Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

Abstract

Over the past decades, advances in plant biotechnology have allowed the development of genetically modified maize varieties that have significantly impacted agricultural management and improved the grain yield worldwide. To date, genetically modified varieties represent 30% of the world's maize cultivated area and incorporate traits such as herbicide, insect and disease resistance, abiotic stress tolerance, high yield, and improved nutritional quality. Maize transformation, which is a prerequisite for genetically modified maize development, is no longer a major bottleneck. Protocols using morphogenic regulators have evolved significantly towards increasing transformation frequency and genotype independence. Emerging technologies using either stable or transient expression and tissue culture-independent methods, such as direct genome editing using RNA-guided endonuclease system as an in vivo desired-target mutator, simultaneous double haploid production and editing/haploid-inducer-mediated genome editing, and pollen transformation, are expected to lead significant progress in maize biotechnology. This review summarises the significant advances in maize transformation protocols, technologies, and applications and discusses the current status, including a pipeline for trait development and regulatory issues related to current and future genetically modified and genetically edited maize varieties.

Keywords: gene editing; genetic modification; maize; morphogenic regulator-mediated transformation; plant biotechnology; plant transformation.

Figure 1

Standard protocol for B104 maize transformation. (A) Growth of donor plants for immature embryo production under greenhouse-controlled conditions. (B) Ears are harvested 10–16 d after pollination. (C) The immature zygotic embryo reaches the ideal size of 1.2–2mm. (D) Isolated immature embryos in co-cultivation. (E) Immature embryo transiently expressing the gus reporter gene. (F) Embryos in resting media seven days after Agrobacterium infection. (G) Calli induction on selection I medium. (H) Compact type I callus in selection II medium. (I,J) Regeneration of transformed plants. (K) Transformed plantlets with roots and shoots are grown in the penumbra room. (L) T₀ transgenic individuals rooted in the soil in an acclimation room. (M) T₀ plants grown at the greenhouse. (N) Flowering and pollination of T₀ plants at the greenhouse. (O) Harvesting of T₁ seeds. The complete process, from infection to T₁ seed production, takes approximately 6–8months. The images are not to scale.

Figure 2

Schematic representation of the morphogenic regulator-mediated maize transformation (MRMT). (A) Comparison between standard transformation (top) and MRMT (bottom) protocols. Tissue culture phases are indicated by different colours. By skipping the callus culture step, MRMT shortens the time needed for in vitro tissue culture. Note that although not specified, a selective agent is used in the MRMT culture media to allow regeneration of transformed embryos only. (B) Schematic representation of generic T-DNA present in MRMT-based vectors. In addition to the genetic payload of interest, T-DNA harbours morphogenic regulators (MRs) and a recombinase (CRE). Upon a given stimulus, CRE excises the MRs from the construct. The time period and culture media are based in Coussens et al. (2012) and Raji et al. (2018) for the standard protocol and in Masters et al. (2020) for the MRMT protocol.

Figure 3

Schematic representation of the desired-target mutator (DTM) maize transformation. (A) The CRISPR-Cas cassette can be transformed into a nonrecalcitrant inbred line for trans editing in an elite recalcitrant inbred line. (B) Pollen carrying a CRISPR-Cas cassette designed to target gene(s) of interest was used to pollinate the elite maize line. (C) The target gene is directly edited via trans-acting CRISPR-Cas. (D) The delivery of RNP, which is expressed by the sperm cell, directly into the egg cell of the elite line (gametophytic expression) or expression of RNP in the zygote after gamete fusion (zygotic expression) generates a hybrid edited embryo. (E) After trans editing, subsequent crossings are needed to obtain CRISPR-Cas-free plants with the original receptor genetic background and homozygous to the desired mutation. The schematic illustration view in (D) was adapted from Jacquier et al. (2020).

Figure 4

Haploid-inducer-mediated genome editing (IMGE) or simultaneous double haploid production and editing (HI-Edit) in maize. (A) The haploid inducer line harbouring a CRISPR-Cas cassette is used to pollinate the maternal elite line of any genotype. (B) The haploid progeny, which are typically sterile, were screened for CRISPR-Cas-induced mutations (~3%) and subsequently treated with a doubling agent to produce fertile doubled haploids. (C) Edited doubled haploid lines with improved agronomic traits are obtained after self-pollination. The zoomed view of trans genome editing and the maternal haploid formation processes occurring in B are shown in D-E. (D) After trans-acting CRISPR-Cas, and fertilization, the unstable paternal chromosome from haploid-inducer pollen is lost. (E) The formed embryo is nontransgenic (Cas-free) and has a doubled chromosome to recover the homozygous edited diploid elite plant.

Figure 5

Agricultural biotechnology pipeline for trait development in maize. General overview of main activities and estimates of maximum (light colours) and minimum (dark colours) costs and development time of each pipeline phase: discovery, proof-of-concept, early development, advanced development and prelaunch. Estimates are based on Mcdougall (2011) and Mumm (2013).