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. 2024 Apr 2;13(7):1013.
doi: 10.3390/plants13071013.

Agrobacterium-Mediated Transformation of the Dwarf Soybean MiniMax

Min Shao  1 , Kent F McCue  1 , James G Thomson  1
Affiliations

Agrobacterium-Mediated Transformation of the Dwarf Soybean MiniMax

Min Shao et al. Plants (Basel). .

Abstract

This study aims to establish an Agrobacterium-mediated transformation system for use with the 'MiniMax'soybean cultivar. MiniMax is a mutant soybean whose growth cycle is around 90 days, half that of most other soybean varieties, making it an optimal model cultivar to test genes of interest before investing in modification of elite lines. We describe an efficient protocol for Agrobacterium-mediated transformation using MiniMax seeds. It uses a modified 'half seed' regeneration protocol for transgenic soybean production, utilizing the rapid generation MiniMax variety to obtain T1 seeds in approximately 145 days. Addition of phloroglucinol (PG) to the regeneration protocol was key to obtaining high-efficiency rooting of the regenerated shoots. Transfer to soil was accomplished using an organic soil amendment containing nutrients and mycorrhiza for plants to thrive in the greenhouse. This combination of genotype and stimulants provides a transformation protocol to genetically engineer MiniMax seeds with a transgenic lab-to-greenhouse production efficiency of 4.0%. This is the first report of MiniMax soybean whole plant transformation and heritable T1 transmission. This protocol provides an ideal resource for enhancing the genetic transformation of any soybean cultivar.

Keywords: MiniMax cultivar; mycorrhiza fungi; phloroglucinol; regeneration; soybean; transformation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Flow chart of Agrobacterium-mediated transformation using Minimax mature half-seed transformation through to harvest of T1 seeds. Approximately 145-day procedure.
Figure 2
Figure 2
Agrobacterium-mediated soybean transformation workflow. The workflow protocol requires up to nine weeks from seeds to transgenic seedlings (A–G). (A) Agrobacterial culture, (B) (I) Sterile seeds after hydration, (II) seed in ‘half-seed’ configuration ready for transformation). (C) Half-seed co-cultivation with filter paper, (D) Spectinomycin resistant tissues after two weeks (green (red arrows) vs. white shoots), (E) Spectinomycin-resistant tissues on elongation medium, (F) Spectinomycin-resistant shoots ready for transfer to RM, (G) Rooted transgenic shoots on RM, (H) Rooted transgenic shoots growing in soil, (I) Transgenic soybean plants in the greenhouse. (J) Putative transgenic T0 plants vegetative stage. (K) Identification of T1 mCherry3 positive seeds (pink seeds-red arrows point out examples). (L) T1 transgenic plants in reproductive stage.
Figure 3
Figure 3
mCherry3 (mCh3) expression in transgenic soybean seeds. (A) Schematic diagram of the JGT44 T-DNA (Spec: spectinomycin resistance; mCh3: mCherry3; Rluc: Renilla Luciferase; Bag4: Arabidopsis derived Bcl-2-associated athanogene 4; GmUbi3, AtUbi10, and db35S: promoters), (B) Representative mCherry3 fluorescence images of dry mature seeds of wild type (WT) and JGT44 transgenic lines (M1, M2, M6, M9, M10 and M16), (C) PCR analysis of genomic DNA of putative transgenic soybean plants using BAG4 gene and Spec gene primers. The length of PCR productions is 681 bp and 550 bp, respectively. M: DNA ladder; P: JGT44 gDNA; N: Wild type soybean gDNA; M1, M2, M6, M9, M10, and M16 are representative transformed soybean plants.
Figure 4
Figure 4
Transgene copy number and Rluc expression. (A) Transgene copy number as determined by ddPCR, (B) Rluc luminescence for regenerated lines, quantified in photons per second (p/sec). Y-axis: JGT44 transgenic lines (M1, M2, M6, M9, M10 and M16).
See this image and copyright information in PMC

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