Advancing Crop Transformation in the Era of Genome Editing

Abstract

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

Figure 1.

Current Bottlenecks in Applying Genome Editing to Crop Functional Genomics and Crop Improvement. The main bottleneck is in plant transformation and regeneration. A secondary bottleneck is in the delivery of genome editing reagents to plant cells to produce the intended effects.

Figure 2.

Sorghum (Sorghum bicolor) Is a Crop Recalcitrant to Transformation and Regeneration. Starting left and proceeding clockwise are representations of steps and time required for each step in the method, from growth of donor plants to provide target immature embryos to the harvesting of mature seed. Times required at each step is indicated as d (days), wk (weeks), and mo (months). Similar protocols and timelines prevent the high-throughput transformation and genome editing for most important U.S. crops.

Figure 3.

Important Historical Milestones in Plant Transformation. Since its beginning in 1977, the pace of crop transformation technology development has not been linear. In recent years, the genome editing revolution begs for crop transformation improvements to enable greater food security.

Figure 4.

Overview of Agrobacterium-Mediated Transformation to Generate a Transgenic Plant. Phenolic compounds secreted by wounded plants are perceived by the Agrobacterium VirA/VirG two-component sensing system, resulting in induction of virulence (vir) genes. Among these genes, virD1 and virD2 form a site-specific nuclease that nicks the T-DNA region at border sequences. In nature, T-DNA resides on the Ti-(tumor inducing) or Ri-(root inducing) plasmid (1), but in the laboratory, T-DNA can be “launched” from binary vectors (2) or from the bacterial chromosome (3). VirD2 covalently links to single-strand T-DNA and leads T-strands through a Type IV secretion system (composed of VirB and VirD4 proteins) into the plant. Other transferred virulence effector proteins are VirE2 (a single-strand DNA binding protein proposed to coat T-strands in the plant cell) and VirD5, VirE3, and VirF (not pictured). Within the plant, VirD2/T-strands likely form complexes with VirE2, other Vir effector proteins, and plant proteins. These complexes target the nucleus. Once inside the nucleus, proteins must be stripped from T-strands, which can replicate to a double-strand nonintegrated form (transient transformation). T-DNA can integrate into the plant chromosomes, resulting in stably transformed cells. These cells can be regenerated to plants harboring and expressing transgenes.