An improved ternary vector system for Agrobacterium-mediated rapid maize transformation

Ajith Anand,Michael Miller,Kevin E Mcbride,Ning Wang,Mo Hua,Todd J Jones,Emily Wu,Steven H Bass,Narayana Annaluru

doi:10.1007/s11103-018-0732-y

Ajith Anand, Michael Miller + Show 7 more

Open Access

https://doi.org/10.1007/s11103-018-0732-y

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Abstract

Key messageA simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation.The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86–103%) raw transformation frequency in an elite maize inbred.

Highlights

Agrobacterium-mediated plant transformation is a preferred method for plant genetic engineering since it was known to reliably transfer large (30–150 kb) DNA fragments into plants to generate events with low transgene copy number (Gelvin 2003, 2009; Komari et al 2004)
The complexity of plasmid construction was later solved through the development of the ‘binary vector’ system, where the transfer DNA (T-DNA) is separated on a smaller, independent episome from the virulence genes encoded by a disarmed Ti plasmid (Bevan 1984; Hoekema et al 1983)
Since the observed usable event (UE) frequency in PH2RT was lower with a ternary vector containing plasmid pPHP70298, we evaluated the accessory plasmid pPHP71539 (Fig. 1c) designed to have additional vir genes (VirD, VirE operons) to improve UE frequency on maize transformation

Summary

Introduction

Agrobacterium-mediated plant transformation is a preferred method for plant genetic engineering since it was known to reliably transfer large (30–150 kb) DNA fragments into plants to generate events with low transgene copy number (Gelvin 2003, 2009; Komari et al 2004). The pVIR plasmids have many desirable features, such as small size, enhanced vector stability, an improved bacterial selectable marker, and amended vir genes (operons virC, virD and virE) for improved T-DNA delivery (Anand et al 2017). Introduction of these accessory plasmids in trans with the T-DNA binary vectors in the same Agrobacterium strain facilitated the generation of highly versatile ternary vectors (Supplementary Fig. 1). We identified unique ORI-by-ORI combinations with relatively high raw transformation frequencies: the percent of infected embryos producing stable events was 86–103% This represents a six-to-seven-fold improvement over conventional random transformation (13–14%) using the plasmid pSB1 in an elite maize inbred

Results and discussion

Experimental procedures

Compliance with ethical standards