Suhandono Sony, Chahyadi Agus
Pharmaceutical Biology Research Group, School of Pharmacy, Institut Teknologi Bandung, West Java, Indonesia.
Plant Molecular Biology, School of Life Sciences and Technology, Institut Teknologi Bandung, West Java, Indonesia.
Pharmacogn Mag. 2014 Jan;10(Suppl 1):S176-80. doi: 10.4103/0973-1296.127372.
Artemisinin, a sesquiterpene lactone endoperoxide isolated from the medicinal plant Artemisia annua L., is a choice and effective drug for malaria treatment. Due to the low yield of artemisinin in plants, there is a need to enhance the production of artemisinin from A. annua and biotechnological technique may be one of the methods that can be used for the purpose.
To study the transformation efficiency of Agrobacterium tumefaciens in A. annua that could be applied to enhance the production of artemisinin by means of transgenic plants.
The factors influencing Agrobacterium-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain and effect of organosilicone surfactants. Three strains of A. tumefaciens, that is, LBA4404, GV1301, and AGL1 harboring the binary vector pCAMBIA 1303 have been used for transformation. The evaluation was based on transient β-glucuronidase (GUS).
Plant cell cultures were inniatiated from the seeds of A. annua using the germination Murashige and Skoog medium. A. tumefaciens harboring pCAMBIA were tranformed into the leaves of A.annua cultures from 2-week-old-seedling and 2-month-old-seedling for 15 min by vacuum infiltration. Transformation efficiency was determinated by measuring of blue area (GUS expression) on the whole leaves explant using ImageJ 1.43 software. Two organosilicon surfactants, that is, Silwet L-77 and Silwet S-408 were used to improve the transformation efficiency.
The transformation frequency with AGL1 strain was higher than GV3101 and LBA4404 which were 70.91, 49.25, and 45.45%, respectively. Effect of organosilicone surfactants, that is, Silwet L-77 and Silwet S-408 were tested on A. tumefaciens AGL1 and GV3101 for their level of transient expression, and on A. rhizogenes R1000 for its hairy root induction frequency. For AGL1, Silwet S-408 produced higher level of expression than Silwet L-77, were 2.3- and 1.3-fold, respectively. For GV3101, Silwet L-77 was still higher than Silwet S-408, were 1.5- and 1.4-fold, respectively. However, GV3101 produced higher levels of expression than AGL1. The area of GUS expression spots of AGL1, LBA4404, and GV3101 strains was 53.43%, 41.06%, and 30.51%, respectively.
A. tumefaciens AGl1 strain was the most effective to be transformed in to A. annua than GV3101 and LBA4404 strain. Surfactant Silwet S-408 produced the highest efficiency of transformation.
青蒿素是从药用植物黄花蒿中分离得到的一种倍半萜内酯过氧化物，是治疗疟疾的首选有效药物。由于植物中青蒿素产量较低，需要提高黄花蒿中青蒿素的产量，生物技术可能是实现这一目标的方法之一。
研究根癌农杆菌对黄花蒿的转化效率，以便通过转基因植物提高青蒿素产量。
探索影响根癌农杆菌介导的黄花蒿转化的因素，以优化转化体系，包括根癌农杆菌菌株和有机硅表面活性剂的作用。使用携带双元载体pCAMBIA 1303的三株根癌农杆菌，即LBA4404、GV1301和AGL1进行转化。基于瞬时β-葡萄糖醛酸酶（GUS）进行评估。
使用发芽的Murashige和Skoog培养基从黄花蒿种子中建立植物细胞培养物。将携带pCAMBIA的根癌农杆菌通过真空渗透法转化到2周龄和2月龄幼苗的黄花蒿培养物叶片中15分钟。使用ImageJ 1.43软件通过测量全叶外植体上的蓝色区域（GUS表达）来确定转化效率。使用两种有机硅表面活性剂，即Silwet L-77和Silwet S-408来提高转化效率。
AGL1菌株的转化频率高于GV3101和LBA4404，分别为70.91%、49.25%和45.45%。测试了有机硅表面活性剂Silwet L-77和Silwet S-408对根癌农杆菌AGL1和GV3101的瞬时表达水平以及对发根农杆菌R1000的毛状根诱导频率的影响。对于AGL1，Silwet S-408产生的表达水平高于Silwet L-77，分别为2.3倍和1.3倍。对于GV3101，Silwet L-77仍然高于Silwet S-408，分别为1.5倍和1.4倍。然而，GV3101产生的表达水平高于AGL1。AGL1、LBA4404和GV3101菌株的GUS表达斑点面积分别为53.43%、41.06%和30.51%。
与GV3101和LBA
