An efficient tissue-culture-free soybean genetic transformation technology using the extremely simple cut-dip-budding strategy

Dear Editor,

Soybean (Glycine max) is one of the world’s most important economic and oilseed crops. It plays a critical role in food security, livestock feed supply, and the vegetable oil industry.1 Genetic transformation is a fundamental technology in soybean gene function research and molecular breeding. However, conventional soybean transformation methods rely on plant tissue culture, which is labor-intensive, time-consuming, costly, low in efficiency, and highly genotype dependent. These limitations have constrained both basic research and biotechnological breeding progress in soybean. With the rapid development and application of gene editing and transgenic technologies, the development of tissue culture-independent plant transformation methods has become an important focus of research on soybean. Our recently developed cut-dip-budding (CDB) gene delivery system enables genetic transformation without tissue culture, fully exploiting the intrinsic regenerative potential of plant tissues to produce gene-edited or transgenic plants. Through the simple process of cutting plants, dipping them in engineered Agrobacteria, and budding of transgenic plant shoots in soil, the CDB system allows direct delivery of genes without tissue culture and under non-sterile conditions, providing an extremely simple and efficient strategy for genetic improvement in various plant species.2,3 In this study, we explored whether the CDB strategy could be adapted for soybean to achieve efficient, tissue culture-free genetic transformation. Establishing a robust tissue culture-independent gene delivery system to achieve transgenesis and heritable gene editing remains a major challenge in soybean. Building upon the principles of the CDB system, we examined various soybean tissues for their shoot regeneration potential, tested different engineered Agrobacteria, and optimized plant culture conditions, resulting in the successful development of a robust and efficient soybean genetic transformation method.

The CDB technology was developed based on the regenerative potential of certain in vitro tissues from various plant species. For example, sweet potato, Taraxacum kok-saghyz, Ailanthus altissima, Aralia elata, Clerodendrum chinense, Coronilla varia, Clerodendrum bungei, Clerodendrum yunnanense, Clerodendrum canescens, Clerodendrum colebrookianum, Lycium chinense, Rubus rosifolius, Anemone hupehensis, and Broussonetia papyrifera can regenerate shoots from Agrobacterium rhizogenes-transformed hairy roots2,4; dandelion and Rehmannia glutinosa can regenerate shoots from root segments;5 Salvia miltiorrhiza and Kalanchoe blossfeldiana can regenerate shoots from cut petioles;3,5,6 Sansevieria trifasciata can regenerate shoots from cut leaves;6 Polygala tenuifolia can regenerate shoots from cut hypocotyls.5 To identify the explant tissues capable of shoot regeneration, we evaluated various soybean explants for regenerative capacity under soil culture conditions. Using the soybean cultivar Qihuang 34 (QH34) (a major elite soybean cultivar in central China), we tested the regeneration potential of cotyledonary petioles, true leaf petioles, compound leaf petioles, stems, and hypocotyls by inserting wounded segments into moist vermiculite. However, all tested in vitro tissues produced only roots, but there was no visible shoot regeneration.

Interestingly, some in situ tissues exhibited a strong potential for shoot regeneration. Seedlings with the cotyledon axillary region wounded or with primary leaves excised, or imbibed seeds with the cotyledon axillary region wounded or with the plumule excised, were found to regenerate shoots (Figure 1A). Since imbibed seeds with the plumule excised are easy to prepare (Figures 1A–1C), they were chosen as explants for developing a soybean CDB gene delivery system.

Soybean seeds were soaked in tap water for 12–16 h for imbibition, after which the seed coat and a cotyledon was removed, with the embryo staying attached to the remaining cotyledon. Both plumules were then removed by gentle lifting with the blade tip of a scalpel (Figure 1C). In conventional soybean genetic transformation protocols, sugar is included in the culture medium, which would cause microbial contamination under non-sterile conditions. We replaced the medium with a solution containing 10 mM MgCl2 and 10 mM MES (pH 5.4).

The prepared explants were immersed in a suspension of Agrobacterium tumefaciens (strain Gv3101 or EHA105) carrying a plasmid of interest with a RUBY reporter gene for visual monitoring of shoot regeneration. Vacuum infiltration was applied at −0.08 MPa for 15 min, and the explants in Agrobacterium suspension were incubated at room temperature with gentle shaking (∼50 rpm) for 1 h in the dark. After infection, the explants were vertically inserted into soil and kept in the dark for 2 days, with the soil pot covered by a transparent lid to maintain humidity. The soil was a mix of nutrient soil and vermiculite (4:3, v/v) and was supplemented with 0.02% (w/v) carbendazim (Guoguang, PD85150-35) to prevent fungal growth on the explants. Subsequently, the explants were transferred to a growth chamber for growth at 26°C/22°C (day/night) under a 14/10 h light-dark photoperiod.