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Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants

  • Genetic Transformation and Hybridization
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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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Abbreviations

CI3:

Callus induction medium

CP:

Coat protein

CPR:

Chlorophenol red

2,4-D:

2,4-dichlorophenoxyacetic acid

IBA:

Indole-3-butyric acid

ID50 dose:

Selection dose required to achieve 50% growth inhibition

manA :

Escherichia coli phosphomannose isomerase gene

MS:

Murashige and Skoog medium

nptII:

Neomycin phosphotransferase II

PMI:

Phosphomannose isomerase (EC 5.3.1.8)

SCMV:

Sugarcane mosaic virus

SrMV:

Sorghum mosaic virus

TDZ:

Thidiazuron

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Acknowledgments

The authors wish to thank Prof. T. E. Mirkov for the gift of plasmids ubi-eut and pAHC17. The authors thank Drs. Altpeter and Wofford for critical reading of the manuscript. This research was supported by the Florida Agricultural Experiment Station.

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Authors and Affiliations

  1. Department of Agronomy, Genetics Institute, University of Florida, Gainesville, FL, 32611-0300, USA

    Mukesh Jain, Kudithipudi Chengalrayan, Ahmed Abouzid & Maria Gallo

  2. Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, Gainesville, FL, 32611-0300, USA

    Maria Gallo

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  1. Mukesh Jain
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  2. Kudithipudi Chengalrayan
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  3. Ahmed Abouzid
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  4. Maria Gallo
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Correspondence to Maria Gallo.

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Communicated by J. C. Register

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Jain, M., Chengalrayan, K., Abouzid, A. et al. Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants. Plant Cell Rep 26, 581–590 (2007). https://doi.org/10.1007/s00299-006-0244-0

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  • DOI: https://doi.org/10.1007/s00299-006-0244-0

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