Effect of two key treatments on the transformation efficiency of fenugreek hairy roots
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摘要: 葫芦巴（Trigonella foenum-graecum L.）是薯蓣皂素源植物之一，本文以来源于中国山西省的一年生草本植物葫芦巴为实验材料，研究发根农杆菌菌液浓度与超声波辅助处理两个因素对葫芦巴发根转化率的影响。结果显示，随着菌液浓度升高，发根数和发根转化率均逐渐升高，中浓度与低浓度菌液相比，发根数量和转化率分别升高了2.58和3.90倍；利用高浓度菌液侵染获得的发根数量和发根转化率最高，分别是低浓度菌液的7.33和4.32倍。在超声波处理实验中，超声（工作频率40 KHz，超声功率180 W）处理30 s与经未超声处理的对照组相比，发根数量及转化率略有下降；当超声处理60 s时，发根转化率明显上升，为对照组的2.48倍。葫芦巴发根体系的建立可为生产薯蓣皂素以及解析薯蓣皂素合成路径提供一个关键的技术平台。Abstract: Plant hairy roots are an important platform for the synthesis of valuable secondary metabolites and for the functional analysis of natural product biosynthetic pathways. Diosgenin is a precursor of more than three hundred sterol drugs, but knowledge regarding how it is biosynthesized in plants remains limited. Fenugreek (Trigonella foenum-graecum L.) is a plant with high diosgenin content. In this study, we used a fenugreek variety from Shanxi Province in China to investigate the effects of two key factors on fenugreek hairy root formation. The two key factors were the density of Agrobacterium rhizogenes and co-incubation supplemented with ultrasonic treatment. Hairy root number and its transformation frequency were improved under increasing A. rhizogenes density, with OD600 values from 1.1 to 4.4. Compared with that measured at the OD600 value of 1.1, the incubation at 2.2 and 4.4 led to better performance, with hairy root numbers increasing 2.58 and 7.33 times, respectively, and root transformation frequency improving 3.90 and 4.32 times, respectively. During the co-incubation of A. rhizogenes with fenugreek explants, 30 s ultrasonic treatment (operating frequency 40 KHz, ultrasonic power 180 W) slightly decreased the efficiency of hairy root induction, whereas 60 s treatment increased the efficiency by 2.48 times compared with incubation without ultrasonic treatment. The established fenugreek hairy root system provides a platform for the synthesis of diosgenin and a strong basis for the analysis of diosgenin biosynthesis pathways in the future.
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Keywords:
- Fenugreek /
- Hairy root /
- Agrobacterium rhizogenes
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