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

Abstract

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 NO 3 − and the ratio of NH 4 + to K + had significant effects on the morphology of plantlets derived directly from germinated transgenic embryos. During early development, optimal tissue morphology required a NH 4 + /K + ratio of 1:1 with 20 mM [NO 3 − ], 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 NH 4 NO 3 (15 mM NH 4 NO 3 and 25 mM KNO 3), 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.

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

“…The T-DNA in all four constructs also carried a CRE expression cassette under the control of the Zm-Globulin1 (Glb1) promoter (Liu et al 1998), to enable auto-excision of the morphogenic genes and the CRE gene flanked by directly oriented loxP sites ( Fig. 1), which has been used previously for excision of the morphogenic genes Zm-Wus2 and Zm-Bbm (Chu et al 2019). Transformation results for the four vectors are shown in Table 1.…”

An Efficient Gene Excision System in Maize

“…This approach selected directly for excised events and was expected to increase QE frequency. A similar construct design was previously used to optimize tissue culture conditions for recovering high quality maize transgenic events ( Chu et al., 2019 ). A schematic design of the construct is depicted in Figure 2A and the quality excised product in Figure 2B .…”

“…We developed a method for regenerating events that are free of morphogenic genes using an excision-activated marker gene approach ( Chu et al., 2019 ). Developmentally regulated promoters Glb1 and Ole that are active during late embryo development ( Kriz et al., 1990 ; Anand et al., 2017b ), were used to express Cre , leading to auto-excision.…”

“…Briefly, maize immature embryos (1.5–2 mm) were harvested and used for Agrobacterium -mediated transformation, using the media, selection and regeneration methods described previously ( Lowe et al., 2018 ; Chu et al., 2019 ; Hoerster et al., 2020 ). For selection, 0.1 mg/L imazapyr was supplemented to somatic embryo formation medium or 150 mg/L G418 was substituted for imazapyr.…”

“…These experiments were performed on two inbreds, HC69 and PHR2HT. The previously described maize transformation protocol ( Chu et al., 2019 ; Hoerster et al., 2020 ) was used with the transformation stage presented in Figure 4 . After 3 weeks of selection in maturation media supplemented with either 0.1 mg/L imazapyr ( HRA ) or 150 mg/L G418 ( NPTII ), the plates with somatic embryos were subjected to heat shock treatment in a pre-heated incubator at different temperatures with 70% relative humidity for different time periods.…”