粗柠檬（Citrus jambhiri Lush.）是南亚最常用的柑橘属砧木。它对盐胁迫极为敏感，这降低了全球许多地区柑橘作物的生长和产量。酵母耐盐基因（HAL2）的过表达导致转基因植物耐盐水平提高。
利用携带质粒pJRM17的根癌农杆菌LBA4404菌株对粗柠檬进行转化。在含有100 mg L(-1)卡那霉素以及各250 mg L(-1)头孢噻肟和万古霉素的培养基上筛选转基因芽，以有效抑制农杆菌生长。含有200 μM乙酰丁香酮（AS）的Murashige和Skoog（MS）培养基被证明是转化的最佳接种和共培养培养基。添加3 mg L(-1) 6-苄基腺嘌呤（BA）的MS培养基显示转化外植体的再生效率最高。基于PCR和Southern印迹分析对转化植株进行最终筛选。
已通过根癌农杆菌成功将β-葡萄糖醛酸酶（GUS）和HAL2基因转入粗柠檬。还研究了影响基因转化和再生效率的各种因素。
