A microsatellite linkage map for the cultivated strawberry (Fragaria × ananassa) suggests extensive regions of homozygosity in the genome that may have resulted from breeding and selection.

The linkage maps of the cultivated strawberry, Fragaria × ananassa (2n = 8x = 56) that have been reported to date have been developed predominantly from AFLPs, along with supplementation with transferrable microsatellite (SSR) markers. For the investigation of the inheritance of morphological characters in the cultivated strawberry and for the development of tools for marker-assisted breeding and selection, it is desirable to populate maps of the genome with an abundance of transferrable molecular markers such as microsatellites (SSRs) and gene-specific markers. Exploiting the recent release of the genome sequence of the diploid F. vesca, and the publication of an extensive number of polymorphic SSR markers for the genus Fragaria, we have extended the linkage map of the 'Redgauntlet' × 'Hapil' (RG × H) mapping population to include a further 330 loci, generated from 160 primer pairs, to create a linkage map for F. × ananassa containing 549 loci, 490 of which are transferrable SSR or gene-specific markers. The map covers 2140.3 cM in the expected 28 linkage groups for an integrated map (where one group is composed of two separate male and female maps), which represents an estimated 91% of the cultivated strawberry genome. Despite the relative saturation of the linkage map on the majority of linkage groups, regions of apparent extensive homozygosity were identified in the genomes of 'Redgauntlet' and 'Hapil' which may be indicative of allele fixation during the breeding and selection of modern F. × ananassa cultivars. The genomes of the octoploid and diploid Fragaria are largely collinear, but through comparison of mapped markers on the RG × H linkage map to their positions on the genome sequence of F. vesca, a number of inversions were identified that may have occurred before the polyploidisation event that led to the evolution of the modern octoploid strawberry species.

Adventitious shoot regeneration from seven commercial strawberry cultivars (Fragaria x ananassa Duch.) using a range of explant types.

The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration--those needed to carry out transformation experiments--with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector.

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.

Adventitious Embryogenesis and the in vitro culture of Apple Seed Parts.

Immature apple seeds from four scion cultivars, Bramley, Cox, Greensleeves and Spartan, and four rootstocks, M.9, M.25, M.26 and M.27 (Malus púmila Mill.), were collected at 30 and 50 days post-anthesis, dissected into nucellus, endosperm and zygotic embryo and cultured in vitro. The basal media of Linsmaier and Skoog (LS) and Murashige and Tucker (MT) were compared with hormone-containing media for their effects on adventitious embryogenesis, nucellus and endosperm callus formation and zygotic embryo development. Nucellar tissues from 30-day-old seeds formed callus only in the presence of an auxin, 2,4-D or NAA, and a cytokinin, BA. Concentrations of 4.4 × 10(-6)M and 2.2 × 10(-5) M were effective. Adventitious embryos arose from the micropylar ends of the nucellus or endosperm in 50-day-old seeds at a frequency of 0-23 % depending on the cultivar. The number of adventitious embryos varied from 1 to 9 per seed. Generally the inclusion of growth regulators had no beneficial effects and the inclusion of malt extract at 500 mg · l(-1) to the basal media was inhibitory. Embryos could be induced to undergo shoot proliferation for subsequent plantlet production. Endosperm callus growth was obtained on both basal and hormone-supplemented media in excised 50-day-old seeds. The frequency of callus formation was cultivar and media dependent and ranged from 0-80%. Growth on LS media was prolific and the hormone-autotrophic nature of this callus has persisted after more than a year in culture. Excised zygotic embryos from 50-day-old seeds could be stimulated to produce multiple shoots from single embryo shoot apices on media containing 4.4 × 10(-6)M and 2.2 × 10(-5) M BA. This effect was reduced by the inclusion of 500 mg·1(-1) casein hydrolysate. Secondary adventitious embryogenesis could also be induced on the cotyledon surface of both adventitious and zygotic embryos at specific combinations of NAA and BA. On basal media zygotic embryos developed into seedlings in vitro and the addition of GA3 sometimes enhanced the process.