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Hemp (Cannabis sativa L.).

Mistianne Feeney1, Zamir K Punja

  • 1Okanagan Biotechnology Inc., Summerland, British Columbia, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|October 13, 2006
PubMed
Summary

Researchers successfully transformed hemp cells using Agrobacterium tumefaciens and a phosphomannose isomerase (PMI) selectable marker. This method achieved a high average transformation frequency, enabling efficient genetic modification of Cannabis sativa L.

Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Genetics

Background:

  • Hemp (Cannabis sativa L.) is a valuable crop with growing interest in genetic improvement.
  • Efficient transformation methods are crucial for developing improved hemp varieties.
  • Agrobacterium-mediated transformation is a common technique for plant genetic engineering.

Purpose of the Study:

  • To establish and optimize an Agrobacterium-mediated transformation protocol for hemp (Cannabis sativa L.) suspension culture cells.
  • To utilize the phosphomannose isomerase (PMI) gene as a selectable marker for efficient screening of transformed cells.
  • To determine the transformation efficiency of the developed method.

Main Methods:

  • Hemp suspension culture cells were transformed using Agrobacterium tumefaciens strain EHA101 with the binary plasmid pNOV3635, containing the phosphomannose isomerase (PMI) gene.

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  • Transformed cells were selected on a mannose-containing medium, leveraging the PMI gene's ability to metabolize mannose.
  • PMI expression was screened using a chlorophenol red assay, and the presence of the PMI gene was confirmed via polymerase chain reaction and Southern hybridization.

    • Main Results:

    • The phosphomannose isomerase (PMI) selectable marker system effectively distinguished transformed from non-transformed hemp cells.
    • Successful confirmation of the PMI gene in putatively transformed callus lines using molecular techniques.
    • An average transformation frequency of 31.23% +/- 0.14 was achieved across all experiments, with a broad range from 15.1% to 55.3%.

    Conclusions:

    • Agrobacterium-mediated transformation using the phosphomannose isomerase (PMI) selectable marker is an effective method for genetic modification of hemp (Cannabis sativa L.) suspension cultures.
    • The established protocol demonstrates a high and reliable transformation frequency, suitable for future genetic studies and breeding programs.
    • This method provides a robust tool for advancing research and development in hemp biotechnology.