Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrid) plants |
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Authors: | Mukesh Jain Kudithipudi Chengalrayan Ahmed Abouzid Maria Gallo |
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Institution: | (1) Department of Agronomy, Genetics Institute, University of Florida, Gainesville, FL 32611-0300, USA;(2) Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, Gainesville, FL 32611-0300, USA |
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Abstract: | For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based “positive” selection system has been used
to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic
callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing
13.6 μM 2,4-D, 20 g l−1 sucrose and 3 g l−1 mannose. Plant regeneration was obtained on MS basal medium with 2.5 μM TDZ under similar selection conditions, and the regenerants
rooted on MS basal medium with 19.7 μM IBA, 20 g l−1 sucrose, and 1.5 g l−1 mannose. An increase in mannose concentration from permissive (1.5 g l−1) to selective (3 g l−1) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four
vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses
showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis. |
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Keywords: | Mannose Virus resistance PMI Positive selection SCMV Sugarcane |
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