Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators

Abstract

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Keywords: Agrobacterium; CRISPR/Cas9; T-DNA; gene editing; maize; morphogenic genes; tissue culture; transformation.

Figure 1

Efficiency and variability of B104 maize transformation. (A) Timelines in months of B104 transformation protocols. Key steps are indicated. (B) Transformation efficiency of the platform in 2016 using method 1 and in 2020 using method 2. The number of transgenic plants scoring positive for presence of BIALAPHOS RESISTANCE (BAR) by lateral flow assays is plotted per starting immature embryo. Efficiency is plotted for both the total number of T0 transgenic plants obtained (left) and independent events (derived from discrete immature embryos, right). n = 35 for method 1, n = 19 for method 2, ^***p < 0.001 (Student t-test). Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, average is indicated as a cross. (C) Heat maps showing transformation efficiency (independent transgenics) per ear ordered chronologically per experiment for 2016 (method 1) and 2020 (method 2). In each experiment, embryos derived from three to five ears were used.

Figure 2

Transformation of B104 using morphogenic genes. (A) Diagrams of the T-DNAs used. RB, right border; pZmPLTP, maize PHOSPHOLIPID TRANSFER PROTEIN promoter; pZmAXIG1, IAA25 auxin-inducible promoter; ZmBBM, maize BABY BOOM; ZmWUS2, maize WUS2, MGC, morphogenic cassette; BAR, BIALAPHOS RESISTANCE; pZmUBI, maize UBIQUITIN-1 promoter; and LB, left border. (B,C) B104 immature embryos five days after transformation with control (B) or pLAPAU9 (C) and stained for GUS expression. Blue arrows indicate somatic proembryos expressing GUS transgene. (D,E) Representative images of tissue culture 11 days on maturation II after transformation with control (D) or pLAPAU9 (E). Orange arrows indicate wild type appearing shoots, while red arrows indicate abnormal shoots.

Figure 3

HIGHLY-RESISTANT ACETOLACTATE SYNTHASE (HRA) as a selection marker for morphogenic regulator-assisted B104 transformation. (A) Diagrams of the T-DNAs used. pZmAXIG1, ZmIAA25 auxin-inducible promoter; pZmPLTP, maize PHOSPHOLIPID TRANSFER PROTEIN promoter; ZmBBM, maize BABY BOOM; ZmWUS2, maize WUSCHEL2, MGC2, morphogenic cassette 2; SbALS, Sorghum bicolor ACETOLACTATE SYNTHASE promoter; HRA, HIGHLY-RESISTANT ACETOLACTATE SYNTHASE; tStPinII, Solanum tuberosum PinII terminator; pZmUBI, maize UBIQUITIN-1 promoter; RB, right border; and LB, left border. (B) Representative images of B104 immature embryos after transformation with pLAPAU14 or a control, 7 days on maturation II media with imazapyr selection. (C) Representative images before hardening. (D) Images of 10 random T0 plants taken at the same day are shown for pLAPAU14. Letters indicate individual T0 plants. Images were taken 99 days after transfer to soil. Green (fertile) or red (infertile) bars below the plants indicate capacity to produce seeds after backcrossing with wild-type.

Figure 4

Relation between morphogenic regulator copy number and overall plant growth and fertility. Images of 10 random T0 plants taken at the same day are shown for pLAPAU14 (A) and pLAPAU16 (B). The T-DNA structure is shown schematically as in Figure 3, except HRA represents pSbALS::HRA:tStPinII. Letters indicate individual T0 plants and are ranked from left to right according to their estimated pZmPLTP::ZmBBM copy number (shaded green). The number in the image indicates the days after transfer to soil the pictures were taken. Green (fertile) or red (infertile) bars below the plants indicate capacity to produce seeds after backcrossing with wild-type.

Figure 5

Loss of VIRESCENT YELLOW-LIKE (VYL) function in B104 results in a pale-yellow phenotype. (A) Genomic structure of the B104 VYL (Chr.9_ClpP5) gene and location of the sgRNA. Dark green boxes designate exons; light green boxes, UTRs; solid lines, introns; and white arrows gene orientation. (B) Diagram of the T-DNA used for CRISPR/Cas9 gene editing in maize. LB, left border; BAR, BIALAPHOS RESISTANCE, pZmUBI, maize UBIQUITIN-1 promoter; and RB, right border. (C) Loss-of-function phenotype of T0 regenerants during the final tissue culture step of maize transformation. Edited T0 plants are indicated with an arrow. (D–F) Seven-day-old seedling phenotype of wild-type, knock-out (+1;+1, homozygous 1 bp insertion) and a weak allele (−9;−9, homozygous 9 bp deletion).

Figure 6

Use of morphogenic regulators for efficient gene editing in B104. (A) The pLAPAU17 construct allowing direct cloning of a spacer sequence. The T-DNA structure is shown schematically as in Figure 4. (B) For eight independent T0 lines, the editing efficiency is plotted at VYL (Chr.9_ClpP5; green bars) and Chr.1_ClpP5 (red bars). (C) Representative image of a T0 plant (line B) showing albino mosaicism. (D) The targeted B104 genomic sequences of VYL and Chr.1_ClpP5. PAM is highlighted in dark green, spacer in light green, and the Cas9 cut site is indicated with a triangle. Mismatched bases in the spacer are highlighted in orange. The reading frame is marked.