Marc De Block

Expression of foreign genes in regenerated plants and in their progeny.

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non‐oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co‐integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co‐cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non‐oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (KmR and CmR). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non‐oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.

Two T-DNA's co-transformed intoBrassica napus by a doubleAgrobacterium tumefaciens infection are mainly integrated at the same locus.

SummaryHypocotyl explants of threeBrassica napus varieties were infected with two nopaline typeAgrobacterium strains each carrying a distinct disarmed T-DNA containing different selectable markers. Selection was done for only one of the markers, after which the regenerated plants were screened for the presence of the second marker. High co-transformation frequencies of both T-DNAs were obtained (39%–85% of the transformants). Where the two T-DNAs were integrated linked, they were usually present in an inverted orientation relative to each other; in all of the cases observed the two right borders were adjacent. Tandem orientations occurred less frequently. The T-DNAs were mainly integrated as intact copies and deletions did not often occur. The co-transformation system described favors a genetically linked integration of the two T-DNAs (78%), although in a single transformed plant both linked and unlinked copies of both T-DNAs may be present.

Silencing of poly(ADP-ribose) polymerase in plants alters abiotic stress signal transduction

Transgenic plants with reduced poly(ADP-ribose) polymerase (PARP) levels have broad-spectrum stress-resistant phenotypes. Both Arabidopsis thaliana and oilseed rape (Brassica napus) lines overexpressing RNA interference-PARP constructs were more resistant to various abiotic stress treatments in laboratory and greenhouse experiments without negative effects on growth, development, and seed production. This outperforming stress tolerance was initially attributed solely to a maintained energy homeostasis due to reduced NAD+ consumption. We show that in PARP2-deficient Arabidopsis plants, the observed abiotic stress resistance can also be explained by alterations in abscisic acid levels that facilitate the induction of a wide set of defense-related genes.

Energy use efficiency is characterized by an epigenetic component that can be directed through artificial selection to increase yield

Quantitative traits, such as size and weight in animals and seed yield in plants, are distributed normally, even within a population of genetically identical individuals. For example, in plants, various factors, such as local soil quality, microclimate, and sowing depth, affect growth differences among individual plants of isogenic populations. Besides these physical factors, also epigenetic components contribute to differences in growth and yield. The network that regulates crop yield is still not well understood. Although this network is expected to have epigenetic elements, it is completely unclear whether it would be possible to shape the epigenome to increase crop yield. Here we show that energy use efficiency is an important factor in determining seed yield in canola (Brassica napus) and that it can be selected artificially through an epigenetic feature. From an isogenic canola population of which the individual plants and their self-fertilized progenies were recursively selected for respiration intensity, populations with distinct physiological and agronomical characteristics could be generated. These populations were found to be genetically identical, but epigenetically different. Furthermore, both the DNA methylation patterns as well as the agronomical and physiological characteristics of the selected lines were heritable. Hybrids derived from parent lines selected for high energy use efficiencies had a 5% yield increase on top of heterosis. Our results demonstrate that artificial selection allows the increase of the yield potential by selecting populations with particular epigenomic states.

Chloroplast transformation by Agrobacterium tumefaciens

A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co‐cultivation in tobacco protoplasts followed by selection with 10 μg/ml chloramphenicol. The chloramphenicol‐resistant plants derived from these selected calli were unable to transmit the CmR phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos‐cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Ti‐plasmid vectors can be used to introduce genes in chloroplasts.

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.

