Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Abstract

In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

Target pollen isolation using automated infrared laser-mediated cell disruption

“…In the case of bombardment, pollen germination occurs before the expression of the transgene; therefore, pollen germination and pollination of isolated pollen cannot be studied (Nagahara et al, 2021). Transgenic 35Sp::H2B-tdTomato was used for pollen germination analysis, and AtUBQ10p::tdTomato pollen grains were used for pollination analyses as target pollen expressing fluorescent proteins.…”

“…Pollen germination is possible not only in the media but also on the pistil. We have previously reported that bombarded pollen can elongate pollen tubes on the pistil and reach the ovule (Nagahara et al, 2021). Our results suggest that pollination with isolated pollen may achieve efficient seed production from the target pollen (Figure 3).…”

“…After IR laser irradiation, the disrupted pollen bulk population was collected through the filter and pollinated to N. tabacum pistil (emasculated before 2 or 2 d). Immediately, the pollinated style was excised with a razor and placed horizontally on the pollen germination medium solidified with 1% (w/v) NuSieve GTG Agarose (Lonza, Switzerland), followed by incubation at 25–30°C for 18–24 hr under humid and dark conditions (Nagahara et al, 2021). Pollen tubes emerging from the cut end of the pistil were observed under an inverted fluorescence microscope (Eclipse Ti2-E; Nikon).…”

“…In this study, wild-type and transgenic N. benthamiana , AtUBQ10p::H2B-mClover , 35Sp::H2B-tdTomato , AtUBQ10p::tdTomato , and wild-type Nicotiana tabacum ‘Petit Havana SR1’ described in a previous study were used (Nagahara et al, 2021). Seeds were sterilised for 10 min using a solution containing 1% (v/v) sodium hypochlorite (Fujifilm Wako Pure Chemical, Osaka, Japan) and 0.02% (v/v) Triton X-100 (Nacalai Tesque, Kyoto, Japan).…”

“…Biolistic delivery, also referred to as particle bombardment, is widely used to transiently introduce and express exogenous genes in cells (Sanford, 2000). Recently, we reported a method for the biolistic delivery of genes into the pollen of Nicotiana benthamiana , which is easy to handle and has abundant pollen grains (Nagahara et al, 2021). However, the efficiency of gene introduction into pollen by bombardment is low, and pollen isolation using antibiotic-resistance genes cannot be used (Hoffmann et al, 1988).…”