Low Inoculum, Long Co-Culture Agrobacterium-Mediated Transformation of Plants
T2016-109 A method developed at The Ohio State University gives tremendous improvements in transformation rates of plant tissues using low inoculation levels of Agrobacterium, coupled with long co-culture periods of the bacterium with plant tissue.
Most plant biotechnology laboratories utilize Agrobacterium for production of transgenic plants, and most of the transgenic plants that are in farmer's fields today were generated using Agrobacterium-mediated transformation. Although production of transgenic crop plants is now routine and is often automated, improvements in the efficiency of transgenic plant production are still needed for both basic research in crop improvement and in the production of commercial transgenic plants. The standard protocol for production of transgenic plants involves the inoculation of specific target plant tissue with Agrobacterium, co-culture of the bacteria with the plant tissue for 2-3 days, washing of the plant tissue to remove the bacteria, and finally culture of the plant tissue on a medium containing antibiotics to remove or kill the residual Agrobacterium. This standard protocol is inefficient because the high inoculation ratio/low cultivation time may result in overgrowth of bacteria, tissue death, and difficulty in eliminating the bacteria. Therefore, a method for producing transgenic plants using low inoculum levels of Agrobacterium might help to reduce or eliminate problems associated with bacterial overgrowth and the resultant damage to the target tissue.
Researchers at The Ohio State University, led by Dr. John Finer, have established a protocol to increase transformation rates using low inoculation levels of Agrobacterium with a long co-culture period. This approach, which uses 10-100 bacteria or less per tissue piece (standard is 100,000-1,000,000 bacteria per tissue piece), allows long-term inoculation (1-2 weeks) of the tissue with Agrobacterium, leading to large increases in transformation rates. Since the low inoculum approach allows a long term co-culture period, targets that would either be sensitive to or overtaken by this bacterium can now be used. In addition to inoculation of excised tissue, this improvement may allow seeds, seedlings, plantlets, plants, pollen, flowers, and other target tissues to be used, which was previously not possible or resulted in very low transformation rates.
- Agriculture biotechnology
- DNA sequencing and genome editing
- Most plant biotechnology laboratories perform plant transformation
- New target tissues may be used that were previously not suitable for transformation
- The improvement could lead to multifold increases in transformation of some plants, a benefit to companies and academic laboratories.