Rice Genetics IV - IRRI books - International Rice Research Institute

Achieving synthetic apomixisThe minimum number of transgenes required for IRRI’s approach to apomixis isthree (Fig. 3). One transgene is required to induce an adventitious embryo in thenucellus; it will take the form of a chimera of a nucellus-specific promoter and thecoding region of an embryo-inducing gene. A second transgene is required to ablatethe sexual embryo. It will be a chimera of an embryo-specific promoter and a geneencoding an ablating protein. However, it will also contain a green fluorescent protein(GFP) gene located between the promoter and the ablating gene to block transcription.Two lox sites in tandem (arrows in Fig. 3) will flank the GFP gene to allowits removal by cre recombinase. GFP will be driven by a suitable promoter and GFPfunction can be monitored to check on the success of excision. The third transgeneconsists of the cre recombinase controlled by a meiosis-specific promoter. In a relatedapproach, Zuo et al (2001) placed GFP downstream from a pair of lox sites todemonstrate cre recombinase-mediated gene activation.The first and second transgenes will be in one parent (male parent in Fig. 3),whereas the third transgene will be located in the other parent (female). Prior to pollinationby the breeder, the cre recombinase will be expressed during meiosis in themale parent but will have no DNA substrate on which to work. After pollination, thecre recombinase will be expressed during meiosis and will immediately remove theGFP gene from the second construct via recombination at the lox sites. The ablatinggene will be expressed in all subsequent zygotic embryos, thus killing them. It willnot be expressed in adventitious nucellar embryos because the lineage leading to eachsuccessive adventitious embryo does not pass through meiosis. Figure 4 shows thelocation of the three transgenes before and after manual hybridization. Because of theaction of the cre recombinase, the ablating gene becomes activated but only inpostmeiotic cell lineages. To avoid ablating the endosperm, it is essential that theembryo-specific promoter is not expressed in the pollen, the central cell, or the endosperm.From the foregoing outline, it will be evident that our two objectives (induction ofthe adventitious embryo and ablation of the zygotic embryo) consist of several dis-Male parentNucellus-specificpromoterEmbryo-inducinggeneTermEmbryo-specificpromoterGFPgeneAblating proteingeneTerm= lox site for cre recombinaseFemale parentMeiosis-specificpromotercre recombinasegeneTermFig. 3. At a minimum, three constructs will be required to generate theproposed synthetic form of apomixis for hybrid rice. See text for details.Molecular tools for achieving synthetic apomixis in hybrid rice 387