Alam Pravej, Khan Zainul Abdeen, Abdin Malik Zainul, Khan Jawaid A, Ahmad Parvaiz, Elkholy Shereen F, Sharaf-Eldin Mahmoud A
Sara Alghonaim Research Chair (SRC), Biology Department, College of Science and Humanities, Prince Sattam bin Abdulaziz University (PSAU), Alkharj, 11942, Kingdom of Saudi Arabia.
Department of Biosciences, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
3 Biotech. 2017 May;7(1):26. doi: 10.1007/s13205-016-0593-5. Epub 2017 Apr 11.
Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L (BAP) and 0.5 mg L (NAA) while 80% shoot percentage obtained with 4.0 mg L (BAP) and 0.05 mg L (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.
长春花是一种重要的药用植物，以其抗菌、抗癌、拒食、抗不育、抗糖尿病等药理特性而闻名。这种植物已合成了130多种生物活性化合物，如长春碱、长春多灵和长春新碱。人们已经对优化再生和转化方案进行了广泛研究。所描述的大多数方案都既费力又耗时。由于再生和遗传转化方案复杂，这些生物活性分子的产量较低，无法在全球范围内商业化。在此，我们优化了高效的再生和转化方案，以缩短时间并通过农杆菌介导和超声辅助转化（SAAT）方法提高转化频率。在本研究中，下胚轴外植体对转化芽的最大产量反应最佳。在添加1.0 mg/L（苄氨基嘌呤）和0.5 mg/L（萘乙酸）时愈伤组织诱导率为52%，而在添加4.0 mg/L（苄氨基嘌呤）和0.05 mg/L（萘乙酸）时芽诱导率为80%。显微镜研究表明，在转化pRepGFP0029构建体后，绿色荧光蛋白（GFP）的表达明显定位于长春花的叶片组织中。因此，基于GFP定位揭示了转化效率。SAAT方法的转化效率为6.0%，而传统方法为3.5%。此外，PCR分析证实了nptII基因整合到了长春花转化植株中。
