Current gene delivery methods for maize are limited to specific genotypes and depend on timeconsuming and labor-intensive tissue culture techniques.Here,we report a new method to transfect maize that is culture-free a...Current gene delivery methods for maize are limited to specific genotypes and depend on timeconsuming and labor-intensive tissue culture techniques.Here,we report a new method to transfect maize that is culture-free and genotype independent.To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%,transfection was performed at cool temperature using pollen pretreated to open the germination aperture(40%–55%).Magnetic nanoparticles(MNPs)coated with DNA encoding either red fluorescent protein(RFP),β-glucuronidase gene(GUS),enhanced green fluorescent protein(EGFP)or bialaphos resistance(bar)was delivered into pollen grains,and female florets of maize inbred lines were pollinated.Red fluorescence was detected in 22%transfected pollen grains,and GUS stained 55%embryos at 18 d after pollination.Green fluorescence was detected in both silk filaments and immature kernels.The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts(29%–74%)quantitated by polymerase chain reaction,and 5%–16%of the T1 seedlings showed immunologically active EGFP or GUS protein.Moreover,1.41%of the bar transfected T1 plants were glufosinate resistant,and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization.These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.展开更多
基金funded by Innovation Program of Beijing Academy of Agriculture and Forestry Sciences (KJCX20200407, KJCX20200204, KJCX20200205)Beijing Postdoctoral Research Foundation (2018-ZZ-062)+1 种基金Beijing Academy of Agriculture and Forestry Sciences Postdoctoral Research Foundation (2018ZZ-004)the Program of Beijing Municipal Commission of Science and Technology (Z171100001517001)
文摘Current gene delivery methods for maize are limited to specific genotypes and depend on timeconsuming and labor-intensive tissue culture techniques.Here,we report a new method to transfect maize that is culture-free and genotype independent.To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%,transfection was performed at cool temperature using pollen pretreated to open the germination aperture(40%–55%).Magnetic nanoparticles(MNPs)coated with DNA encoding either red fluorescent protein(RFP),β-glucuronidase gene(GUS),enhanced green fluorescent protein(EGFP)or bialaphos resistance(bar)was delivered into pollen grains,and female florets of maize inbred lines were pollinated.Red fluorescence was detected in 22%transfected pollen grains,and GUS stained 55%embryos at 18 d after pollination.Green fluorescence was detected in both silk filaments and immature kernels.The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts(29%–74%)quantitated by polymerase chain reaction,and 5%–16%of the T1 seedlings showed immunologically active EGFP or GUS protein.Moreover,1.41%of the bar transfected T1 plants were glufosinate resistant,and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization.These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.
