Agrobacterium-mediated Transformation Of Anti-fungal Genes Into Maize Shoot Apical Meristem Using A Double T-DNA Plant Expression Vector

Maize (Zea mays L.) is one of the most important crops in the world for its importantrole it plays both in agriculture and industry. But it is constantly subjected to invasive fungaldiseases (northern leaf blight, southern leaf blight, ear and stalk rots, etc.) during its growthwhich has seriously affected its yield and quality. In normal years, these diseases makesmaize yield lost by10%and the percentage may be increased by30-40%in their popularyears. However, it is very difficult for conventional breeding to develop an elite maizematerial with the resistance to all the fungi disease. Therefore, we can use transgenic methodto transfer some genes related to the resistance of these disease into maize genome in orderto obtain resistance materials.By this way we can accelerate the process for diseaseresistance breeding.Chitin and β-1,3-dextran is the main ingredient of most of the fungal cell wall, andchitinase (chitinase, Chi, EC3.2.1.14) β-1,3-and glucanase (Beta-1,3-glucanase, Glu, EC3.2.1.39) catalytic hydrolysis reaction of the two, respectively. Chitinase and β-1,3-glucanasehas a very strong synergistic effects, the chitinase production is often accompanied by β-1,3-glucanase and the formation of other disease-resistant proteins.What is more, they canwork together to play the role of defensing pathogenic fungal infection in plants. Sointroducing the two hydrolase genes Chi and Glu into plant genome will greatly enhanceplant resistance.Selection of transgenic receptor is an important part in maize transformation, selectingthe appropriate receptor materials can highly improve the transformation and regenerationefficiency. Agrobacterium mediated transformation of the wounded shoot apical meristem(SAM) of germinated seedlings circumvented the long period of tissue culture steps andlimitation of different seasons, moreover, many elite inbred lines which are recalcitrant incallus induction can be efficiently transformed by this method.With the development of the transgenic technology, the security problems produced bythe marker gene (such as antibiotic resistance genes and herbicide resistance gene) intransgenic crops have aroused people’s attention. The development of safe genes used in thescreening of transformed plants or methods to produce marker-free transgenic crops becomes a new direction of plant transgenic research. The double T-DNA expression vectorcan achive this goal efficiently.In order to get the fungal disease resistant maize germplasm materials.we haveconstructed a double T-DNA plant expression vector carried a fusion gene, then transformedinto maize shoot apical meristem mediated byAgrobacterium, the results were as follows:1. The pCAMBIA1300-EPSPS was used as the basic vector, after removal of theunnecessary restriction site, we abtained the intermediate vectors pCAMBIA1300-EPSPS/HEand pCAMBIA1300/HX.2. The boundary district was amplified using the polymerase chain reaction (PCR)technology, the abtained fragment was connected into the Sph I site of the intermediatevector pCAMBIA1300-EPSPS/HE, after transformed into the competent cells of DH5α，weabtained a double T-DNAplant expression vector pCAMBIA1300-EPSPS-2T.3. An optimized transformation system for Agrobacterium mediated transformation ofshoot apical meristem was using an optimal concentration of bacterial culture of LBA4404(the value of OD600was0.6) for infection, addition of150μM acetosyringone (AS) in thebacterial suspension, the whole infection process was carried out in a vacuum desiccatorswith a negative pressure of50KPa for12minutes.4. After identified by herbicide screening procedure and PCR detection,13transformedplants were obtained among the33herbicide-resistant plants, and the overall transformationefficiency was2.6%.The preliminary evidences showed that the foreign genes had beenintroduced into the maize genome.