V. N. Iyer

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

SummaryA survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.

Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L.

SummaryAgrobacterium-mediated transformation of thin cell layer explants (Klimaszewska and Keller 1985) yielded large numbers of transgenic plants of a major Canadian rapeseed cultivar Brassica napus ssp. oleifera cv Westar. The morphology and fertility of these plants were indistinguishable from controls. The Ti plasmid vector, pGV3850 (Zambryski et al. 1983) was used as a cis vector and as a helper plasmid for the binary vector pBin19 (Bevan 1984). Selectable marker genes that conferred resistance to high levels of kanamycin (Km) on Nicotiana tabacum were less efficient in the selection of transgenic B. napus. At low levels of Km (15 μg/ml) large numbers of transgenic plants (50%) were identified among the regenerants by nopaline synthase activity and several of these were confirmed by Southern blot analyses. Only a small number were resistant to higher levels of Km (80 μg/ml). Preliminary analyses indicated that resistance to Km was transmitted to the selfed progeny. Chimeric chloramphenicol acetyl transferase genes were ineffective biochemical markers in transgenic B. napus.

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-β-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

Detection of gene regulatory signals in plants revealed by T-DNA-mediated fusions

A binary vector, pPRF120, was designed to detect T-DNA insertions within transcriptionlly active areas of the plant genome. Linked to the right-border repeat, the vector contains a promoterless β-glucuronidase (GUS) gene which can, upon integration into chromosomes, be activated by cis-acting regulatory elements. The vector also incorporates a chimeric marker gene conferring resistance to kanamycin to ensure recovery of gene fusions regardless of the extent of their tissue-specific or developmentally regulated expression, and to permit analysis of the frequency of plants which express the promoterless reporter. Approximately 1000 transgenic tobacco plants harboring pPRF120 were regenerated. Analysis of 52 individuals indicated that more than 80% contain single, intact copies of the T-DNA, regardless of their ability to express the promoterless GUS gene. Screening of leaf tissue from the 1000 pPRF120 transformants revealed that ca. 5% of the plants contained GUS activity. Fluorogenic and histological GUS assays were used to visualize and quantify tissue- and cell-specific gene expression. The potential usefulness of pPRF120 in comparison to other vectors designed to generate in vivo gene fusions is discussed.

In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

SummaryGenes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.

Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea.

SummaryBrassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.

Induction of efficient cell division in alfalfa protoplasts.

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2–10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.

Microinjection: An Experimental Tool for Studying and Modifying Plant Cells

The conviction that DNA is the genetic material of cells has its origins in a set of experiments in which purified DNA was transferred and expressed in bacterial cells. It is therefore no surprise that since these initial experiments with bacteria, methods of transferring DNA and other molecules into eukaryotic cells have been explored with varying degrees of success and usefulness. The most direct way of delivering a population of molecules into a cell or cell compartment is to microinject it mechanically (Celis, 1984; Celis et al., 1986). Techniques to accomplish this have been developed for frog oocytes (Gurdon et al., 1971; Kressman et al., 1978; Rusconi and Schaffner, 1981), insect embryos (Rubin and Spradling, 1982), animal egg cells (Brinster et al., 1985; Gordon et al., 1980; Hammer et al., 1985; Wagner et al., 1981) and mammalian somatic cells (Capecchi, 1980; Diacumakos, 1973; Graessman et al., 1980a, b). The anatomical organization of plant cells and the experimental difficulties associated with their manipulation in culture prevented a facile transposition of such techniques from animal to plant cells (Steinbiss et al., 1984). Fortunately, this situation is changing rapidly. It is now possible to microinject some plant cells and various protoplast types successfully (Crossway et al., 1986; Reich et al., 1986 a, b, c). It is the intent of this contribution to review these methodologies so that they can be extended, improved upon and become easily accessible to the community of plant cell and molecular biologists. Through this discussion we hope that the experimental advantages offered by microinjection become apparent.