DRL1, a Homolog of the Yeast TOT4/KTI12 Protein, Has a Function in Meristem Activity and Organ Growth in Plants

The DEFORMED ROOTS AND LEAVES1 (DRL1) gene is single copy in the Arabidopsis genome, and based on overall amino acid similarity and conservation of functional domains, the DRL1 protein is homologous with yeast TOT4/KTI12. TOT4/KTI12 associates with Elongator, a multisubunit complex that binds the RNA polymerase II transcription elongation complex. Recessive mutations at the DRL1 locus caused defective organ formation indicative of disorganized shoot, inflorescence, flower, and root meristems. DRL1 is a putative ATP/GTP binding protein; in addition, calmodulin binding activity was demonstrated in vitro for the C terminus of the DRL1 protein. Phenotypic and genetic data position DRL1 relative to regulatory loci for leaf development, in which it acts early. We identified Arabidopsis homologs for the six Elongator components and hypothesize that DRL1 regulates transcription elongation through a putative plant Elongator. Upregulation of the ANGUSTIFOLIA transcript in the strong drl1-2 allele supports this model.

The cell biology of plant transformation: Current state, problems, prospects and the implications for the plant breeding

SummaryThe DNA delivery systems which are routinely used to introduce genes into crop plants are Agrobacterium tumefaciens, electroporation and particle bombardment. The differences and similarities between these different transformation techniques are outlined. The influence of the cell biological approach, and more specifically the impact of the state of the plant cell at the moment of transformation, on the genotype and phenotype of the regenerated transgenic plant is analysed. In this respect phenomena such as position effects, gene silencing, co-suppression, epistasis, co-transformation and somaclonal variation are discussed. The relevance of these factors for plant breeders is discussed.

Transforming petals into sepaloid organs in Arabidopsis and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner

Oilseed rape (Brassica napus L.) genotypes with no or small petals are thought to have advantages in photosynthetic activity. The flowers of field-grown oilseed rape form a bright-yellow canopy that reflects and absorbs nearly 60% of the photosynthetically active radiation (PAR), causing a severe yield penalty. Reducing the size of the petals and/or removing the reflecting colour will improve the transmission of PAR to the leaves and is expected to increase the crop productivity. In this study the ‘hairpin’ RNA-mediated (hpRNA) gene silencing technology was implemented in Arabidopsis thaliana (L.) Heynh. and B. napus to silence B-type MADS-box floral organ identity genes in a second-whorl-specific manner. In Arabidopsis, silencing of B-type MADS-box genes was obtained by expressing B. napus APETALA3 (BAP3) or PISTILLATA (BPI) homologous self-complementary hpRNA constructs under control of the Arabidopsis A-type MADS-box gene APETALA1 (AP1) promoter. In B. napus, silencing of the BPI gene family was achieved by expressing a similar hpRNA construct as used in Arabidopsis under the control of a chimeric promoter consisting of a modified petal-specific Arabidopsis AP3 promoter fragment fused to the AP1 promoter. In this way, transgenic plants were generated producing male fertile flowers in which the petals were converted into sepals (Arabidopsis) or into sepaloid petals (B. napus). These novel flower phenotypes were stable and heritable in both species.

Engineered fertility control in transgenic Brassica napus L.: Histochemical analysis of anther development

Cytological and histochemical analyses were performed on developing anthers of wild-type, transgenic male-sterile and fertility-restored Brassica napus plants. Male sterility resulted from the expression of the barnase gene under the control of the tobacco-derived tapetumspecific promoter pTA29. Fertility was restored to male sterile plants by expressing the barstar gene, which encodes a barnase-specific inhibitor protein. In addition, the tissue specificity of the pTA29 promoter in B. napus was studied in transgenic plants expressing pTA29:gus fusions. In B. napus, the pTA29 promoter not only directed the expression of genes to the tapetum, but also, although more weakly, to the vascular tissue region of the anther filament at the late uninucleate and early binucleate stage. The pTA29:barnase gene was expressed in the tapetum at the vacuolated-microspore stage. This resulted in the disappearance of RNA from the tapetum. Degradation of RNA in the tapetum was immediately followed by a complete loss of RNA in the developing microspores. Following lysis of the microspores in the sterile anthers, the pTA29:barnase gene was expressed in the vascular tissue region of the anther filament. This expression resulted in a deposition of wound callose in the phloem, followed by a precocious wilting of the whole anther. Finally, in the fertility-restored plants the cytological and histochemical patterns of anther development were identical to those of wild-type anthers.