| In recent years, crop transgenic breeding has received extensive attention for breakingthe species isolation，improving the target traits accurately, and breeding inefficient. Aefficient and stable method of genetic transformation is the precondition and key of enhancingtransgenic breeding efficiency. To Explore a fast and convenient genetic transformationmethod of wheat not only helps to expand the approaches of wheat transgenic breeding, butalso contributes to speed up the breeding new varieties of wheat.This study is aiming to establish a pollen electroporation transformation system andparticle bombardment system of another callus for common wheat by optimizing the mainparameters of transformation for obtaining transgenic plants of wheat. First, The pollens werecollected in the pollen germination buffer mixed with Gus gene and treated withelectroporation, and then pollinated Jimai19to obtain transgenic plants; The putativetransformed plants were confirmed by PCR, Southern, histochemical staining of Guspositive reaction. Secondly, Gus gene was transformed into another callus of wheat varietyXinchun9by particle bombardment. The3rd day after particle bombardment, Gus transientexpression of another callus was detected and the regenerated plant was confirmed by PCRand PCR-Southern. Finaly, the particle bombardment system with another callus as receptorswas found.The main research results are as follows:1. The average of Trypan Red and FDA staining rate was up to58.67%，by the pollenelectroporation transformation of wheat, with the pulse field strength is6kV/cm, it suggestedthat6kV/cm is the best pulse field strength; FDA staining rate was up to92.27%, keepingthe pollens on ice in4hours after electroporation, this is higher than kept the pollens in roomtemperature （69.97%）. It indicated that keeping the pollens on ice can effectively prolong theviability of the pollen in vitro; the highest seed setting was obtained, up to9.82%by theproper pollinating condition: pollen density5×106pollens·mL-1and pollinating on the5th dayafter castration.2.112folwers were pollinated by the pollen electroporation transformation of wheat,, and2positive plants were detected by PCR, and the transformation efficiency is1.79%in T1;In T2,19positive plants were detected by PCR, and the positive efficiency is40.43%;Southern blot proved that two T2transgenic wheat lines showed positive signals of Gus gene.Meanwhile, histochemical staining showed that the blue color was detected in young roots,leaves of the transgenic plants. Exogenous gene Gus can be integrated into the genomeof common wheat by the pollen electroporation transformation method and stable expressin progeny of transgenic plant.3. The3rd day after particle bombardment of wheat another callus, the Gus transientexpression of the different treatments were compared to show that the ratio average oftransient expression was the highest（up to91.11%）, when the transformation parameterswere target distance6cm and Gus:Bar=3:1; However, there were211wheat another callus tobe transformed by particle bombardment, and3positive plants were detected by PCR, whichwas the highest transformation efficiency（up to1.42%），when the transformation parameterswere target distance6cm and Gus:Bar=1:1. Exogenous gene Gus can be integrated into thegenome of common wheat by particle bombardment of wheat another callus which wasdetected by PCR-Southern.This research is basically established the pollen electroporation transformation of wheatand particle bombardment system of wheat anther callus, which laid a foundation for thetransformation of related functional genes in the future studying. |
