Use of DoE methodology to optimize the regeneration of high-quality, single-copy transgenic Zea mays L. (maize) plants

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The maize Agrobacterium tumefaciens-mediated transformation process normally takes about 10 to 15 wk from embryo infection and co-cultivation to sending “T0” plants to the greenhouse (GH). A new method was developed using the maize transcription factors Babyboom (BBM) and Wuschel2 (WUS2), to stimulate direct transgenic embryo formation and plant regeneration, that bypasses the need for prolonged tissue culture and regeneration from callus. In the present study, a design of experiment (DoE) method was used to test 10 factors to optimize the quality of somatic embryo maturation, root formation, and subsequent plantlet survival, without compromising the molecular event quality. The concentration of NO3− and the ratio of NH4+ to K+ had significant effects on the morphology of plantlets derived directly from germinated transgenic embryos. During early development, optimal tissue morphology required a NH4+/K+ ratio of 1:1 with 20 mM [NO3−], of 14.2 μM 6-benzylaminopurine (BAP), the highest concentration tested, and a low light intensity of 50 μmol m−2 s−1. Later development of rooted shoots required additional macronutrients with reduced NH4NO3 (15 mM NH4NO3 and 25 mM KNO3), reduced BAP (7.4 μM), and 1 μM abscisic acid (ABA) and at a higher light intensity of 140 μmol m−2 s−1. Using the optimized parameters, the frequency of plants sent to the GH was improved by twofold compared with the current process and the number of single-copy T-DNA events was doubled.