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

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.

Nano-enabled agriculture: How do nanoparticles cross barriers in plants?

“…Besides directly interfacing with plants, nanomaterials are also used as platforms to deliver cargo to plants. For example, Fe 3 O 4 NPs ( Zhao et al., 2017b ; Wang et al., 2022a ) and single-walled carbon nanotubes ( Demirer et al., 2019 ; Kwak et al., 2019 ) have been used to deliver DNA to enable transgenic events in non-model plant species such as wheat and cotton. Clay nanosheets ( Mitter et al., 2017 ), DNA nanostructures ( Zhang et al., 2019a ), gold nanoclusters ( Zhang et al., 2021 ), and AuNPs ( Zhang et al., 2022 ) have been used to deliver siRNA to enable RNA silencing in plants.…”

“…Engineered nanomaterials have been proposed as ideal platforms for leading the agri-technology revolution ( Lowry et al., 2019 ). For example, efforts have been made to deliver genetic materials such as DNA ( Torney et al., 2007 ; Lakshmanan et al., 2013 ; Zhao et al., 2017a ; Demirer et al., 2019 ; Kwak et al., 2019 ; Wang et al., 2022a ), RNA ( Mitter et al., 2017 ; Zhang et al., 2019a , 2022 ; Demirer et al., 2020 ), and proteins ( Ng et al., 2016 ; Santana et al., 2020 ; Wang et al., 2021 ; Watanabe et al., 2021 ) to plants. Using a plant nanobionics approach, bomb-detecting plants (plants as detectors of explosive chemicals) ( Wong et al., 2017 ), light-emitting plants ( Kwak et al., 2017 ), and smart plant sensors ( Giraldo et al., 2019 ) have been developed.…”

The Promising Nanovectors for Gene Delivery in Plant Genome Engineering

“…Similar methods have also been reported in maize. It has been found that pollen can absorb DNA nanoparticles with high ability under specific conditions; maize embryos have been confirmed to contain GUS genes transferred by magnetic nanoparticles [ 73 ]. Similarly, the offspring of transgenic maize displayed the desired features of foreign genes vividly showed as the Figure 3 .…”

“…However, studies have shown that foreign DNA delivered into cotton and maize pollen cells using magnetic nanomaterials indeed integrated into the genome. Moreover, the expression of reporter genes, such as GUS and GFP, have been detected in progeny seedlings [ 72 , 73 ], demonstrating the stable integration and inheritance of exogenous DNA. However, detailed analyses, such as the identification of the integration sites and the border sequences of the integrated foreign DNA, remain to be conducted.…”

CRISPR/Cas9 Technology and Its Utility for Crop Improvement

“…Hence, the transformation of pollen grains to modify the maize genome may be completed within several hours and can be performed in the field. This novel transformation system may be applicable for crops lacking an established tissue culture regeneration system (e.g., some fruit trees and other horticultural crops) [ 143 ]. This method may expand the range of crops that can be genetically modified by the CRISPR/Cas9 system.…”