High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers

Bower R., Elliott A.R., Potier B.A.M. and Birch R.G. (1996) High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Molecular Breeding, 2 3: 239-249.

Author Bower R.
Elliott A.R.
Potier B.A.M.
Birch R.G.
Title High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers
Journal name Molecular Breeding   Check publisher's open access policy
ISSN 1380-3743
Publication date 1996
Sub-type Article (original research)
Volume 2
Issue 3
Start page 239
End page 249
Total pages 11
Subject 1102 Cardiovascular Medicine and Haematology
1108 Medical Microbiology
1110 Nursing
1300 Biochemistry, Genetics and Molecular Biology
1303 Specialist Studies in Education
1305 Biotechnology
Abstract Transient expression of the maize anthocyanin regulatory elements, R and C1, 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 of R and C1 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 of luc, aphA or bar 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. When luc and aphA 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.
Keyword Anthocyanin
Antibiotic G418
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import
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