Efficient and genotype independent maize transformation using pollen transfected by DNA‐coated magnetic nanoparticles

• Published in: Journal of integrative plant biology 2022-06, Vol.64 (6), p.1145-1156
• Main Authors: Wang, Zuo‐Ping, Zhang, Zhong‐Bao, Zheng, Deng‐Yu, Zhang, Tong‐Tong, Li, Xiang‐Long, Zhang, Chun, Yu, Rong, Wei, Jian‐Hua, Wu, Zhong‐Yi
• Format: Article
• Language: English
• Subjects: Bar gene; Corn; Culture techniques; Deoxyribonucleic acid; DNA; Embryos; exogenous genes; Filaments; Fluorescence; Gene transfer; Genetic transformation; Genomes; Genotype; Genotype & phenotype; genotype independence; Genotypes; Germination; Glufosinate; Green fluorescent protein; Hybridization; Inbreeding; magnetic nanoparticles; Magnetite Nanoparticles; maize inbred lines; Nanoparticles; Plants, Genetically Modified - genetics; Pollen; Pollen - genetics; pollen aperture; pollen transfection; Pollination; Polymerase chain reaction; Proteins; Red fluorescent protein; Seedlings; Silk; Tissue culture; Transfection; Zea mays - genetics
• ISSN: 1672-9072; 1744-7909
• DOI: 10.1111/jipb.13263

ABSTRACT 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. The success, stability, and repeatability of transfection of maize pollen with magnetic nanoparticles was improved by using low temperature during transfection to maintain pollen viability and pretreatment to open the pollen aperture.