RESUMO
Following the production of transgenic plants, the selectable marker gene(s) used in the process are redundant, and their retention may be undesirable. They can be removed by exploiting segregation among the progeny of co-transformants carrying both the selectable marker gene and the effector transgene. Here we show that the doubled haploid technology widely used in conventional barley breeding programmes represents a useful means of fixing a transgene, while simultaneously removing the unwanted selectable marker gene. Primary barley co-transformants involving hpt::gfp (the selectable marker) and gus (a model transgene of interest) were produced via Agrobacterium-mediated gene transfer to immature embryos using two respective T-DNAs. These plants were then subjected to embryogenic pollen culture to separate independently integrated transgenes in doubled haploid progeny. A comparison between 14 combinations, involving two Agrobacterium strains carrying various plasmids, revealed that the highest rate of independent co-transformation was achieved when a single Agrobacterium clone carried two binary vectors. Using this principle along with Agrobacterium strain LBA4404, selectable marker-free, gus homozygous lines were eventually obtained from 1.5 per 100 immature embryos inoculated. Compared to the segregation of uncoupled T-DNAs in conventionally produced progeny, the incorporation of haploid technology improves the time and resource efficiency of producing true-breeding, selectable marker-free transgenic barley.
Assuntos
Hordeum/genética , Agrobacterium tumefaciens/genética , Sequência de Bases , Cruzamento , DNA Bacteriano/genética , DNA de Plantas/genética , Expressão Gênica , Genes de Plantas , Marcadores Genéticos , Proteínas de Fluorescência Verde/genética , Haploidia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas Geneticamente Modificadas , Ploidias , Proteínas Recombinantes/genética , Transformação GenéticaRESUMO
Methods of the Agrobacterium-mediated transformation of bread wheat (Triticum aestivum L.) have been improved in recent years so that genetic engineering can be routinely used for functional genomics as well as for wheat breeding. In the protocol described here, immature embryos of the spring-type model genotype Bobwhite SH 98 26 have been used. Preculture and temperature pretreatment of these explants have led to the reproducible generation of transgenic plants at efficiencies between 5 and 15%. Whereas primary transgenic plants regenerated in vitro commonly show reduced fitness and fertility, no apparent variations with regard to morphology and grain set in their transgenic progeny as compared to wild-type counterparts were observed.
Assuntos
Agrobacterium/genética , Técnicas de Transferência de Genes , Plantas Geneticamente Modificadas , Sementes/genética , Transformação Genética , Triticum/genética , DNA Bacteriano , Testes Genéticos , FenótipoRESUMO
Barley is a major crop species, and also has become a genetic model for the small grain temperate cereals. A draft barley genome sequence has recently been completed, opening many opportunities for candidate gene isolation and functionality testing. Thanks to the development of customizable endonucleases, also site-directed genome modification recently became feasible for higher plants, which marks the beginning of a new era of genetic engineering. The development of improved binary vectors and hypervirulent Agrobacterium tumefaciens strains has raised the efficiency of genetic transformation in barley to a level where the technique has become relatively routine. The transformation method described here involves immature barley embryos cocultivated with Agrobacterium after removal of their embryo axis. Critical adjustments to the protocol have included the supplementation of the cocultivation medium with the polyphenolic signaling compound acetosyringone at comparatively high concentration and the use of cysteine to reduce the extent of cellular oxidation upon agroinfection. In addition, the use of liquid, rather than solid, cocultivation medium promotes the throughput of the method. The protocol has delivered well over 10,000 transgenic barley plants over the past 10 years. Routine transformation efficiency, calculated on the basis of the recovery of independent transgenics per 100 explants, has reached about 25 % in cultivar (cv.) "Golden Promise". The protocol has proven effective for more than 20 barley cultivars, although some adjustments to the culture conditions have had to be made in some cases. The transformation efficiency of cv. "Golden Promise" remains higher than that of any other cultivar tested.