Hemp (Cannabis sativa L.).

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Efficient genetic transformation of hemp (Cannabis sativa L.) is crucial for crop improvement and functional genomics.
• Agrobacterium tumefaciens-mediated transformation is a widely used method for plant genetic engineering.

Purpose of the Study:

• To establish and optimize an Agrobacterium tumefaciens-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 hemp cells.

Main Methods:

• Hemp suspension culture cells were transformed using Agrobacterium tumefaciens strain EHA101 with the binary plasmid pNOV3635, containing the phosphomannose isomerase (PMI) selectable marker.
• Transformed cells were selected on mannose-containing medium, and PMI expression was screened using a chlorophenol red assay.
• Genomic DNA from putative transformants was analyzed for the presence of the PMI gene via PCR and Southern hybridization.

Main Results:

• The phosphomannose isomerase (PMI) gene enabled efficient selection of transformed hemp cells, as they could metabolize mannose.
• A high average transformation frequency of 31.23% (±0.14) was achieved across all experiments, with individual frequencies ranging from 15.1% to 55.3%.
• PCR and Southern hybridization confirmed the stable integration of the PMI gene into the hemp genome.

Conclusions:

• Agrobacterium tumefaciens-mediated transformation using the phosphomannose isomerase (PMI) selectable marker provides an effective and efficient method for genetic modification of hemp (Cannabis sativa L.) suspension cultures.
• The established protocol offers a reliable approach for generating transgenic hemp lines with potential applications in research and industry.
• This study significantly advances the genetic engineering capabilities for Cannabis sativa L., paving the way for future crop development.