Arlette Reynaerts

Vectors for cloning in plant cells

Publisher Summary This chapter discusses vectors for cloning in plant cells. The chapter presents recent octopine Ti plasmid-derived vectors and their use to transfer and express foreign genes in plants. Plant cells transformed with this avirulent tDNA retain their intrinsic capacity to redifferentiate into complete plants. Moreover, the tDNA vectors contain a dominant selectable marker gene, mainly a chimeric neomycin phosphotransferase gene that provides the transformed plant cell with a selectable kanamycin resistance trait.. pGV2260 contains the intact oir region, which encodes Ti plasmid functions necessary for tDNA transfer and/or integration. These functions can act in trans to the tDNA. The chapter discusses cointegration vectors and binary vectors. The chapter includes the steps to introduce a foreign gene into a Ti plasmid vector. The chapter also discusses the expression of chimeric genes in plant cells, plant transformation, the transformation of tobacco leaf fragments, the analysis of gene expression in transformed plants, neomycin phosphotransferase assay, and tDNA organization in transformed plants.

The bar gene as selectable and screenable marker in plant engineering

Publisher Summary This chapter explains the use of the bar gene as a selectable marker in plant transformation, as a screenable marker in tissue culture and plant breeding, and as a reporter gene in plant molecular biology. The bar gene has served as a useful assayable marker gene in plant molecular biology. To guarantee correct translation initiation in plants, an ATG initiation codon was introduced instead of the GTG codon used in the streptomyces strain and the second codon was changed to introduce an NcoI site at the 5’ end of the coding region. Chimeric gene constructs, containing the bar coding region under control of different promoters, have been transferred to several crops. The bar gene has also been successfully used as a selectable marker in some plant species, using PPT or bialaphos as a selective agent. Transgenic plants were resistant to herbicide applications in the greenhouse and in field conditions.

Transgenic expression of two marker genes under the control of an Arabidopsis rbcS promoter: sequences encoding the Rubisco transit peptide increase expression levels

SummaryChimeric gene constructs were made in which two reporter genes, the neo and bar genes, encoding neomycin phosphotransferase II and phosphinothricin acetyl transferase, respectively, were placed under the control of the promoter of ats1A, one of four genes encoding the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (SSU) in Arabidopsis thaliana. In one set of constructs the fusions were made at the initiation codons, while in the second set the sequences encoding the ats1 A transit peptide were included. Significantly higher steady-state levels of RNA and protein were observed in leaves of transgenic plants varrying the latter constructions. Individual transgenic plants varied in their degree of tissue specific expression of the chimeric genes as well as in absolute levels of expression. Preliminary results suggest that the ats1 A promoter may be only weakly responsive to phytochrome.

cry IA(b) transcript formation in tobacco is inefficient.

Chimaeric PCaMV35Scry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5′ splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

Engineered genes for fertility control and their application in hybrid seed production

Abstract A new hybrid system based on genetically engineered genes for fertility control has been applied to several vegetable crops. Genic male sterile ( S ) lines were produced by expressing the ribonuclease genes Rnase T1 from Aspergillus oryzae or barnase from Bacillus amyloliquefaciens under the control of a tapetum specific promoter originally isolated from tobacco. Restorer ( R ) lines for the barnase S lines can be obtained by expressing the gene coding for barstar, the intracellular inhibitor of barnase under control of the same promoter. S lines are maintained either by linkage of the ms gene to the herbicide resistance gene bar or by isolating homozygous male steriles using the R lines. In this paper we describe how the engineered fertility control system can be used for the production of hybrid chicory and cauliflower and we demonstrate the potential of this system to produce novel hybrid crops such as hybrid lettuce.

Selectable and screenable markers

The transfer of foreign DNA to plant cells can be achieved in a variety of ways that are described in several chapters of this manual. This chapter describes the various selectable and screenable marker genes that are presently used in plant transformation experiments. Screenable markers have been widely used in gene constructs to study the regulation of plant gene expression.

Quantification of Transient Expression Levels of Genes Transferred to Plant Protoplasts by Electroporation

A simple and fast procedure for the quantitative analysis of gene expression directed by chimeric constructs in plants has been developed. The problem of ‘inter-experiment’ variability has been reduced by the inclusion of an internal standard. Different chimeric constructs have been evaluated in Nicotiana tabacum protoplasts using this system and compared with expression in transformed tobacco plant tissues. The system has also been tested in Brassica oleracea protoplasts.

[11] – The bar Gene as Selectable and Screenable Marker in Plant Engineering

Publisher Summary Plants can be transformed by using Agrobacterium-based T-DNA vectors or by direct uptake of DNA. However, for the selection of transformed cells or tissues, selectable marker genes are required. This chapter describes the use of the bialaphos resistance gene as a selectable marker in plant transformation, as a screenable marker in tissue culture and plant breeding, and as a reporter gene in plant molecular biology. The bar gene is a part of the biosynthesis pathway of bialaphos. The strains streptomyces hygroscopicus and streptomyces viridochromogenes produce bialaphos, a tripeptide antibiotic. It consists of phosphinothricin (PPT), which is an irreversible inhibitor of glutamine synthetase, and two L-alanine residues. Glufosinate ammonium, the chemically synthesized PPT, and bialaphos are used as nonselective herbicides. The bar gene codes for phosphinothricin acetyltransferase converts PPT with high affinity into a nonherbicidal acetylated form by transferring the acetyl group from acetyl-CoA on the free amino group of PPT.