AUTHOR=Wang Ning , Arling Maren , Hoerster George , Ryan Larisa , Wu Emily , Lowe Keith , Gordon-Kamm William , Jones Todd J. , Chilcoat N. Doane , Anand Ajith 

TITLE=An Efficient Gene Excision System in Maize

JOURNAL=Frontiers in Plant Science

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.01298

DOI=10.3389/fpls.2020.01298

ISSN=1664-462X

ABSTRACT=<p>Use of the morphogenic genes <italic>Baby Boom</italic> (<italic>Bbm</italic>) and <italic>Wuschel2</italic> (<italic>Wus2</italic>), 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 <italic>Bbm</italic>/<italic>Wus2</italic> has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of <italic>Bbm</italic>/<italic>Wus2</italic> 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 (<italic>Cre</italic>) to excise morphogenic genes. The use of developmentally regulated promoters, such as <italic>Ole</italic>, <italic>Glb1</italic>, <italic>End2</italic>, and <italic>Ltp2</italic>, to drive <italic>Cre</italic> 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. <italic>Hsp17.7</italic>, <italic>Hsp26</italic>) 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.</p>