Study On Genetic Transformation Of Agrobacterium-mediated Rf4Restore Gene In Maize

Maize is one of the important food crops, and plays an important role onagricultural production and national economic development. Transgenic approach is aneffective means for modified maize germplasm. The utilization of cytoplasim male sterility(CMS) is the main way for using hybrid, while heterosis is an approach to improve highgrain yield and quality of several crops. In maize, the Rf4restorer gene is one importantgene for Cms-C type. In this study, the transformation technology based onAgrobacterium-mediated was modified, and the candidate gene for Rf4was transformated tomaize by means of Agrobacterium-mediated genetic transformation. The main resultsobtained were as follows:This experiment researched the receptor transformation systems using several genotypesin maize, including inbred lines Zong3,87-1, HIIA, and crosses87-1*Zong3, Zong3*87-1and87-1*HIIA. The results showed that embryo callus induction rate of difference genotypeimmature embryo had significant difference. The callus induction rate and embryonic callusinduction rate of Zong3*87-1and87-1*HIIA were97.5%,65.5%and98.4%,70.9%respectively; but embryonic induction rate of inbred lines87-1and the cross87-1*Zong3were low, the lowest was only20.2%. The callus induction rate of inbred lines Zong3andHIIA was at the middle level. Therefore, the inbred line Zong3, HIIA, and crosses87-1*Zong3as well as87-1*HIIA could be used as good transgenic receptors. Theimmature embryos of1.0-2.0mm in length were optimal for callus induction, when reachingthe length, the immature embryos of different genotypes had different embryo age.1-2mg/lof2,4-D concentration could be conducive to callus induction.1.0mg/L of6-BAconcentration could be benefit to the seedling callus differentiation, and the suitable NAAconcentration for root regenerated was0.6mg/L.This experiment demonstrated that a series of factors could affect the transformationprocess by using the cross Zong3×87-1callus as receptor. The best transformed conditionswere the bacterial concentration OD600of0.6, infection time of10min, co-culturetemperature of22℃, and cultured for3days. The PPT test also identified the criticalscreening concentration of the material was8mg/L, and the concentration was the most conducive resistant callus screening.The callus of cross Zong3×87-1was used as infecting receptor material for transformedthe Agrobacterium EHA105with Rf4candidate gene, and a total of656transgenic seedlingswere obtained. For detection of true transgenic plants, the marker of Bar gene and theprimer of terminator NOS were used, and10transgenic seedlings were identified.6of theidentified planted the seeds. Combination of T1and T2of molecular detection and fertilitysurvey results, drawed Rf4RANi of silence genes into transgenic plants can lead to malesterility, it could be a candidate genes Rf4restoring genes.