| Pyrethrum (Tanacetum cinerariifolium) is an economically important herbacious plant for the natural source of pyrethrins. Pyrethrins derived from pyrethrum flower serve as high effective, widely used, low toxic and eco-friendly botanical pesticide which is widely used in pest control on the field of agriculture, horticulture, home and other aspects. The average content of pyrethrins is only 1.5%-1.8% that falls short of global market demand. While traditional breeding methods exist defects such as breeding long time, distant hybridization affinity, wasting lot of manpower and material resources et al. Useing these breeding methods can not get pyrethrum lines for high pyrethrins content and yield. The development of biotechnology that provides a new approach to increase the content of pyrethrins within a short time. However, up to now, little information has been published concerning transgenic technology to transfer the key genes which are related to the biosynthesis of the pyrethrins to improve the content and quality of pyrethrins.CDS (Chrysanthemyl Diphosphate Synthase) is a key enzyme that regulates the first step of pyrethrins biosynthasis. This preliminary study of this paper showed that the tissue specific result of the Rubisco promoter and CDS promoter, and also optimized the transformation system using leaf as explants using Rubisco promoter then CDS over expression was cloned in to vector under the control of Rubisco promoter, in addition, we also studied on the pyrethrins content repeatedly subculture in vitro of pyrethrum. The main results and conclusions were as follows:1. Based on previous Agrobacterium-mediated transformation system, finally we obtained four of Rubisco::GUS resistant seedlings and six of CDS::GUS resistant seedlings, but three Rubisco::GUS plants and five CDS::GUS plants showed positive in the PCR detection for the NPTⅡ target gene. The X-Gluc staining result showed that the GUS gene with Rubisco promoter specially expressed in the green tissue, GUS activity was highly observed in the leaf of transgenic plants; on the other hand, the blue spots were only observed in the trichome of CDS::GUS transgenic plants and no GUS staining observed in other tissue.The result suggesting that CDS is a trichome tissue-specific promoter.2. This experiment optimized the pyrethrum genetic transformation system and at the same time we transformed the Rubisco::CDS overexpress vector into pyrethrum, the specific results are as follows:(1) Agrobacterium-mediated genetic transformation system using leaf as explants were optimized further by adjusting the concentration of AS and delay time. The result showed that 200 μmol/L acetosyringone in co-culture medium and about three days delay of selection obtained the highest frequency of transformation buds. The average frequency of Kan-resistence shoots was 9.81% under the 200μmol/L acetosyringone treatment; on other hand resistant shoots regeneration rate was high 8.41% with three days delay incubation.(2) Use the leaf as explants to transformed Rubisco::CDS gene into Pyrethrum genome with NPTⅡ gene as resistant marker. PCR amplification was carried out to detect the CDS gene, finally obtained 15 positive plants. At the same time, in order to detect the relative expression level of CDS and fluorescence real-time quantitative PCR was carried out with GAPDH as reference gene. And the result showed that compared with the wild-type pyrethrum, CDS gene relative expression level increased in the transgenic pyrethrum plants, the expression level of CDS gene was about 20 times than the wild-type pyrethrum plants.3. The contents of pyrethrins, pyrethrins I and pyrethrinsⅡ of pyrethrum leaves were determined by RP-HPLC, and the growth vigor of pyrethrum plantlets in vitro with 5 subculture times were also analyzed.The results showed that the content of pyrethrins, pyrethrins I and pyrethrins II of the leaves were increased during the first three subculture times, and reached the highest respectively 1.997 mg/g,1.609 mg/g and 0.388 mg/g at the third time. At the fourth and fifth subculture time, the pyrethrins content decreased significantly. Meanwhile, subculture times had an impact on the vigor and roots growth of plantlets. The average root number and root length were both decreased, but the plantlets at the third subculture time were more vigorous with stronger roots than the first subculture time. At the fifth subculture time, the plantlets showed slender foliage, slower growth phenomenon than other four subculture times. Indicated that the leaves of pyrethrin content of genotype of ’F1-10-4’ genotype was strongest growth vigor at the third subculture time. Further subculture time did not improve leaves’ pyrethrins contents and growth of pyrethrum, and it could increase labor cost. |
PDF Full Text Request