A fast and genotype-independent in planta Agrobacterium-mediated transformation method for soybean

Abstract

Efficient genotype-independent transformation and genome editing are highly desirable for plant biotechnology research and product development efforts. We have developed a novel approach to enable fast, high-throughput, and genotype-flexible Agrobacterium-mediated transformation using the important crop soybean as a test system. This new method is called GiFT (genotype-independent fast transformation) and involves only a few simple steps. The method uses germinated seeds as explants, and DNA delivery is achieved through Agrobacterium infection of wounded explants as in conventional in vitro-based methods. Following infection, the wounded explants are incubated in liquid medium with a sublethal level of selection and then transplanted directly into soil. The transplanted seedlings are then selected with herbicide spray for 3 weeks. The time required from initiation to fully established healthy T0 transgenic events is about 35 days. The GiFT method requires minimal in vitro manipulation or use of tissue culture media. Because the regeneration occurs in planta, the GiFT method is highly flexible with respect to genotype, which we demonstrate via successful transformation of elite germplasms from diverse genetic backgrounds. We also show that the soybean GiFT method can be applied to both conventional binary vectors and CRISPR-Cas12a vectors for genome editing applications. Analyses of T1 progeny demonstrate that the events have a high inheritance rate and can be used for genome engineering applications. By minimizing the need for tissue culture, the novel approach described here significantly improves operational efficiency while greatly reducing personnel and supply costs. It is the first industry-scale transformation method to utilize in planta selection in a major field crop.

Keywords: genome editing; genotype-flexible transformation; herbicide selection; in planta regeneration; soybean.

Figure 1

Diagram of T-DNA regions of binary transformation vectors with ALS and EPSPS marker genes. RB, right border; LB, left border; prCMP, Cestrum yellow leaf curling virus (CMP) promoter; prGmEF1, soybean translation elongation factor EF-1 alpha/Tu promoter; prGmUbi1, soybean ubiquitin-1 promoter; prAtEF1a, Arabidopsis thaliana translation elongation factor EF-1 alpha/Tu promoter; prFMV, full-length figwort mosaic caulimovirus promoter; prSMAS, Agrobacterium tumefaciens superMAS promoter; t35S, cauliflower mosaic virus 35S terminator; prMt51186, a modified promoter from a Medicago truncatula gene; tGmEF, soybean translation elongation factor EF-1 terminator; tNOS, nopaline synthase terminator; tGmEPSPS, soybean EPSPS terminator; tMt51186, a modified terminator from a Medicago truncatula gene; eNOS, A. tumefaciens NOS enhancer; eFMV, FMV enhancer; AmCyan, Anemonia manjano cyan fluorescent protein; mEPSPSpt, plastid-targeted soybean codon-modified class 2 A. tumefaciens EPSPS conferring glyphosate resistance; NtALS, tobacco ALS gene with double mutations (W191A and W568L) conferring sulfonylurea resistance; Cry1Ab, truncated maize codon-modified Bacillus thuringiensis Cry1Ab gene; cMOV3Aa, maize-modified B. thuringiensis MOV3Aa gene; PMI, Escherichia coli phosphomannose isomerase gene; Cry1Ba, truncated mutant of insecticidal delta-endotoxin Cry1Ba gene; LbCas12a, Arabidopsis codon-optimized Lachnospiraceae bacterium ND2006 LbCas12a with D156R mutation and Pots (ST-LS) intron (26357), the maize codon-optimized wild-type version with iAtBAF60 intron (27962), or the maize codon-optimized version with C965S mutation and iAtBAF60 intron (27963); crRNA, CRISPR RNA comprising the scaffold and spacer (gRNA) sequence targeting the soy safe harbor region GmSH2; HH, hammerhead ribozyme; HDV, hepatitis delta virus ribozyme.

Figure 2

Soybean in planta and GiFT transformation. (A) Axillary meristem region of soybean seedling infected with Agrobacterium. (B) A transgenic shoot expressing CFP regenerated from the axillary meristem region of a seedling 2 weeks post-infection with Agrobacterium. (C) A transgenic shoot developed from an in planta-transformed seedling (right) and non-transgenic shoots (left) developed after 4 weeks of selection with 100 μM glyphosate. (D) Seed imbibition overnight. (E) Explant preparation. (F) Explants wounded by sonication. (G) Co-cultivation in a dry plate. (H) Co-cultivation in an airtight container for 3–5 days. (I) Germinated explants in liquid recovery medium after co-cultivation. (J) Developed explants 2 days post-recovery under light. (K) Developed explants in culture vessels with pre-selection medium containing corresponding agents. (L) Developed explants transferred to soil for selection with corresponding agents in the greenhouse. (M) Transgenic shoots with fully developed non-transgenic roots 3 weeks after selection in the greenhouse. (N) Chimeric leaf sections from transgenic shoots. (O) T1 immature embryos expressing fluorescent protein (AmCyan) under bright light. (P) Transgenic immature embryos expressing fluorescent protein (AmCyan) observed using a CFP filter. Scale bar, 2 cm.

Figure 3

Flowchart and timeline of the soybean GiFT transformation method. The transformation process starts with imbibing mature dry seeds in sterile water overnight. Cotyledon explants with epicotyl are prepared by removing the seed coat, one of the cotyledons, and the primary leaf primordia. The explants are further wounded and inoculated with Agrobacterium suspension. After 3–5 days of co-cultivation, the explants are incubated with liquid medium containing the herbicide selection agent for 1 week, then transplanted into soil for continuous herbicide selection for an additional 3 weeks. Leaves of regenerated shoots are sampled for TaqMan assays to determine the copy number of transgenes and gene-editing target sequences. The complete GiFT transformation process takes about 35 days.

Figure 4

Comparison of transformation and escape frequencies with different transformation methods and selectable markers using the elite line 06KG. (A) Average transformation frequency. (B) Average escape frequency. Different lowercase letters indicate statistically significant differences (P < 0.05).