Open AccessHigh efficiency Agrobacterium ‐mediated site‐specific gene integration in maize utilizing the FLP ‐ FRT recombination system
Author(s) -
Anand Ajith,
Wu Emily,
Li Zhi,
TeRonde Sue,
Arling Maren,
Lenderts Brian,
Mutti Jasdeep S.,
GordonKamm William,
Jones Todd J.,
Chilcoat Nicholas Doane
Publication year - 2019
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.13089
Subject(s) - agrobacterium , heterologous , transformation (genetics) , biology , agrobacterium tumefaciens , gene , genetics , transgene
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.