Transform Fine Inbred Lines Of Maize By Floral-dip Method Of Genetic Transformation

Plant genetic transformation technology has become an extreme important aid for genetic improvement of crops.Currently,there are two major categories of plant genetic transformation:vector without exogenous genes directly into transformation and Agrobacterium-mediated transformation.Floral dip by Agrobacterium-mediated transformation is a in planta permeability transformation.It is a simple method of genetic transformation which is a non-tissue culture or non-plant regeneration process transformation.However,the application in maize genetic transformation is rarely reported.This study tried to use the floral-dip by Agrobacterium-mediated transformation to transform maize in fields and for the first time to use the method on male inflorescence of maize.Preliminary explored whether the Floral dip by Agrobacterium-mediated method can be used for genetic transformation of maize.The main results obtained were as follows:1,Pointed out that the period of maize floral dip method as follows:when the female inflorescence hold out 5～10cm,the flowers on the main axis of male inflorescence to blossom out less than 30%.The method was to be transformed into the female flowers of maize or male inflorescence dipped into the culture medium containing Agrobacterium or sprayed into the culture medium containing Agrobacterium in the plant's female / male inflorescence;for female flowers motif could also extract the culture medium using a syringe to insert between the bract and the ear,then pushed it into,until the culture medium overflowed from top of the ear.2,The culture medium containing Agrobacterium make the N6 medium as the basic nutrients.The appendages were surfactants CSO40,Triton X-100,acetosyringone,a purity of 92%of the GA,6 - Benzyl amino purine gland Yin,growth hormone,hydrolysis of casein,L-proline,and mannitol(to replace the sugar).3,GFP expression observed results showed that:the male and female inflorescence of maize were sensitive to Agrobacterium;the TO generation of seeds of transformed male and female flowers both had the GFP expression,which thing showed that the T-DNA carried by gfp gene had been integrated into the plant genome;the optimal cell concentration of Agrobacterium transformation was OD₆₀₀ = 1.0.4,Through hygmycin selection,the result showed resistance of seedlings of male-positive rate of 12.9%higher than the seedlings of the female flower positive rate of 3.3%.5,The result of PCR of positive plants showed that:T-DNA carrying exogenous gene hpt had been integrated into the plant genome.Based on the above findings,the conclusions can be drawn:Agrobacterium-mediated transformation floral dip can be used for the genetic transformation of maize,and had a higher conversion efficiency.It is a new method of genetic transformation of maize.