Botrytis cinerea-resistant marker-free Petunia hybrida produced using the MAT vector system

Abstract

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-autotransformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected leaf explants of P. hybrida were cultured on hormone-and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic-and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida.

Plant Tissue Culture: Current Status and Opportunities

“…In addition, marker free transgenic plants of Petunia hybrida were produced using multi-auto-transformation (MAT) vector system. The plants exhibited high level of resistance to Botrytis cinerea, causal agent of gray mold [71].…”

Histological analysis and transcription profiles on somatic embryogenesis in interspecific hybrids of <i>Elaeis guineensis × E. oleifera</i>

“…Dehydrins are late embryogenesis abundant (LEA) proteins related to the acquisition of desiccation tolerance but, in addition, they have been clearly related to stress responses, as well as defensins [24,[35][36][37][38]. The differences among the expression profiles of those two genes and that of the LIPID TRANSFER PROTEIN led us to conclude that transcription of this last was not related to stress in our experiments.…”

Recent Progress in Enhancing Fungal Disease Resistance in Ornamental Plants

“…Transgenic Petunia hybrida, overexpressing the endochitinase gene from Trichoderma harzianum, alone or in combination with osmotin, exhibited resistance to B. cinerea [114]. Khan et al (2011) developed transgenic petunia plants overexpressing the wasabi defensin (WD) gene from Wasabia japonica [115]. Expression of the AMP defensin increased resistance to B. cinerea in marker-free transgenic petunias.…”