Establishment Of Agrobacterium-Mediated Anther Transformation System Of Wucai (Brassica Campestris L.) In Planta

Wucai?Brassica campestris L.ssp.Chinensis?is a biennial herb and belongs to a variety of Brassica subspecies of Cruciferae.Because of its high vitamin content,Wucai is also called vitamin dish.As one of the most important varieties of leaf vegetables in Jianghuai River Basin,wucai is cultivated in most areas of China,especially in Anhui,Jiangsu,Shanghai and other places.It is an ideal vegetable for regulating"winter shortage"and"spring light".In recent years,some traditional breeding methods are used to improve the quality and yield of Wucai,such as heterosis breeding method and so on.However,these traditional breeding methods need more time,and have low efficiency and uncertain breeding results,which limit the development of breeding in Wucai.The transgenic technology provides a possible approach to the directional breeding for Wucai.Agrobacterium-mediated method is the one of the most commonly method used in plant genetic transformation.This method has several advantages,simple and convenient operation,low cost and short cycle,which can be applied to develop the new varieties.However,the method of genetic transformation based on tissue culture needs heavy workload and has low efficiency.The method of Agrobacterium-mediated anther transformation in planta is simple and easy,in which the tissue culture is not necessary.Thus,this method can save a lot of time and provide an effective and rapid way to obtain transgenic plants for crop genetic improvement.In this study,a novel and efficient Agrobacterium-mediated anther transformation method in planta was developed based on the optimization of several factors that affect anther transformation and the biological mechanism of genetic transformation was analyzed.Main research results:1.Several factors affecting anther transformation were optimized.The best results in terms of GUS expression were obtained from the sepals and petals-peeled buds of the size3-5 mm that were inoculated with Agrobacteria using the liquid droplet inoculation method and dark cultured for 2 days.In addition,the Agrobacteria were suspended in CCM?p H 5.8?,which contained MS medium,2.0 mg?L^-1 BAP,40 mg?L^-1 As,0.03%Silwet,0.5 g?L^-1 MES and 5%sucrose.Applying this optimized procedure,the transient expression frequency were 91.59%.2.The biological mechanism of Agrobacterium-mediated anther transformation method was analyzed.The GUS histochemical assay revealed that the male germ line was the predominant target in this transformation.