Factors Affecting the Level of Kanamycin Resistance in Transformed Sunflower Cells

Nutter, Robert C.; Everett, Nicholas P.; Pierce, Dorothy A.; Panganiban, Lucy; Okubara, Patricia A.; Lachmansingh, Rosanna; Mascarenhas, Desmond; Welch, H. Gilbert; Mettler, Irvin J.; Pomeroy, Lucille; Johnson, Jill L.; Howard, John A.

doi:10.1104/pp.84.4.1185

Cited by 11 publications

(1 citation statement)

References 14 publications

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“…While the kind of resistance is determined by the coding region of the chimeric gene, the level of resistance can be influenced by the transcription signals, as illustrated by Nutter et al (21). Thus, it is necessary to test several gene constructs in order to find one that permits transformed cells to outgrow nontransformed cells.…”

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Evaluation of Selectable Markers for Rice Transformation

Dekeyser

Claes²,

Marichal³

et al. 1989

Plant Physiol.

145

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A variety of expression systems and selection regimes have been developed to transform plants such as tobacco, petunia, and tomato. We investigated several of these to determine whether the promoters and selectable markers used in dicotyledonous plants are suitable for selecting transformed rice callus. We compared transient expression driven by constitutive and regulated promoters in rice (Oryza sativa) protoplasts and found that the 2' promoter of the octopine T-DNA is approximately 3 to 4 times more efficient than the CAMV 35S promoter, 10 times more efficient than the nos promoter and the 1' promoter, and more than 100 times better than two other regulated plant promoters. Similar results were obtained in tobacco (Nicotiana tabacum) protoplasts with the exception that the nos promoter was expressed nearly 10 times better in rice. Further studies demonstrated that rice callus growth is sensitive to low concentrations of methotrexate, phosphinothricin, and bleomycin, and to moderate concentrations of G418 and hygromycin, but is only partially inhibited by relatively high concentrations of kanamycin. Finally, we tested the ability of stably introduced resistance genes to protect callus against some of the selective agents. Genes that inactivated phosphinothricin or G418 permitted transformed calli to grow almost unimpeded on toxic concentrations of these selective agents. However, a gene conferring resistance to methotrexate could not be used to select for activily growing transformants. Southem analysis of the transformed cell lines demonstrated that 50% of the transformants contained a single plasmid copy and that nearly all integrated copies showed rearrangements. These results on the use of selectable markers in rice should facilitate efforts to obtain transformants of this important grain.

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confidence: 99%