Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Mariliis Tark-Dame; Blaise Weber; Mara de Sain; Damar Tri Anggoro; Rechien Bader; Aimee Walmsley; Rurika Oka; Maike Stam

doi:10.3791/57295

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Method Article

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

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DOI:

10.3791/57295

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March 28th, 2018

Mariliis Tark-Dame¹ , Blaise Weber¹ , Mara de Sain¹ , Damar Tri Anggoro¹ , Rechien Bader¹ , Aimee Walmsley¹ , Rurika Oka¹ , Maike Stam¹

¹Swammerdam Institute for Life Sciences, University of Amsterdam

Summary

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

Abstract

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2–0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Introduction

When generating transgenic plants, usually the objective is to have the integrated transgene(s) stably expressed. This can be achieved by intact single copy integrations of a transgene. Multiple integrations can lead to increased expression of a transgene, but also to gene silencing. Silencing of transgenes is more likely if inserted sequences are arranged in tandem or inverted repeats¹^,²^,³^,⁴. Binary vectors are used as shuttles in Agrobacterium-mediated transformation experiments to deliver the sequences of interest into plant geno....