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.

Improving Agrobacterium -mediated transformation frequency and event quality by increasing binary plasmid copy number and appropriate strain selection is reported in an elite maize cultivar. Agrobacterium-mediated maize transformation is a well-established method for gene testing and for introducing useful traits in a commercial biotech product pipeline. To develop a highly efficient maize transformation system, we investigated the effect of two Agrobacterium tumefaciens strains and three different binary plasmid origins of replication (ORI) on transformation frequency, vector backbone insertion, single copy event frequency (percentage of events which are single copy for all transgenes), quality event frequency (percentage of single copy events with no vector backbone insertions among all events generated; QE) and usable event quality frequency (transformation frequency times QE frequency; UE) in an elite maize cultivar PHR03. Agrobacterium strain AGL0 gave a higher transformation frequency, but a reduced QE frequency than LBA4404 due to a higher number of vector backbone insertions. Higher binary plasmid copy number positively correlated with transformation frequency and usable event recovery. The above findings can be exploited to develop high-throughput transformation protocols, improve the quality of transgenic events in maize and other plants.

No abstract

Summary An efficient Agrobacterium ‐mediated site‐specific integration ( SSI ) technology using the flipase/flipase recognition target ( FLP / FRT ) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line ( RTL ) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom ( Bbm ) and Wuschel2 ( Wus2 ) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL 1 resulted in higher transformation frequency than strain LBA 4404 THY ‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL 1; 0.65% in LBA 4404 THY ‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.

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.

1Use of the morphogenic genes Baby Boom (Bbm) and Wuschel2 (Wus2), along with new ternary 2 constructs, has increased the genotype range and the type of explants that can be used for maize 3 transformation. In addition, altering the ectopic expression pattern for Bbm/Wus2 has resulted in 4 rapid maize transformation methods that are faster and applicable to a broader range of inbreds. 5However, expression of Bbm/Wus2 can compromise the quality of regenerated plants, leading to 6 sterility. We reasoned excising morphogenic genes after transformation but before regeneration 7 would increase production of fertile T0 plants. We developed a method that uses an inducible 8 site-specific recombinase (Cre) to excise morphogenic genes. The use of developmentally 9 regulated promoters, such as Ole, Glb1, End2 and Ltp2, to drive Cre enabled excision of 10 morphogenic genes in early embryo development and produced excised events at a rate of 25%-11 100%. A different strategy utilizing an excision-activated selectable marker produced excised 12 events at a rate of 53.3%-68.4%; however, the transformation frequency was lower (12.9%-13 49.9%). The use of inducible heat shock promoters (e.g. Hsp17.7, Hsp26) to express Cre, along 14 with improvements in tissue culture conditions and construct design, resulted in high frequencies 15of T0 transformation (29%-69%), excision (50%-97%), usable quality events (3.6%-14%), and 16 few escapes (non-transgenic; 14%-17%) in three elite maize inbreds. Transgenic events produced 17 by this method are free of morphogenic and marker genes.