Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar

Abstract

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.

Developing a flexible, high‐efficiency Agrobacterium‐mediated sorghum transformation system with broad application

“…To assess event quality, T0 plants were generated using pPHP38332(CIV), pPHP78152(ternary) or pPHP78233(ternary) as detailed in Experimental Procedures. T0 plants were subjected to detailed molecular event characterization as previously reported (Wu et al ., ; Zhi et al ., ). As represented in Table , 54% and 65% of the events produced with the ternary constructs were determined to be single‐copy insert, backbone‐minus quality events, which was comparable to quality event frequency determined for the CIV construct pPHP38332 (66%) with a relatively smaller T‐DNA (Table ).…”

“…The integrated copy number of the T‐DNA and the vector backbone in these transgenic plants were determined by a series of qPCR analyses (Wu et al ., ; Zhi et al ., ). The transgenic plants carrying a single copy of the intact T‐DNA integrations without vector backbone were defined as ‘quality events’.…”

Agrobacterium tumefaciens-mediated transformation of drumstick (Moringa oleifera Lam.)

“…tumefaciens strain Different A. tumefaciens strains exhibit different effects on plant transformation frequency and transgenic event quality [25]. Do et al [26] made a comparison of three immature A. tumefaciens strains, AGL1, EHA101 and GV3101, regarding the infection of sorghum and found that AGL1 improves the transformation efficiency.…”

Maize Somatic Embryogenesis: Agronomic Features for Improving Crop Productivity

“…Authors proposed that this method is suitable for the propagation of recalcitrant commercial maize inbreeds. Zhi et al (2015) improved the number and quality of transformation events, using a combination of Agrobacterium strain and incrementing the binary plasmid copy number. This resulted in increase of the transformation frequency.…”