Study On Agrobacterium-Mediated Transformation Of Narcissus Tazetta Var. Chinensis And NTLEAFY Gene Expression During The Flower Differentiation

Narcissus tazetta var. chinensis is a famous traditional Chinese Flower with higheconomic and ornamental value. As a homologous triploid plant, it is difficult to createdesirable varieties by traditional hibridization. For thousand years, only two varieties such as'jinzhanyintai' and 'yulinglong' have cultured in China. As the development of biotechnology,transgenic technology provides an effective way for the Chinese narcissus varieties innovation.The estabilishment of genetic transformation system and the exploration the potential geneticresources in the narcissus is a necessary prerequisite for genetic engineering breeding ofnarcissus.Peduncle was the best explants compared with other organs in this study. The bestexplants was peduncles. It has three advantages in easy sterilization, easy for callus inductionand subculture and the time of duration short. The media for callus induction and shootsregeneration from peduncles were based of MS+30g·L^-1sucrose with1g·L^-1NaH2PO4+0.003g·L^-16-BA+0.001g·L^-1NAA+0.2g·L^-1Ad and the rate of callus induction was43.33%. The process of callus from peduncles to the bulbs bud differentiation required30～35d, which was shorter50d than that from other explants. Shoots were regenerated bypeduncles callus for histological examination in the process of callus from peduncles todifferentiation.Agrobacterium tumefaciens(LBA4404) was the best for infection of peduncles callusamong four kinds of Agrobacterium stains.100mg·L^-1AS was good for A. tumefaciensactivation.15days co-cultivation and3days preculture were good for transformation.Cefotaxime was suit for inhibitting A. tumefaciens and the concentration was400mg·L^-1.Positive shoots got through the above conditions by GUS coloration analysis from peduncles.The results of PCR detection and Southern blot showed that NTLEAFY were inserted intonarcissus genome through the above-mentioned methods. The observation started from early July and went through seven phases, which were leafbud formation, inflorescence primordium differentiation phase, spathe primordia informationphase, sepal differentiation phase, petal differentiation phase, stamen differentiation phase andpistil differentiation phase and is over at pistil fromed in mid-september. Tissues were selectedin each phases and real-time quantitative PCR was used to analyze NTLEAFY gene expression.The expression level of NTLEAFY gene in the period of petal differentiation phase was muchhigher than the initial period. In the scales the gene expression level fell gradually during theflower bud differentiation with higher expression in the inflorescences. The results suggestedthat NTLEAFY gene controled flowering time and related to the flower development.