A Practical Approach to Obtain Phytase Transgenic Maize Germplasm Via Ultrasound-mediated Pollen Transformation

Title A Practical Approach to Obtain Phytase Transgenic Maize Germplasm Via Ultrasound-mediated Pollen Transformation

As one of most important cereal crops in the world, maize is consumed for food,feed and industrial raw materials. For forage usage, maize is rich in phosphorusmainly existed in the form of phytate. Monogastric animals, however, can not utilizethe most stored phosphorous in maize due to the shortage of phytase, resulting inphosphate pollution problems caused by the excretion of phosphorus in manure. It istherefore necessary to improve the availability of phosphorus form with enhancingphytase content of maize seeds using biotechnology Engineering.In this study, five different elite inbred lines, which are Zhengdan958, line78559, Si487, Zheng58and Nongxi531, respectively, were selected as explants, andthe expressional vector harboring phytase gene was constructed with herbicide as aselectable marker gene. The foreign phytase gene was introduced into the genome ofmaize by ultrasound-mediated pollen transformation and the transgenic maize plantsexpressing phytase gene were obtained after herbicide selection and molecularidentification.The main results are shown as follows:1. Construction of expression vector for genetic transformation. Based on thebackbone of vector PCAMBIA3300, the GUS gene in this vector is replaced withforeign phytase gene (phy), while35S promoter is changed by ubi promoter which isfeasible for maize transformation, and finally the vector was constructed successfullyverified by digestion.2. Optimization of sucrose concentration. The pollen grains of five maize inbredlines are dipped in sucrose solution for screening suitable concentrations in which thepollen grains can germinate normally. The results showed that when the sucroseconcentration is15%, the germination efficiency of pollen grains from five maizeinbred lines exhibited better than that in other different concentrations in which some pollen grains failed to germinate, and others germinated poorly or even rupture.3. Achievement of herbicide-resistant transgenic plants. Putative transgenicplants were sprayed using600ppm and achieve herbicide-resistant transgenic plants.4. PCR results investigated that the exotic phy gene and herbicide-resistantselectable marker gene already integrated into the genome of maize. Moreover, RT-PCR amplified positive bands of phy gene from several transgenic plants exhibitedthat phy gene expressed at least at transcriptional level.5. The expression of bar gene in putative transgenic plants was tested by Quick-strip Kit and some of them showed positive, meaning bar gene was translated intoprotein normally.In summary, this protocol for ultrasound-mediated pollen transformation cantransfer the foreign gene(s) into the genome of explants and is feasible for maizegenetic transformation with the advantages of simple operation, more efficiency andlower input. These findings will be the solid ground to pave the new pathway forpotential large-scale genetic transformation of maize and other proper plants.

Category Crops
Keywords Maize, phytase, pollen, Transformation, ultrasound,
FileType PDF
Pages 193
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