Current gene delivery methods for maize are limited to specific genotypes and depend on time‐consuming 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.

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Background: Biotechnological engineering of maize to introduce favorable new traits relies on delivery of foreign DNA into its cells. Current gene delivery methods for maize is limited to specific genotypes, and depend on time-consuming and labor-intensive processes of tissue culture. Results: Here, we report a new method to transfect maize that is culture-free and genotype independent. Enhanced green fluorescent protein gene (EGFP) or bialaphos resistance gene (Bar) bound with magnetic nanoparticles (MNPs) was delivered into maize pollens, and female florets of five maize varieties were pollinated. Green fluorescence was detected in 92% transfected pollens and 70% immature embryos. EGFP gene detected by PCR ranged from 29 to 68% in T1 generation of these five transfected varieties, and 7-16% of the T1 seedlings showed immunologically active EGFP protein. Moreover, 1.41% of the Bar transfected T1 plants were glufosinate resistant, and heritable Bar gene was integrated into the maize genome effectively (verified by Southern blot), expressed normally and inherited stably in their progenies.Conclusion: These results demonstrate that exogenous DNA could be delivered into maize efficiently and expressed normally through our genotype-independent pollen transfection system, providing a reliable, fast and large-scale gene delivery choice for most elite maize varieties recalcitrant to tissue culture.