An Efficient Gene Excision System in Maize

Abstract

Use of the morphogenic genes Baby Boom ( Bbm ) and Wuschel2 ( Wus2 ), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. Further optimizing the expression pattern for Bbm / Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm / Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase ( Cre ) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole , Glb1 , End2 , and Ltp2 , to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25–100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53–68%; however, the transformation frequency was lower (13–50%). The use of inducible heat shock promoters (e.g. Hsp17.7 , Hsp26 ) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29–69%), excision (50–97%), usable quality events (4–15%), and few escapes (non-transgenic; 14–17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes

“…In this study a maize heat shock protein gene promoter was used to induce the expression of the cre gene which resulted in an excision efficiency of 33 and 40%, respectively, for Blackwell and Summer. This removal frequency is comparable to previous reports in which a heat shock induced Cre-Lox excision system was used ( Chong-Pérez et al, 2012 ; Wang et al, 2020 ).…”

An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos

“…To suppress the non-transgenic plant growth, a higher concentration of bialaphos is recommended for the first maturation medium (13329B3). Alternatively, other fast-acting herbicide resistance genes and selection agents such as HRA gene with imazapyr ( Lowe et al, 2018 ; Masters et al, 2020 ) or antibiotics selection system NPTII gene with G418 ( Wang et al, 2020 ) can be adapted to this protocol.…”

Current status and prospects of plant genome editing in Australia

“…Therefore, additional studies to understand more about protoplast regeneration are required for genome editing and the delivery of effective materials (RNPs or donor DNA) to achieve transgene-free crops (Woo et al 2015;Lin et al 2018). Additional methods have been developed to eliminate the CRISPR-Cas9 constructs including, using the cre-loxP system to excise the integrated fragments after editing, antibiotic marker-free transformation strategies through CRISPR-Cas9 genes transient expression in T 0 , and germline injection of Cas9 protein and guide RNA (Schwartz and Sternberg 2014;Aliaga-Franco et al 2019;Veillet et al 2019;Wang et al 2020). It is more important for vegetatively propagated crops (such as sugarcane and banana) to obtain transgene-free genome editing plants in the T 0 generation because it is hard to eliminate CRISPR-Cas9 constructs through self-crossing or out-crossing in these species.…”