Minimal handling and super-binary vectors facilitate efficient, agrobacterium-mediated, transformation of sugarcane (Saccharum spp. hybrid)

Anderson, David J. and Birch, Robert G. (2012) Minimal handling and super-binary vectors facilitate efficient, agrobacterium-mediated, transformation of sugarcane (Saccharum spp. hybrid). Tropical Plant Biology, 5 2: 183-192. doi:10.1007/s12042-012-9101-1


Author Anderson, David J.
Birch, Robert G.
Title Minimal handling and super-binary vectors facilitate efficient, agrobacterium-mediated, transformation of sugarcane (Saccharum spp. hybrid)
Journal name Tropical Plant Biology   Check publisher's open access policy
ISSN 1935-9756
1935-9764
Publication date 2012
Sub-type Article (original research)
DOI 10.1007/s12042-012-9101-1
Volume 5
Issue 2
Start page 183
End page 192
Total pages 10
Place of publication New York, NY, United States
Publisher Springer
Collection year 2013
Language eng
Abstract Agrobacterium-mediated transformation (AMT) of sugarcane has been limited by low transformation efficiency, high variability between experiments and genotype specificity. We tested combinations of parameters that have been useful in other recalcitrant plant systems, aiming to develop an efficient and reproducible method. Applied to elite sugarcane cultivar Q117, key parameters were (i) minimal handling of callus near the time of co-cultivation, (ii) use of a super-binary helper vector with additional virB,C,G gene copies, and (iii) use of Agrobacterium strain AGL1. Transformation efficiency was in the range 0.5 to 3.5 stably transformed, embryogenic-callus-forming lines per gram fresh weight of co-cultivated callus, over six independent callus batches. Addition of 5 μM copper sulphate to the callus-growth medium appeared beneficial in a single further test. Following selection for aminoglycoside resistance conferred by PUbi-aphA, 87% of transformed lines that formed embryogenic callus were regenerable to plants. Southern blot analysis of 24 transgenic lines showed 21% with a single-copy insertion of an intact T-DNA without vector backbone, and a mean transgene copy number of 2.5. Over multiple batches, the AMT protocol approached the transformation efficiency from our routine conditions for particle bombardment of Q117. However, the same parameters were ineffective for AMT of cultivars Q208 and Q172, and yielded a lower transformation efficiency (0.02) with KQ228. As experienced in other systems such as rice, high-efficiency transformation of one recipient genotype may provide useful starting parameters for work towards AMT of additional genotypes.
Keyword Agrobacterium
Saccharum
Sugarcane
Transformation
Transgene integration
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Official 2013 Collection
 
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Created: Tue, 09 Apr 2013, 11:14:04 EST by Emma Lambe on behalf of School of Agriculture and Food Sciences