FLP‐mediated recombination for use in hybrid plant production

Abstract

SummaryWe have studied the feasibility in Arabidopsis of using a site-speci®c recombination system FLP/FRT, from the 2 mm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP sitespeci®c recombinase were crossed with plants transformed with a vector containing kanamycinresistance gene (npt)¯anked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen-and tapetum-speci®c gene, bcp1,¯anked by FRT sites. Upon cross-pollination of¯owers on the same male-sterile plants with pollen from FLPcontaining plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the ®rst time that a site-speci®c recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.

Highly Efficient In Vitro Site-Specific Recombination System Based on Streptomyces Phage φBT1 Integrase

“…In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.Site-specific recombinases have been widely used in genetic engineering: for example, in vitro cloning, plant and mammalian cell genome modification, and gene therapy (3,11,12,15,16). Nearly all site-specific recombinases can be classified as tyrosine recombinases, also known as the integrase family, or serine recombinases, also known as the resolvase/invertase family, based on comparisons of amino acid sequences and different mechanisms of catalysis; these two types use tyrosine or serine, respectively, to attack the DNA sugar-phosphate backbone (14,18,20).…”

Pollen Sterility—A Promising Approach to Gene Confinement and Breeding for Genetically Modified Bioenergy Crops

“…CMS has been observed in over 150 plant species including maize, rice, and wheat [70,71]. Detasseling (for maize) or emasculation procedures and/or CMS systems are lacking or deficient in many important crops still grown as inbred or partially domesticated varieties [36,72,73]. Given the accumulated knowledge of CMS systems and the desirability of developing male sterility systems lacking the shortcomings of most CMS systems, a number of strategies for engineering male sterility have been developed to generate male-sterile plants as breeding materials and for gene confinement strategies [2,73,74].…”

Site-specific recombination in Arabidopsis plants promoted by the Integrase protein of coliphage HK022

“…So far we have only shown recombination reactions in the cis configuration, such reactions can be useful to eliminate undesired genes, such as genes for antibiotic resistance or genes that cause male sterility, as is already practiced by using the Cre-lox and FLP-frt systems (Luo et al, 2000;Hoa et al, 2002;Ow, 2002;Puchta, 2003). For the insertion or replacement of genes it still needs to be shown that the Int-catalyzed reactions take place also in the trans configuration (each att site on a different DNA molecule).…”