Citation: Shao, M.; McCue, K.F.; Thomson, J.G. Agrobacterium- Mediated Transformation of the Dwarf Soybean MiniMax. Plants 2024, 13, 1013. https://doi.org/10.3390/ plants13071013 Academic Editor: Vincent G.M. Bus Received: 30 January 2024 Revised: 25 March 2024 Accepted: 26 March 2024 Published: 2 April 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Protocol Agrobacterium-Mediated Transformation of the Dwarf Soybean MiniMax Min Shao, Kent F. McCue and James G. Thomson * USDA-ARS Crop Improvement and Genetics, Western Regional Research Center, Albany, CA 94710, USA; min.shao@usda.gov (M.S.); kent.mccue@usda.gov (K.F.M.) * Correspondence: james.thomson@usda.gov 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: soybean; MiniMax cultivar; transformation; regeneration; phloroglucinol; mycorrhiza fungi 1. Introduction Soybean (Glycine max L. Merrill) is a globally significant source of nutrition, both as whole seed and when processed for protein and oil. Improving nutritional content, disease resistance, and environmental tolerance requires the ability to efficiently introduce modified and novel genes conferring new traits and phenotypes. Many advanced biotechnology techniques require the ability to genetically alter the genome and regenerate whole plants from callus or tissue culture. Further analysis and segregation of desirable traits also requires the ability to rapidly produce seeds for future generational analysis. Regeneration of soybean subsequent to Agrobacterium transformation has been historically difficult and time consuming. In addition, the typical generation time of soybean (~270 days) makes it difficult to rapidly obtain seeds for analysis. MiniMax soybean [Glycine max (L.) Merr.] is a variety developed by the USDA that exhibits compact growth (22 cm) and early maturity (73–85 days), allowing cultivation of more plants in a shorter time in a smaller space [1]. This variety is an ideal model for testing gene delivery and stacking techniques for the ultimate production of improved elite soybean varieties. The process typically requires ~270 days and has a success rate of 2–10%, depending on cultivar and technique employed [2–5]. Here we report the development of a transformation protocol involving organogenic regeneration in the MiniMax variety that requires ~145 days for T1 seed generation (Figure 1). For plant transformation we utilized the GAANTRY system [6] to facilitate introduction of a selectable marker, a visible reporter gene, and an assayable reporter gene on a single T-DNA (Figure 2). The combination of Plants 2024, 13, 1013. https://doi.org/10.3390/plants13071013 https://www.mdpi.com/journal/plants