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

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.

Auxin independent1 (Axi1) as an endogenous gene for copy number determination in transgenic tobacco

“…As expected, the wild type tobacco did not show amplification of the Bar gene (Table 1). Transgenic single copy events are desirable because they follow a Mendelian segregation pattern, which means that the possibility of gene silencing is lower (Collier et al 2017, Tark-Dame et al 2018. Our results corroborated those of Šubr et al (2006) who also used the Axi1 gene as an endogenous control to determine the copy number for the Potato virus A (PVA) P3 gene in transgenic tobacco.…”