The Selection And Identification Of Transgenic Eustoma Grandiflorum With Bean Chitinase Gene And The Antifungal Determination

Eustoma grandiflorum(Raf.)Shinn.,also known as prairie gentian,is a biennial herbaceous ornamental plant.It is one of the popular potted and cut flowers with high ornamental and commercial value.However,lisianthus was vulnerable to a variety of pathogenic fungi since it was introduced and cultivated in many regions of the world.Therefore,in this study the antifungal bean chitinase gene was transfered into 'Double Mariachi Pink' lisianthus leaf to cultivate lisianthus with enhanced antifungal ability.The results as follows:Firstly,the optimal concentration of cytokinin promoting shoot regeneration and the lowest concentration of kanamycin inhibiting shoot regeneration were determined to establish the optimal transformation system.Then,the agrobacterium LBA 4404 containing plasmid pBI121-Chi(the vector carrying the bean chitinase gene)stored at-80? refrigerator was activated and confirmed by colony PCR.The positive agrobacterium was dilutioned to infect lisianthus leaf blade.The 76 lines of lisianthus obtained by direct and delayed selection under Km selection pressure were proliferated and 42 lines of non-rooted lisianthus were acquired finally.Next,the 42 lines of non-rooted lisianthus were put into rooting medium for further rooting selection and 27 rooted lines were obtained ultimately.Subsequently,the rooted lines were confirmed by PCR and another PCR two months later and 11 positive lines were obtained finally.Of these 11 positive lines,five lines growing well and forming stable lines were chosed and confirmed by RT-PCR.As a result,three transcripted normally lines were identified.Finally,chitinase activities of these three transgenic lines were measured via dinitrosalicylic acid(DNS)method.The inhibitory effects on three indication fungi(Soybean fusarium 5A,5E and boytrytis cinerea)and the risistance to boytrytis cinerea of the detached leaves were determined subsequently.The results indicated that chitinase activity of these three transgenic lines(0.215U?0.225U?0.286U)were higher than non-transformed line(0.133U)and significantly different.The inhibitory effects of three transgenic lines(the size of the inhibition zone)and non-transformed line were significantly different and the inhibition zone diameter of the line 26 was longer than the other lines.The non-transformed line had an onset on the 4th day,but the three transgenic lines on the 6th day.On the 8th day,the lesion areas of the three transgenic lines(60.4,53.3,23.6 mm2)were significantly different compared with the control(128.2 mm2).