Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

• Main Authors: Wang Quan, Xing Shihai, Pan Qifang, Yuan Fang, Zhao Jingya, Tian Yuesheng, Chen Yu, Wang Guofeng, Tang Kexuan
• Format: Article
• Language: English
• Published: BMC 2012-06-01
• Series: BMC Biotechnology
• Subjects: <it>Catharanthus roseus</it>; <it>Agrobacterium tumefaciens</it>; Deacetylvindoline-4-<it>O</it>-acetyltransferase; Regeneration; Vindoline
• Online Access: http://www.biomedcentral.com/1472-6750/12/34

<p>Abstract</p> <p>Background</p> <p>As a valuable medicinal plant, Madagascar periwinkle (<it>Catharanthus roseus</it>) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in <it>C. roseus</it>, the establishment of an efficient genetic transformation method is required.</p> <p>Results</p> <p>To develop a genetic transformation method for <it>C. roseus</it>, <it>Agrobacterium tumefaciens</it> strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report <it>β</it>-glucuronidase (<it>GUS</it>) gene and a selectable marker neomycin phosphotransferase II gene (<it>NTPII</it>). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, <it>A. tumefaciens</it> infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of <it>GUS</it> gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, <it>DAT,</it> which encodes deacetylvindoline-4-<it>O</it>-acetyltransferase, was transferred into <it>C. roseus</it> using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of <it>DAT</it> increased the yield of vindoline in transgenic plants<it>.</it></p> <p>Conclusions</p> <p>In the present study, we report an efficient <it>Agrobacterium</it>-mediated transformation system for <it>C. roseus</it> plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of <it>A. tumefaciens</it> mediated transformation and regeneration of <it>C. roseus</it>. More importantly, the <it>C. roseus</it> transformation system developed in this work was confirmed in the successful transformation of <it>C. roseus</it> using a key gene <it>DAT</it> involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.</p>