Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants.
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 microM 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 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM 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.
Jain
Mukesh
M
Department of Agronomy, Genetics Institute, University of Florida, Gainesville, FL 32611-0300, USA.
Chengalrayan
Kudithipudi
K
Abouzid
Ahmed
A
Gallo
Maria
M
D015139:Blotting, Southern; D002678:Chimera / Q000235:genetics*; D047108:Embryonic Development; D008358:Mannose / Q000378:metabolism*; D008359:Mannose-6-Phosphate Isomerase / Q000378:metabolism*; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives / Q000378:metabolism; D030821:Plants, Genetically Modified; D010957:Plasmids; D012038:Regeneration; D031786:Saccharum / Q000196:embryology / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D012641:Selection, Genetic; D046509:Tissue Culture Techniques; D014170:Transformation, Genetic; D019076:Transgenes
C007016:chlorophenol red; D008359:Mannose-6-Phosphate Isomerase; D010637:Phenolsulfonphthalein; D008358:Mannose