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Two disarmed Ti-binary vectors in Agrobacterium tumefaciens have been used to produce viable transgenic strawberry plants. Fertile strawberry plants with a normal phenotype were regenerated after transformation with pBIN6, which carries genes for nopaline synthase (nos) and neomycin phosphotransferase (nptII) (conferring kanamycin resistance). The transfer and expression of the two genes was confirmed by Southern blot analysis, the detection of nopaline synthase (NOS) activity in vegetative and reproductive tissues and rooting in vitro in the presence of kanamycin. The nos gene continued to be expressed in glasshouse-grown plants many months after removal from in vitro growth conditions. After selfing the RO plants nos segregated in the R1 progeny according to a 3:1 Mendelian ratio. In in vitro germinated seedlings there was complete correlation between the presence of nopaline synthase activity and the ability of leaf segments to produce callus on a medium containing kanamycin. Transgenic clones that exhibited an abnormal phenotype associated with cytokinin overproduction were produced when plants were transformed with pSS1, a derivatives of pBIN19 carrying both the nptII gene and the ipt gene (encoding the enzyme isopentenyltransferase). Shoots of these clones grew on hormone-free medium, could not be induced to root and their growth was unaffected by the presence of 50 μg/ml kanamycin in hormone-free media.

Summary Several factors have been examined for their effects on the frequency of shoot regeneration from apple leaf discs and strips. Up to 90 % of leaves from micropropagated cultures of the cultivar Greensleeves could be induced to form adventitious shoots when cut into strips. On a per leaf basis 4–7 shoots regenerated over a 2 to 8 week period. Cut strips gave higher regeneration frequencies than 7 mm diameter discs when data were expressed on this basis. Six different combinations of the growth regulators BA and NAA were assessed for their effects on shoot organogenesis and 2.0 mg 1 -1 BA and 0.5 mg .1 -1 NAA were optimal. Levels of regeneration were increased by having abaxial rather than adaxial surface in contact with the regeneration medium, although this effect was dependent on the presence of 0.1 or 1.0 mM putrescine but not the DNA methylation inhibitor 5-azacytidine. Similarly, leaf discs excised from rooted in vitro plantlets gave significantly increased levels of regeneration compared to unrooted controls but, again, only when the same concentrations of the diamine were present. Shoots were still able to regenerate after X-irradiation up to 3 Krads. At 5 Krads no regeneration was observed. The cultivars Tuscan, M.9 and M.25 could also be made to regenerate shoots from leaf discs but the frequency varied from a few percent to 60 % depending on the cultivar. Once shoots had been regenerated they were easily micropropagated, rooted and acclimatised.