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.

Overexpression of the Transcription Factor GROWTH-REGULATING FACTOR5 Improves Transformation of Dicot and Monocot Species

“…Even though there has been considerable progress enabling in vitro organogenesis and somatic embryogenesis in a variety of crops, transformation is often restricted to very few crop varieties, limiting the application of genome editing and transformation to crop improvement ( Altpeter et al., 2016 ). Expression of developmental genes to boost regeneration has been reported as promising strategy to overcome this hurdle in transformation of plants of agricultural interest ( Nagle et al., 2018 ; Gordon-Kamm et al., 2019 ). Here, we have discovered that the overexpression of the transcription factor GRF5 from Arabidopsis and/or its homologs increases transformation efficiency in sugar beet and maize.…”

“…Several genes encoding developmental regulators have been described to improve the regeneration efficiency in various plant species ( Heidmann et al., 2011 ; Horstman et al., 2017 ; Gordon-Kamm et al., 2019 ). For example, the constitutive expression of BABY BOOM ( BBM ), a transcription factor of the AP2/ERF family with diverse functions in plant development, can promote cell proliferation and ectopic embryo formation in cotyledons and leaves of Arabidopsis ( Boutilier et al., 2002 ).…”

“…Additional developmental genes have been proposed as candidates to further improve regeneration and transformation technologies in monocots and dicots ( Gordon-Kamm et al., 2019 ; Kausch et al., 2019 ). For example, overexpression of an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION1 (WIND1) increased callus-induction and shoot regeneration in rapeseed, tomato, and tobacco ( Iwase et al., 2015 ).…”

TRA1: A Locus Responsible for Controlling Agrobacterium-Mediated Transformability in Barley

“…To identify candidate TRA1 gene(s), first we looked for barley orthologs of genes already known to influence Agrobacteriummediated transformation. The ectopic over-expression of genes involved in cell growth and differentiation has been used to improve transformability in dicotyledonous species (reviewed in Gordon-Kamm et al, 2019) and in monocots (Lowe et al, 2016). In cereals, the over-expression of either of the genes WUSCHEL or BABY BOOM stimulates Agrobacterium-mediated transformation in maize, rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and sugarcane (Saccharum officinarum L.) (Lowe et al, 2016).…”

A chimera including aGROWTH-REGULATING FACTOR(GRF) and its cofactorGRF-INTERACTING FACTOR(GIF) increases transgenic plant regeneration efficiency

“…The GRF4-GIF1 technology results in fertile and normal transgenic plants without the need of specialized promoters or transgene excision, overcoming some of the limitations of transformation technologies with other morphogenic genes (Supplementary Table 8). Because GRF4-GIF1 likely operates at a later stage of meristem differentiation and stem cell proliferation 28 than Bbm-Wus2 [6][7][8] , there is potential to combine both technologies and have synergistic effects in the regeneration efficiency of recalcitrant genotypes. A concurrent and independent work showed that overexpression of Arabidopsis AtGRF5 and AtGRF5 homologs positively enhance regeneration and transformation in monocot and dicot species not tested here 34 .…”