The Screening And Detection Of Transgenic Lisianthus With A Tobacco β-1,3-glucanase Gene And The Identification For Their Resistance To Botrytis Cinereas

Lisianthus (Eustoma grandiflorum Griseb.), an ornamental crop, is Gentianceae biennial herbs, which has high economic, viewing and admiring value, and was ranked as one of the popular cut flowers.However, like many other crops or flowers, the production of Eustoma grandiflorum is challenged by a variety of potential pathogens including fungis, bacteria and virus. Among them, the diseases of lisianthus caused by fungal pathogens accounted for over80%of the total. Therefore, it was very important to cultivate antifungal varieties of lisianthus. In the research, a construct with tobacco β-1,3-glucanase gene was transformed into’Double Mariachi Pink’Eustoma grandiflorum by Agrobacterium tumefaciens mediated transformation system, in order to cultivate new and antifungal varieties.Firstly, the expression vector pBI121with the β-1,3-glucanase gene, named pBI121-glu, was extracted from Escherichia coli stored at-80℃in a refrigerator. Then, pBI121-glu was transformed into Agrobacterium tumefacien LBA4404by electroporation, and used for transformation.Secondly, a total of35resistant seedlings were obtained by kanamycin selection. Then,21transformed seedlings out of35resistant seedlings rooted on half-strength MS medium containing0.1mg/L NAA and15mg/L Km. By three times PCR analysis,5transformants out of21rooted seedlings were PCR-positive plants. These transgenic lines were further confirmed for the transcription of the transgene by reverse transcription (RT)-PCR. And, RT-PCR analysis demonstrates that3transgenic plants out of5PCR-positive transformed plants were positive.Lastly, enzyme activity analysis showed that β-1,3-glucanase activity in the transgenic plants EG6and EG17were0.301U and0.401U respectively, and improved markedly compared with the non-transgenic plants (0.236U). But there was no significant difference between the transgenic plants EG11(0.243U) and non- transgenic plants. By a detached-leaf antifungal activity assay, the transgenic plants EG17strain’s lesion areas (0.43cm2) were significantly less than the control (2.2cm2). However, using in vivo green-house whole-plant assay, there was no significant difference between the transgenic plants and non-transgenic plants in resistance to Botrytis cinereas. This were due largely to low copy number and low expression level of foreign β-1,3-glucanase gene in lisianthus genome. Therefore, in future research, we will focused on improving the expression level of foreign gene in genome, or transforming both foreign β-1,3-glucanase gene and chitinase gene into lisianthus, in order to improve antifungal capability of transgenic lisianthus plants.