Tissue culture protocols for gene transfer and editing in maize (Zea mays L.).

Abstract

Efficient methods for gene transfer to maize were developed in the 1990s, first mediated by particle bombardment and then by Agrobacterium tumefaciens. Both methods can efficiently create high-quality events. Genetically modified varieties were commercialized in 1996 and are now planted in more than 90% of the US corn field. Tissue culture protocols for both methods have been well developed and widely employed. Thus, various factors, including handling before gene delivery, techniques to protect cells during gene delivery, and culture media, have been well optimized for various genotypes. Typical protocols for both methods are herein presented to show major outputs from the studies conducted since the early 1990s. As the bombardment protocols tended to be optimized specifically for limited genotypes, the one for B104, a new public inbred with favorable agronomic characteristics, is shown. The Agrobacterium protocol is suitable for various inbred lines, including B104. These protocols are also useful starting points in the optimization of tissue culture for gene editing. The rate-limiting step in both transformation and gene editing is in tissue culture and plant regeneration from modified cells in elite germplasm. Despite the prolonged efforts, large varietal differences in tissue culture responses remain a serious issue in maize. Recently, protocols using morphogenic regulator genes, such as Bbm and Wus2, have been developed that show a strong potential of efficiently transforming recalcitrant varieties.