Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen.

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.

The S locus glycoprotein and the S receptor kinase are sufficient for self-pollen rejection in Brassica.

Self-incompatibility (SI) is one of several mechanisms that have evolved to prevent inbreeding in plants. SI in Brassica is controlled by the polymorphic S locus complex. Two S locus-encoded proteins are coordinately expressed in the stigma epidermis: the cell wall-localized S locus glycoprotein (SLG) and the plasma membrane-anchored S receptor kinase (SRK). These proteins are thought to recognize a pollen factor that leads to the rejection of self-pollen. Evidence has accumulated that indicates that both proteins are necessary for the ability of the stigma to inhibit self-pollen. However, it has not been possible to prove this necessity definitively or to demonstrate that these genes are sufficient for this phenotype, because previous attempts to transfer this phenotype via transformation have not been successful. In this study, two overlapping S locus genomic clones, which cover approximately 55 kilobases of DNA and contain the SLG, SRK, and an anther-expressed gene in the region common to the two, were introduced into a self-compatible Brassica napus line. The resulting transgenic plants were shown to carry the female part of the SI phenotype, rejecting pollen in a haplotype-specific manner. However, the pollen SI phenotype was not found in any of the transgenic plants. These data show that the SLG and SRK are sufficient for the female side but not the male side of the SI phenotype in Brassica and that there must be an independent pollen S factor encoded outside the cloned region.

A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants.

Self-incompatibility, the rejection of self pollen, is the most widespread mechanism by which flowering plants prevent inbreeding. In Brassica, the S receptor kinase (SRK) has been implicated in the self-incompatibility response, but the molecular mechanisms involving SRK are unknown. One putative downstream effector for SRK is ARC1, a protein that binds to the SRK kinase domain. Here it is shown that suppression of ARC1 messenger RNA levels in the self-incompatible Brassica napus W1 line is correlated with a partial breakdown of self-incompatibility, resulting in seed production. This provides strong evidence that ARC1 is a positive effector of the Brassica self-incompatibility response.

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.

Modification of carbonic anhydrase activity by antisense and over-expression constructs in transgenic tobacco.

The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO2 assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO2 assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity.

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.

Microspore mutagenesis and selection: Canola plants with field tolerance to the imidazolinones.

In vitro microspore mutagenesis and selection was used to produce five fertile double-haploid imidazolinone-tolerant canola plants. The S2 plants of three of the mutants were resistant to at least the field-recommended levels of Assert and Pursuit. One mutant was tolerant to between five and ten times the field-recommended rates of Pursuit and Scepter. Two semi-dominant mutants, representing two unlinked genes, were combined to produce an F1 hybrid which was superior in imidazolinone tolerance to either of the heterozygous mutants alone. Evaluation of the mutants under field conditions indicated that this hybrid and the original homozygous mutants could tolerate at least two times the field-recommended rates of Assert. The field results indicated the mutants were unaffected in seed yield, maturity, quality and disease tolerance. These genes represent a potentially valuable new herbicide resistance system for canola, which has little effect on yield, quality or maturity. The mutants could be used to provide tolerance to several imidazolinones including Scepter, Pursuit and Assert.