Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds

Abstract

CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.

Keywords: CRISPR/Cas9; carbon nanotubes (CNTs); gene editing; phytoene desaturase (PDS); rice (Oryza sativa).

Figure 1

Brightfield and fluorescence stereomicroscope images of rice leaves three days post-infiltration with binary CmYLCV::GFP pDNA-CNT solutions at 2:1, 4:1, or 6:1 pDNA:CNT ratios. Exposure time was 800 ms.

Figure 2

Brightfield and fluorescence stereomicroscope images of rice leaves three days post-infiltration using binary and nonbinary vectors encoding GFP with CmYLCV promoters. The CNT solution consists of a 2:1 pDNA:CNT ratio. Exposure time was 800 ms.

Figure 3

Brightfield and fluorescence stereomicroscope images of excised rice embryos two days post-imbibement: (A) water control (brightfield); (B) water control (fluorescence); (C) CNT-only control; (D) ZmUbi::YFP-CNTs; and (E) CaMV 35S::GFP-CNTs. Solutions consist of 1:3 pDNA:CNT ratios. Fluorescence was observed through a GFP filter at an exposure time of 125 ms.

Figure 4

RNA was extracted from GFP- or YFP-CNT-treated leaf, embryo, and seed tissues, and complementary DNA (cDNA) was synthesized. (A) cDNA was amplified with GFP- or YFP-specific primers following KAPA3G Plant PCR Kit parameters. Expected fragment sizes were 215 bp and 238 bp for GFP and YFP primer sets, respectively. Gel lane numbers with the corresponding reporter gene, source tissue, and number of days submerged in CNT solution are shown at the right; samples with an asterisk did not amplify a detectable product. (B) As an additional precaution to verify proper RNA extraction and cDNA synthesis from the samples with negative results, primers of the housekeeping gene OsActin1 were used to PCR amplify cDNA from samples from lanes 8, 9, 20, and 21 in (A) above, showing successful amplification of OsActin1 from these four samples, although at a low level (expected fragment size: 195 bp). Ladders: GeneRuler 1 kb Plus (Thermo Fisher Scientific, Catalog No. SM1331).

Figure 5

Excised leaves and embryos treated with CNTs carrying NLS-GFP vectors were sectioned, stained with DRAQ5, and imaged under confocal microscope after four days in solution. (A) GFP filter, (B) DRAQ5 filter, and (C) a and b overlay. Image overlays indicate GFP fluorescence overlap with nuclear staining. Sectioning and imaging performed by TAMU MIC.

Figure 6

Rice leaves were punctured with needles and imbibed in CNTs loaded with (A) nonbinary or (B) binary GUSPlus vectors. After three days in solution, GUSPlus enzymatic activity was visualized by histochemical assay [32] and chlorophyll bleaching. Blue coloration indicates GUSPlus activity at the infiltration site of treated leaves. (C) Lack of blue color in a negative control leaf for comparison.

Figure 7

Histochemical detection of GUS expression in rice seeds imbibed in a CNT-plasmid DNA solution. Sterilized rice seeds were soaked overnight in water to initiate germination, then with a osmotic solution of 0.6 mannitol for two hours, followed by a 5-day exposure to a CNT-pDNA solution at 1:3 ratio with a 35S::GUSPlus vector. GUSPlus enzymatic activity was visualized by histochemical assay using standard procedures [32]. (A) Imbibed rice seed in MES delivery buffer (negative control). (B–E) Detection of GUS expression in germinating rice tissues after 5 days in the CNT-pDNA mixture solution.