Efficient,reproducible <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum using heat treatment of immature embryos |
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Authors: | Songul Gurel Ekrem Gurel Rajvinder Kaur Joshua Wong Ling Meng Han-Qi Tan Peggy G Lemaux |
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Institution: | (1) Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720, USA;(2) Present address: Department of Plant Breeding, Sugar Institute, Etimesgut, 06790 Ankara, Turkey;(3) Present address: Department of Biology, Faculty of Science and Literature, Abant Izzet Baysal University, 5380 Bolu, Turkey |
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Abstract: | A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for
example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various
temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal
43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at
43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased
frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs
were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2
to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing
calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar
treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable
transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is
several-fold higher than published frequencies. |
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Keywords: | Agrobacterium Heat treatment Phosphomannose isomerase Sorghum Transformation |
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