Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector.

Dengxiang Du,Fangdong Zhang,Jinjie Guo,Ruchang Jin

doi:10.3390/genes10050374

Abstract

Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

Highlights

Genetic engineering techniques generally involve the addition of gene integrated into a plant leading to the modification of its genome [1]
Fifteen (88%) lines segregated with a 3:1 Mendelian pattern of inheritance, and the other two (12%) lines segregated with non-Mendelian ratios of distortion toward non-regulator seeds
The progeny of each T0 plant showed a complete marker excision, and a high percentage of T1 progeny from each T0 line is expected to contain a marker-free locus, which calli that produced secondary calli, was calculated to be approximately 16.7% at three weeks of culture was confirmed by PCR on the excision footprint (RS)

Summary

Introduction

Genetic engineering techniques generally involve the addition of gene (single or multiple) integrated into a plant leading to the modification of its genome [1]. Selectable marker genes are essential to plant genetic engineering and used in most transformation procedures to simplify the identification of transgenic plants Selectable markers, such as hygromycin, kanamycin, or glyphosate, differentiate transformed cells pass resistance toward antibiotics or herbicides to the transformed cells, whereas untransformed cells and tissues are killed by treatment with lethal compounds [6]. These systems allow relatively straightforward identification and selection of stably incorporated plants [7], and are used to follow the inheritance

Methods

Results

Conclusion