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. 1990 Jul;2(7):591–602. doi: 10.1105/tpc.2.7.591

Transient Gene Expression in Intact and Organized Rice Tissues.

RA Dekeyser 1, B Claes 1, R De Rycke 1, ME Habets 1, MC Van Montagu 1, AB Caplan 1
PMCID: PMC159914  PMID: 12354966

Abstract

Regulated gene expression of chimeric genes has been studied extensively in electroporated protoplasts. The applicability of these assays is limited, however, because protoplasts are not always physiologically identical to the cells from which they are derived. We have developed a procedure to electroporate DNA into intact and organized leaf structures of rice. Optimization of the new gene delivery system mainly involved eliminating explant-released nucleases, prolonging the DNA/explant incubation time, and expanding the pulse time. Using a [beta]-glucuronidase gene under the control of constitutive promoters, we demonstrated that all cell types within a leaf base were susceptible to electroporation-mediated DNA uptake. Although the technique was initially developed for leaf bases of young etiolated rice seedlings, we proved that it was equally applicable both to other monocotyledons, including wheat, maize, and barley, and to other explants, such as etiolated and green sheath and lamina tissues from rice. Transient gene expression assays with electroporated leaf bases showed that the promoter from a pea light-harvesting chlorophyll a/b-binding protein gene displayed both light- and chloroplast-dependent expression in rice, and that the promoter from the Arabidopsis S-adenosylmethionine synthetase gene was, as in transgenic Arabidopsis and tobacco, preferentially expressed in cells surrounding the vascular bundles.

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Selected References

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  1. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  2. Callis J., Fromm M., Walbot V. Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts. Plant Physiol. 1988 Dec;88(4):965–968. doi: 10.1104/pp.88.4.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. De Clercq A., Vandewiele M., De Rycke R., Van Damme J., Van Montagu M., Krebbers E., Vandekerckhove J. Expression and Processing of an Arabidopsis 2S Albumin in Transgenic Tobacco. Plant Physiol. 1990 Apr;92(4):899–907. doi: 10.1104/pp.92.4.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dekeyser R., Claes B., Marichal M., Van Montagu M., Caplan A. Evaluation of selectable markers for rice transformation. Plant Physiol. 1989 May;90(1):217–223. doi: 10.1104/pp.90.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fromm M., Taylor L. P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5824–5828. doi: 10.1073/pnas.82.17.5824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gallie D. R., Lucas W. J., Walbot V. Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell. 1989 Mar;1(3):301–311. doi: 10.1105/tpc.1.3.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huttly A. K., Baulcombe D. C. A wheat alpha-Amy2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts. EMBO J. 1989 Jul;8(7):1907–1913. doi: 10.1002/j.1460-2075.1989.tb03593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ingelbrecht I. L., Herman L. M., Dekeyser R. A., Van Montagu M. C., Depicker A. G. Different 3' end regions strongly influence the level of gene expression in plant cells. Plant Cell. 1989 Jul;1(7):671–680. doi: 10.1105/tpc.1.7.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jefferson R. A., Kavanagh T. A., Bevan M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987 Dec 20;6(13):3901–3907. doi: 10.1002/j.1460-2075.1987.tb02730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones J. D., Dunsmuir P., Bedbrook J. High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J. 1985 Oct;4(10):2411–2418. doi: 10.1002/j.1460-2075.1985.tb03949.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim S. H., Shinkle J. R., Roux S. J. Phytochrome induces changes in the immunodetectable level of a wall peroxidase that precede growth changes in maize seedlings. Proc Natl Acad Sci U S A. 1989 Dec;86:9866–9870. doi: 10.1073/pnas.86.24.9866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lamppa G., Nagy F., Chua N. H. Light-regulated and organ-specific expression of a wheat Cab gene in transgenic tobacco. Nature. 1985 Aug 22;316(6030):750–752. doi: 10.1038/316750a0. [DOI] [PubMed] [Google Scholar]
  13. Peleman J., Boerjan W., Engler G., Seurinck J., Botterman J., Alliotte T., Van Montagu M., Inzé D. Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. Plant Cell. 1989 Jan;1(1):81–93. doi: 10.1105/tpc.1.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Peleman J., Saito K., Cottyn B., Engler G., Seurinck J., Van Montagu M., Inzé D. Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana. Gene. 1989 Dec 14;84(2):359–369. doi: 10.1016/0378-1119(89)90510-6. [DOI] [PubMed] [Google Scholar]
  15. Reiss B., Sprengel R., Will H., Schaller H. A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene. 1984 Oct;30(1-3):211–217. doi: 10.1016/0378-1119(84)90122-7. [DOI] [PubMed] [Google Scholar]
  16. Rhodes C. A., Pierce D. A., Mettler I. J., Mascarenhas D., Detmer J. J. Genetically transformed maize plants from protoplasts. Science. 1988 Apr 8;240(4849):204–207. doi: 10.1126/science.2832947. [DOI] [PubMed] [Google Scholar]
  17. Richards D., Thompson W. K., Pharis R. P. The influence of dwarfing interstocks on the distribution and metabolism of xylem-applied [h]gibberellin a(4) in apple. Plant Physiol. 1986 Dec;82(4):1090–1095. doi: 10.1104/pp.82.4.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Velten J., Velten L., Hain R., Schell J. Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J. 1984 Dec 1;3(12):2723–2730. doi: 10.1002/j.1460-2075.1984.tb02202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vernet T., Fleck J., Durr A., Fritsch C., Pinck M., Hirth L. Expression of the gene coding for the small subunit of ribulosebisphosphate carboxylase during differentiation of tobacco plant protoplasts. Eur J Biochem. 1982 Sep 1;126(3):489–494. doi: 10.1111/j.1432-1033.1982.tb06806.x. [DOI] [PubMed] [Google Scholar]
  20. Wienhues U., Hosokawa K., Höveler A., Siegmann B., Doerfler W. A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells. DNA. 1987 Feb;6(1):81–89. doi: 10.1089/dna.1987.6.81. [DOI] [PubMed] [Google Scholar]

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