The utility of CRISPR in plants has remained limited by the dual difficulties of delivering the molecular machinery to target cells and the use of somatic cell techniques that require tissue culture-based de novo organogenesis. We developed 5-10 nm isodiametric polyplex nanoassemblies, comprising poly [2-(dimethylamino)ethylmethacrylate] PDMAEMA (PD) polycationic linear homopolymers and CRISPR/Cas9 ribonucleoproteins (RNPs), that enable endocytosis-driven RNP uptake into pollen grains. Pollen from wheat plants (genotype Gladius+Sr50), homozygous for monogenic Sr50-mediated resistance to stem rust (Puccinia graminis f. sp. tritici -Pgt), were incubated with RNP/PD nanoassemblies targeting the dominant, Sr50 rust resistance gene. The treated pollen grains were then used to fertilize Gladius+Sr50 florets and the resulting M1 plants were tested for loss of Sr50 function via rust resistance screens. The identification of fully susceptible M1 seedlings indicated that the Sr50 RNPs acted on both alleles, indicating they were transferred via the treated pollen to the zygote. The ability to readily deliver CRISPR RNPs to reproductive cells via biodegradable, polymeric nanocomplexes has significant implications for the efficiency of gene editing in plants.
The use of biotechnology for the genetic improvement of wheat (Triticum aestivum L.) has been hampered by its recalcitrance to standard transformation and regeneration protocols. Male gametes present a potentially useful option for introducing gene edits via clustered regularly interspaced short palindromic repeats (CRISPR). However, the utility of male gametes for introducing genetic improvements would be dependent on the retention of viability after treatment to introduce the CRISPR components. We have studied wheat pollen morphology and its viability in a range of germination media to identify conditions that optimize the viability of in vitro hydrated pollen. The size, shape, and aperture from seven different wheat genotypes were compared using scanning electron microscope (SEM). The SEM results revealed that the pollen of all of the wheat genotypes examined in this study were monoporate; however, a significant variation in the size of the mature pollen grains was observed. The hydrated pollen of the wheat genotypes remained viable for up to five hours at 20 ± 2 °C. Of all of the germination media tested, the medium containing 5% sucrose, 10% PEG4000, 100 mg/L boric acid, 200 mg/L calcium nitrate, 100 mg/L potassium nitrate, and 100 mg/L magnesium sulphate at pH 6.5 achieved the highest percentage of pollen germination after 5 h of hydration. Impedance Flow Cytometry (IFC) provided similar results to the in vitro germination study. This work elucidates important factors that can form the basis for a pollen-based non-genetically modified system for gene editing in wheat.
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