Study Of Agrobacterium Tumefaciens-Mediated Genetic Transformation Of Grape Hyacinth

Grape hyacinth (Muscari) is a perennial monocotyledonous herbaceous flower bulbs, which is belongs to Hyacinthaceae family. It has many small flowers looking 1 ike a string of bells. It also has a long florescence and green leaf stage, which has a high ornamental, ecological and scientific value. Grape hyacinth has a simple genetic basis of pigments formation. As it was known that more than 40 kinds and cultivars, grape hyacinth flower color is varying degrees of blue except for a few white color mutants, which makes it become ideal material model to study monocots blue flower formation. However, since traditional cross-breeding was not only difficult to emasculat e and pollinate artificially but also takes three years at least from seed to flo wering, which limits color-related gene function verification to some extent, thus 1 imiting the use of molecular biotechnology progress color breeding. In this stu dy, optimizing the efficiency of Agrobacterium-mediated transformation of grape hyacin th, to establish efficient genetic transformation system of grape hyacinth and lay the f oundation related gene functional verification of grape hyacinth.In this study, we used material grape hyacinth Muscari armeniacum and Mu scari ’pink sunrise’ to establish plant regeneration system of grape hyacinth, and make sure the kanamycin and hygromycin resistance force of grape hyacinth. Then transferr ing pCAMBIA1301 vector which T-DNA region carrying the GUS and Hyg gene into grape hyacinth callus by Agrobacterium-mediated method and optimizing the conversio n conditions. With the best conditions transformed to grape hyacinth to obtain resistant plants. The main results of the study were as follows:1. Leaves of ’Muscari armeniacum’ and Muscari ’pink sunrise’ of grape hyaci nth were used to induce callus. The best medium for callus differentiation of two spe cies was MS+01mg·L-1TDZ+0.5mg·L-12,4-D+30g·L-1Sucrose+3g·L-1Phytage, which callu s induction rate were 100%,80% respectively. It was had been further studied that th e best medium of callus induced to buds of grape hyacinth, found that Muscari arme niacum best medium was MS+1mgL-16-BA+30g·L-1Sucrose+3g·L-1Phytage, the inductio n rate was 83.33%; The best medium of Muscari ’pink sunrise’ to induce buds was MS+0.1mg·L-16-BA+0.1mg·L-11TDZ+30g·L1Sucrose+3g·L-1Phytage, the induction rate w as 68.75%.2. When inducing the buds to growth roots of grape hyacinth, we found that two species could induce the occurrence of roots, Muscari armeniacum’s roots were t nicker and shouter than the Muscari’pink sunrise"s. Then about 8cm high aseptic wit h roots was transplanted to nutritional bowl and incubated under artificial climate gree nhouse conditions, the survival rate reached 90%.3. Muscari armeniacum and Muscari’pink sunrise’callus was tested for the sensitivity of kanamycin and hygromycin, which was found that grape hyacinth callus was insensitive to kanamycin. Kanamycin couldn’t inhibit the growth of grape hyacint h callus. The concentration of hygromycin resistance screening found that Muscari arm eniacum callus lethal concentration of hygromycin was 210mg·L-1. Muscari’pink sunri se’Callus lethal concentration of hygromycin was 90mg·L-1.4. Different Agrobacterium tumefaciens concentration, different infection time, different co-culture time was used to transform Muscari armeniacum and Muscari’pi nk sunrise’callus, It was indicated that trends of GUS transient expression rate of the two species were very similar, GUS transient expression efficiency of each transform ation processing emerged rise firstly, then down. When the Agrobacterium tumefaciens concentration was 0.5-0.8, GUS transient expression efficiency reached the highest. T herefore, the optimal Agrobacterium tumefaciens concentration ODeoo was 0.5-0.8. Whe n infection 20min, GUS transient expression efficiency reached to maximum, Muscari armeniacum GUS transient expression efficiency was 13.73%, which was 1.3 times co mpared to Muscari’pink sunrise’. When callus was co-cultured 4d, GUS transient expr ession efficiency of Muscari armeniacum was 10.85%, which was less different with Muscari’pink sunrise’. Therefore, the best genetic transformation system of grape hya cinth was OD6oo=0.5-0.8, infection 20min, co-culture 4d.5. Since Agrobacterium was unnatural host of monocots, therefore GUS transi ent expression of this study was so low, in order to improve the efficiency of genetic transformation of grape hyacinth, Muscari armeniacum callus was ultrasonic treated, when the ultrasonic pretreatment 3min, GUS transient expression efficient was 75.36%, which was five times compared to without ultrasonic pretreatment.6. In this study, EHA105 strain was used, which has high efficiency for mon ocotyledons trasformation. It was transformed to grape hyacinth Muscari armeniacum a nd Muscari’pink sunrise’callus with the system OD6oo=0.5-0.8, infection 20min, co-cu lture 4d,80W sonication pretreatment 3min. As results showed that Agrobacterium-me dialed Muscari’pink sunrise’genetic transformation did not get resistant plants, while Agrobacterium-mediaied grape hyacinth Muscari armeniacum transformation obtained 2 07 hygromycin plants, GUS histochemical analysis showed that in different parts of th e resistant plants emerged with varying degrees of blue, there were 7 PCR positive pi ants, suggested that the foreign genes was integrate into the plant genome.