Study On Groundcover Chrysanthemum(Chrysanthemum Morifolium) Transformation Using Pmi As Selective Safety Marker

Transgenic technology has long been widely used to enhance breeding purposes and predictability. But its limit factors were more obvious, one of which is the safety problem of selective marker gene (SMG). SMG does help to identify transformants. But after completion of the identification, SMG not only lose its value, but also poses unwanted security risks for humans and the environment. Therefore, the study of safety transgenic technology is of great significance to protect human health and to maintain the stability of the ecological environment. This study addressed the safety issues of SMG in transgenic chrysanthemum using phosphomannose isomerase gene (pmi) as safety SMG. And the main conclusions can be found as following points:1. Construction of transgenic expression vector pCAMBIA1301-pmi and pCAMBIA1301-pmi-hpt were completed, using phosphomannose isomerase gene as safety marker gene. The above two vectors were transformed into Agrobacterium strain EHA105,and prepared two engineering strains, which were named as EHA105-pl301-pmi and EHA105and-p1301-pmi-hpt that can be used to infect plant.2. The differentiation culture medium of ground cover chrysanthemum cultivar’Beilin Huang’ was optimized as:MS+6-BA1.5mg/L,+NAA1.2mg/L, on which regeneration frequency were stable and differentiation rate up to83.3%; The differentiation culture medium of ground cover chrysanthemum’Jinding Hongxin’was optimized as:MS+6-BA1.0mg/L+NAA0.5mg/L, on which regeneration frequency were stable and differentiation rate up to66.67%.3. The safety transgenic system was established for ground cover chrysanthemum cultivar ’Beilin Huang’:mannose selective concentration for the zero-generation tissue cultured leaves was firstly12g/L mannose+18g/L sucrose to reduce false positive phenomenon then dropped to6g/L mannose+22g/L sucrose to enhance differentiation rate; for the high generation (subcultured more than five times) tissue cultured leaves, was firstly lOg/L mannose+20g/L sucrose to reduce false positive phenomenon then dropped to4g/L mannose+26g/L sucrose to enhance differentiation rate. Simultaneously, ground cover chrysanthemum’Jinding Hongxin’was highly sensitive to mannose, and therefore was inappropriate to mannose positive selection transgenic system.4. Transgenic ground cover chrysanthemums system using pmi as selective safety marker, whose PCR positive rate was12.7%, and positive rate both identified by PCR and chlorophenol red assay was7.5%, while the tranditional hygromycin marker system PCR positive rate was3.2%.Consequently, ground cover chrysanthemums transformation system using pmi as selective safety marker was proved to be more efficient and more safety than traditional transformation hygromycin marker system.For the first time, the safety transgenic system was established for ground cover chrysanthemum cultivar’Beilin Huang’using pmi as selective marker gene, avoiding the involvement of traditional antibiotics or herbicide and the occurrence of safety hazards, while improving the conversion rate. Therefore, this study was carried of vital importance on theoretical value and social benefits.