Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars |
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Authors: | Shiromani W V Basnayake Richard Moyle Robert G Birch |
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Institution: | (1) Botany Department/BIOL, The University of Queensland, Brisbane, 4072, Australia; |
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Abstract: | Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants
of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental
phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set
of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection
steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested
cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three
cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or
light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced
no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants
with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain
several independent transgenic plants per bombardment, with time in callus culture limited to 11–15 weeks. A caution with
this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half
of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a
useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of
other sugarcane cultivars. |
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