Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants

Abstract

Efficient transformation of numerous important crops remains a challenge, due predominantly to our inability to stimulate growth of transgenic cells capable of producing plants. For years, this difficulty has been partially addressed by tissue culture strategies that improve regeneration either through somatic embryogenesis or meristem formation. Identification of genes involved in these developmental processes, designated here as morphogenic genes, provides useful tools in transformation research. In species from eudicots and cereals to gymnosperms, ectopic overexpression of genes involved in either embryo or meristem development has been used to stimulate growth of transgenic plants. However, many of these genes produce pleiotropic deleterious phenotypes. To mitigate this, research has been focusing on ways to take advantage of growth-stimulating morphogenic genes while later restricting or eliminating their expression in the plant. Methods of controlling ectopic overexpression include the use of transient expression, inducible promoters, tissue-specific promoters, and excision of the morphogenic genes. These methods of controlling morphogenic gene expression have been demonstrated in a variety of important crops. Here, we provide a review that highlights how ectopic overexpression of genes involved in morphogenesis has been used to improve transformation efficiencies, which is facilitating transformation of numerous recalcitrant crops. The use of morphogenic genes may help to alleviate one of the bottlenecks currently slowing progress in plant genome modification.

Keywords: embryogenesis; meristem formation; morphogenic; organogenesis; transformation.

Figure 1

Methods for expression of morphogenic genes in plant transformation. WUS is used to exemplify the morphogenic gene expression cassettes, which could be designed for overexpression of the gene, downregulation of a gene, or combinations of genes, while the box labeled “GOI” (Genes Of Interest) represents trait gene expression cassettes. (A) Using a constitutive promoter, such as CaMV35S, to drive expression of a WUS gene results in growth stimulation of cells transformed with the T-DNA, either through somatic embryogenesis or through meristem proliferation. (B) Using an inducible promoter to drive expression of WUS will result in growth stimulation only when the plant tissue is exposed to the inducing stimulus (typically a chemical ligand). (C) Growth stimulation can also be effectively controlled by using a combination of constitutive expression of WUS and inducible expression of CRE recombinase to remove the WUS expression cassette. (D) Transforming the same plant cell with a T-DNA containing the WUS expression cassette and a second T-DNA containing the trait expression cassette will also provide transient growth stimulation sufficient to recover regenerable tissues, such as somatic embryos, without the integration of the morphogenic gene. (E) Using a single T-DNA containing the trait, with the WUS expression cassette outside the T-DNA Left Border sequence, higher numbers of the trait-containing T-DNA are introduced relative to the low numbers of “read-through” sequences containing the WUS gene, providing transient growth stimulation without WUS integration. IND_pro is used to represent chemically inducible promoters, such as the estradiol-, glucocorticoid-, or tetracycline-responsive promoters in 1-B. Promoters that are induced by physical conditions, such as desiccation (e.g., the RAB17 promoter in [17]), are used to control recombinase-mediated excision (as in 1-C). CRE represents the CRE recombinase expression cassette and loxP are the CRE-recombinase target sites. RB and LB represent the right and left T-DNA border sequences, respectively.