Application Of Two Agrobacterium-mediated Transgenic Methods In Kentucky Bluegrass

The aim of this study is to explore the transgetic method of tumefaciens-mediated system through trying different receptor materials of Kentucky bluegrass,included callus and seeds,as we called callus method and seed method.We transfered two plant expression vectors(pTCK303-PpGBF1、pTCK303-PpGAMT1),obtained some types of transgenic plants,and compared these two genetic transformation methods in detail.The main research results of the experiment are as follows.1.In the experiment of seed method,we used the bacterial liquid with OD600 of 0.9,added 3 different receptor materials and vacuum 3 min,shaking at a low speed shaker of 100rpm for 10 min,then transfer them to the filter paper in a petridish for 3days in the dark.Then we using screening liquid selected for 30 days on the filter paper,transfered the green seedlings into soil,extracted total DNA after 30 days,The results showed that the positive rate of PCR identification of seed method was higher when the sprouts of the seeds were infested,finally we obtained 39 PCR-positive plants.2.In the experiment of callus method,we found that when OD600 of the bacterial liquid was 0.3,and the infection time was 15 min,or OD600 of the bacterial liquid was 0.6,and the infection time was 10 min,we can get the best transgetic effect.It can be seen that after infection,the callus growed some new tissue and the Agrobacterium was not overgrowed,the quantity of PCR-positive plants was 2.finally we obtained 2 PCR-positive plants.In this study,we constructed the transgetic system of Agrobacterium using callus method in Kentucky bluegrass,and explored the possibility of using seed method in Kentucky bluegrass transgetic system for the first time.It was found that the seed method can shorten the test cycle of the genetic transformation of Kentucky bluegrass,these research have certain value in this field.