| Plant Physiol. 1992 May; 99(1): 81–88. | PMCID: PMC1080409 |
Regeneration of Transgenic Soybean ( Glycine max) Plants from Electroporated Protoplasts 1Sarwan K. Dhir, 2 Seema Dhir, Michael A. Savka, Faith Belanger, 3 Alan L. Kriz, Stephen K. Farrand, and Jack M. Widholm Department of Agronomy, Plant and Animal Biotechnology Laboratory, University of Illinois, Urbana, Illinois 61801 Department of Plant Pathology, Plant and Animal Biotechnology Laboratory, University of Illinois, Urbana, Illinois 61801 Abstract Transgenic soybean ( Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 10 6 electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10 −4. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants. Full text Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References. Images in this article Click on the image to see a larger version. These references are in PubMed. This may not be the complete list of references from this article. - Chee, Paula P; Fober, Krystal A; Slightom, Jerry L. Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens. Plant Physiol. 1989 Nov;91(3):1212–1218. [PubMed]
- Christou, Paul; Murphy, Jean E; Swain, William F. Stable transformation of soybean by electroporation and root formation from transformed callus. Proc Natl Acad Sci U S A. 1987 Jun;84(12):3962–3966. [PubMed]
- Damm, B; Schmidt, R; Willmitzer, L. Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. Mol Gen Genet. 1989 May;217(1):6–12. [PubMed]
- Fromm, ME; Taylor, LP; Walbot, V. Stable transformation of maize after gene transfer by electroporation. Nature. 319(6056):791–793. [PubMed]
- Gallie, DR; Novak, S; Kado, CI. Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium. Plasmid. 1985 Sep;14(2):171–175. [PubMed]
- Hauptmann, RM; Vasil, V; Ozias-Akins, P; Tabaeizadeh, Z; Rogers, SG; Fraley, RT; Horsch, Robert B; Vasil, Indra K. Evaluation of Selectable Markers for Obtaining Stable Transformants in the Gramineae. Plant Physiol. 1988 Feb;86(2):602–606. [PubMed]
- Lin, Willy; Odell, Joan T; Schreiner, Ronald M. Soybean Protoplast Culture and Direct Gene Uptake and Expression by Cultured Soybean Protoplasts. Plant Physiol. 1987 Jul;84(3):856–861. [PubMed]
- Lyznik, LA; Ryan, RD; Ritchie, SW; Hodges, TK. Stable co-transformation of maize protoplasts with gusA and neo genes. Plant Mol Biol. 1989 Aug;13(2):151–161. [PubMed]
- Southern, EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
- Widholm, JM. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 1972 Jul;47(4):189–194. [PubMed]
|