Herbicide-resistant weeds result from rare genetic mutations that increase in frequency through selection by herbicides. The ability of scientists to make specific edits in weed genomes including the genes for herbicide resistance is becoming feasible. The value of such work is that studying changes in herbicide response due to specific gene edits would greatly further our understanding of potential solutions to the growing herbicide resistance problem. Gene editing processes could also one day be introduced into weed populations to facilitate increased weed control, including the reversion of resistant weeds back to susceptibility, through systems called gene drives. To develop gene drives that reverse herbicide resistance in weeds, laboratory studies need to first be done using weed tissues that do not have the capacity to escape laboratory containment through the production of seed, pollen, or other propagules. Plants grown in tissue culture as undifferentiated cells do not have such capacity, yet still maintain most of the physiological processes that are targeted by herbicides. We previously developed a tissue culture system in waterhemp and used these cultures to generate cells without cell walls (protoplasts), which are useful in gene editing research. The current proposal will investigate the experimental conditions necessary to allow protoplast division and growth. We also propose to begin this same tissue culture research in Palmer amaranth by establishing tissue culture lines of this species. For both weed species, the long-term goal is to evaluate the potential of emerging gene drive technology to reverse herbicide resistance in the field.

1) Test the recovery and growth of waterhemp protoplasts under various conditions.
2) Establish laboratory-grown callus tissue cultures of Palmer amaranth.

1) The ability to grow and study genetic alterations in waterhemp cells without risking unintentional release into the environment, facilitating study of genetic weed control technologies such as gene drives.
2) Expansion of the current waterhemp tissue culture system to Palmer amaranth, including the production of cultured Palmer amaranth lines for future study.

Updated December 6, 2022:
Completed work:
The waterhemp tissue culture cell line used in the current work was originally derived from the stem of a germinating waterhemp seedling. The cell line was first established as callus culture (clumps of undifferentiated cells), which was then used to initiate cell suspension cultures grown in liquid and maintained under laboratory conditions.

Waterhemp protoplasts (cells without cell walls) were prepared by incubating cell suspension cultures in an enzyme solution of 4% Cellulase Onozuka R-10, 0.2% Macerozyme R-10, 6.5% mannitol, and 0.1% CaCl2. Incubation was for 90 minutes at room temperature followed by 90 minutes at 32 C, after which the enzyme solution was replaced by a solution of 0.5 M mannitol, 20 mM KCl, and 4 mM 2-ethanesulfonic acid, pH 5.7-6.0. Protoplast viability was tested by microscopy using fluorescein diacetate (FDA) dye. To further evaluate protoplast health after enzyme digestion, a form of FDA dye that detects oxidation was used to evaluate oxidative stress. The ability of protoplasts to express a transgene after PEG-mediated transformation was also used as a measure of viability.

The following conditions were tested for ability to improve protoplast viability: 1) incubation in enzyme solution for 180 minutes at room temperature; 2) reducing the Cellulase Onozuka R-10 concentration to 1.5% while raising the Macerozyme R-10 concentration to 0.75%; and 3) reducing the mannitol concentration to 2% while adding 3% sucrose.

Preliminary results:
Protoplasts were found to be viable after incubation in the original enzyme solution, but viability then substantially decreased over two days at room temperature. This was consistent with previous research where protoplasts 1) did not recover and grow, and 2) did not express a transgene after PEG-mediated protoplast transformation. Changes in incubation temperature, enzyme concentration, and sugar concentration did not improve protoplast health and the ability to express a transgene.

Oxidative stress created by conditions during protoplast production was considered as a possible reason for insufficient protoplast viability. Microscopic observation of protoplasts treated with an oxidation-sensitive stain did detect substantial oxidative stress in protoplasts. This suggests that inclusion of antioxidants in the enzyme solution might be beneficial, and that using an oxidation-sensitive stain has value in assessment of protoplast health.

Work to be completed:
Results indicating oxidative stress in waterhemp protoplasts are preliminary and still need to be confirmed. Objective 2, the establishment of Palmer amaranth tissue culture lines, also still needs to be completed.

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Herbicide-resistant weeds are decreasing the effectiveness of existing herbicides for soybean production. Alternative weed control strategies need to be explored, including the potential of emerging genetic technologies for weed control. Gene drives are a genetic technology with potential to reverse herbicide resistance in weed populations and/or directly disrupt the ability of weeds to successfully propagate. While gene drives are gaining worldwide interest, much research needs to be done before they would be available for release. As this research progresses, it is important that: 1) problems experienced by North Dakota soybean farmers are included among the priorities, 2) research is performed in a manner that does not risk negative impacts on North Dakota agriculture through unintentional weed escapes, and 3) efforts are directed toward gene drive systems that are most likely to be successful and accepted by the public. Establishing methods for gene drive research in waterhemp and Palmer amaranth, using laboratory-contained tissue cultures and with a focus on reversing herbicide resistance, upholds these priorities and positions North Dakota soybean growers to benefit from this emerging technology.