Phleomycin resistance as a dominant selectable marker for plant cell transformation

Abstract

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance ('Ble') genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however, the highest frequency of rooted plants was obtained when transformation was carried out with the 'Sh Ble' gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.

Regeneration and Transformation of Apple (Malus pumila Mill.)

“…We have now compromised on using 21 days exposure to kanamycin as a suitable time for screening out untransformed cells without subjecting the tissues to overlong exposures to the antibiotic should it interfere with the regeneration process. There are reports that other antibiotics related to kanamycin and equally effective at selection are less inhibitory to the regeneration process viz paromomycin [27] and phleomycin [28].…”

“…Although the growth of Agrobacterium is inhibited at low pH, the addition of glycine betaine has been shown to improve growth and virulence induction at pH 5.2 [34]. Currently Agrobacterium cultures to be used in transformation experiments are grown for 24-36 h in YEP medium at [24][25][26][27][28] oc. The cultures are centrifuged and resuspended in a simplified induction medium (SIM) to an OD of 0.5 (550 nm) and allowed to grow at 26 o C for 5 h to induce virulence.…”

Antibiotic resistance markers for plant transformation

“…Two genes encoding bleomycin-binding proteins [8] have been used to construct dominant bleomycin or phleomycin resistance markers for plant cell transformation: A bleomycin resistance gene from Tn5 [11,18] and one derived from the actinomycete Streptoalloteichus hindustanus [18].…”

Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease.

“…To delete the 3' end of GUS, plasmid p35S-GUS-tn,, was digested with Haell, blunted by T4 DNA polymerase, ligated to Hindlll linkers, and digested with Hindlll to generate the gus3'A fragment, which was ligated to the Hindlll site of pUC19 to give pAR164. The PAR164 Hindlll fragment containing P35s-gus3'A was inserted by ligation at a unique Hindlll site between the left and right border sequences of the pGA-3Sh binary vector (Perez et al, 1989) to give pAR168. Finally, a partial EcoRl digest of pARl62 was cloned into a complete EcoRl digest of pAR168, yielding pAR173 containing the inverted repeat cassette gus3'A-P~-pUC19-P35s-5'Agus;:yh (Figure 1).…”