Abstract
Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×103 cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.
Similar content being viewed by others
References
Arencibia A, Molina PR, de laRiva G, Selman-Housein G: Production of transgenic sugarcane (Saccharum officinarum L.) plants by intact cell electroporation. Plant Cell Rep 14: 305–309 (1995).
Birch RG, Bower R: Principles of gene transfer using particle bombardment. In: Yang N-S, Christou P, (eds) Particle Bombardment Technology for Gene Transfer, pp. 3–37. Oxford University Press, New York (1994).
Bowen B: Anthocyanin genes as visual markers in transformed maize tissues. In: Gallagher SR (ed) GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, pp. 163–179. Academic Press, San Diego, FL (1992).
Bower R, Birch RG: Transgenic sugarcane plants via microprojectile bombardment. Plant J 2: 409–416 (1992).
Bruce WB, Christensen AH, Klein TM, Fromm ME, Quail PH: Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment. Proc Natl Acad Sci USA 86: 9692–9696 (1989).
Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM: Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci USA 90: 11212–11216 (1993).
Chamberlain DA, Brettel RIS, Last DI, Witrzens B, McElroy D, Dolferus R, Dennis ES: The use of the Emu promoter with antibiotic and herbicide resistance genes for the selection of transgenic wheat callus and rice plants. Austral J Plant Physiol 21: 95–112 (1994).
Chia T-F, Chan Y-S, Chua N-H: The firefly luciferase gene as a non-invasive reporter forDendrobium transformation. Plant J 6: 441–446 (1994).
Christensen AH, Sharrock RA, Quail PH: Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18: 675–689 (1992).
Dennehey BK, Petersen WL, Ford-Santino C, Pajeau M, Armstrong CL: Comparison of selective agents for use with the selectable marker genebar in maize transformation. Plant Cell Tissue Organ Cult 36: 1–7 (1994).
Dupuis I, Pace GM: Gene transfer to maize male reproductive structure by particle bombardment of tasel primordia. Plant Cell Rep 12: 607–611 (1993).
Finer JJ, McMullen MD: Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep 8: 586–589 (1990).
Finer JJ, Vain P, Jones MW, McMullen MD: Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11: 323–328 (1992).
Franks T, Birch RG: Gene transfer into intact sugarcane cells using microprojectile bombardment. Aust J Plant Physiol 18: 471–480 (1991).
Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM: Inheritance and expression of chimaeric genes in the progeny of transgenic maize plants. Bio/technology 8: 833–839 (1990).
Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, Adams WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG: Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603–618 (1990).
Hauptmann RM, Vasil V, Ozias-Akins P, Tabaeizadeh Z, Rogers SG, Horsch RB, Vasil IK, Fraley RT: Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species. Plant Cell Rep 6: 265–270 (1987).
Heinz DJ, Mee GWP: Plant differentiation from callus tissue ofSaccharum species. Crop Sci 9: 346–348 (1969).
Hiei Y, Ohta S, Komari T, Kumashiro T: Efficient transformation of rice (Oryza sativa L.) mediated byAgrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282 (1994).
Hunold R, Bronner R, Hahne G: Early events in microprojectile bombardment: cell viability and particle location. Plant J 5: 593–604 (1994).
Jefferson RA: Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Klein TM, Gradziel T, Fromm ME, Sanford JC: Factors influencing gene delivery intoZea mays cells by high velocity microprojectiles. Bio/technology 6: 559–563 (1988).
Last DI, Brettel RIS, Chamberlain DA, Chaudhury AM, Larkin PJ, Marsh EL, Peacock WJ, Dennis ES: Emu: an improved vector for gene expression in cereal cells. Theor Appl Genet 81: 581–588 (1991).
Leduc N, Iglesias VA, Bilang R, Gisel A, Potrykus I, Sautter C: Gene transfer to inflorescence and flower meristems using ballistic micro-targeting. Sex Plant Reprod 7: 135–143 (1994).
Lloyd AM, Walbot V, Davis RW:Arabidopsis andNicotiana anthocyanin production activated by maize regulators R and C1. Science 258: 1773–1775 (1992).
Ludwig SR, Bowen B, Beach L, Wessler SR: A regulatory gene as a novel visible marker for maize transformation. Science 247: 449–450 (1990).
Mudge SR, Lewis-Henderson W, Birch RG: Comparison ofVibrio and firefly luciferases as reporter gene systems for use in bacteria and plants. Aust J Plant Physiol 23: 75–83 (1996).
Perl A, Kless H, Blumenthal A, Galli G, Galan E: Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimisation of culture conditions and DNA-microprojectile procedures. Mol Gen Genet 235: 279–284 (1992).
Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR: Fertile, transgenic oat plants. Bio/technology 10: 1589–1594 (1992).
Taylor PWJ, Ko H-L, Adkins SW, Rathus C, Birch RG: Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids). Plant Cell Tissue Organ Cult 28: 69–78 (1992).
Vain P, McMullen MD, Finer JJ: Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12: 84–88 (1993).
Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil IK: Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Bio/technology 11: 1553–1558 (1993).
Wan Y, Lemaux PG: Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37–48 (1994).
Ye GN, Daniell H, Sanford JC: Optimization of delivery of foreign DNA into higher-plant chloroplasts. Plant Mol Biol 15: 809–819 (1990).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bower, R., Elliott, A.R., Potier, B.A.M. et al. High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Mol Breeding 2, 239–249 (1996). https://doi.org/10.1007/BF00564201
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00564201