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.

Update:
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.

View uploaded report

Update:
Herbicide-resistant waterhemp and Palmer amaranth represent serious concerns for North Dakota soybean growers and other crop producers. Emerging genetic biocontrol technologies to supplement herbicidal weed control or reverse herbicide resistance need to be explored. However, sophisticated genetic research techniques and protocols are necessary to evaluate genetic biocontrol methods, and development of laboratory protocols for genetic research in weeds is behind what has been done in crops.

Conducting research on genetic biocontrol strategies requires safety considerations to ensure that any plants carrying genetic changes are not allowed to reproduce and escape containment before full evaluation and intentional release. Because of this, we believe that initial studies investigating the application of new genetic technologies for weed control should preferably be done on 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 capacity to propagate outside the laboratory, yet they still maintain most of the physiological processes that are targeted by herbicides. In other words, herbicide resistance and susceptibility are traits that can safely be studied in tissue culture.

To advance potential genetic biocontrol technologies for weed control, we previously developed a tissue culture system for waterhemp where callus tissue (clumps of undifferentiated cells), are grown from seedling hypotocols, followed by establishment of cell suspension cultures in liquid media. We then used these waterhemp cultures to generate protoplasts (cells without cell walls), to facilitate genetic transformation. However, introduction of a gene for green fluorescent protein (GFP) did not result in observable gene expression. Further investigation identified significant oxidative stress among the protoplasts, and it is likely that this stress is inhibiting transgene expression.

The objectives of the current project were to: 1) Test the recovery and growth of waterhemp protoplasts under various conditions, and 2) Establish laboratory-grown callus tissue cultures of Palmer amaranth. Testing of waterhemp protoplasts centered around reduction of oxidative stress as related to the source of cell wall digesting enzymes, and their concentrations. Establishment of Palmer amaranth callus cultures was also performed to begin similar work in Palmer amaranth.

Generation of waterhemp protoplasts was done using 1.5% Cellulase Onozuka R-10 enzyme, and 0.75% Macerozyme R-10 enzyme. These enzymes digest cell walls to produce protoplasts, and we sourced each enzyme from at least two different suppliers in order to detect any differences in the production of oxidative stress during protoplast generation. A flow cytometer was used to quantify oxidative stress after staining with a dye that fluoresces when oxidized (fluorescence intensity increases with increased oxidative stress). Oxidative stress was significantly higher (P<0.02), by a factor of 1.5-fold, when enzymes from Source 1 were used compared to Source 2. Enzymes used to produce protoplasts are isolated from plant pathogens, and the level of impurities may differ among suppliers. Due to their association with plant pathogens, these impurities may elicit oxidative stress in plant cells. It is clear that for our waterhemp cell culture line, enzymes from Source 2 should be used in future research due to the generation of significantly less oxidative stress compared to enzymes from Source 1.

Using enzymes from Source 2, we then studied varying amounts of Macerozyme R-10 to see if there was less oxidative stress with lower concentrations of this enzyme. Macerozyme R-10 concentrations of 0.19% (1/4x), 0.38% (1/2x), and 0.75% (1x), did not significantly affect the level of oxidative stress (P>0.5). The effects of reduced concentrations of Cellulase Onozuka R-10 on oxidative stress is currently being studied, but preliminary results do not suggest a significant effect.

It is likely that oxidative stress is affecting the health of our waterhemp protoplasts and their ability to express introduced genes. While commercial sources of enzymes have an impact, it is most likely that alternative methods to reduce oxidative stress, such as the use of antioxidants, will be necessary.

To begin similar tissue culture research in Palmer amaranth, we established callus tissue cultures of this weed using seed that was collected near Valley City, ND, in 2021. Seeds were placed on half-strength MS media with 1.5% sucrose and 0.8% agar in Petri plates, and incubated for 6 days at 32 C to promote germination. Hypocotyls approximately 1 cm in length were harvested and placed in culture bottles with MS media containing auxin (2,4-D) and cytokinin (6-benzylaminopurine). Cultures were then placed in the dark at room temperature, with callus tissue forming after about 2 weeks.

Establishment of Palmer amaranth cultures was successful, with 18 of 19 hypocotyls (95%) forming callus tissue. However, only 6 of 18 calluses were typical and undifferentiated. The other 12 calluses produced roots, which is not desirable. Rooting of callus cultures was not observed in previous research with waterhemp, and indicates that the level auxin/cytokinin ratio might be too high. The 6 calluses with typical, undifferentiated tissue were transferred to fresh media after 2 months, and have continued to grow normally without rooting. These calluses will be used to establish cell suspension cultures in liquid media.

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 biocontrol technologies for weed control. However, genetic research investigating weeds is far behind that of crops, and much work needs to be to develop tools for weed genetics. As research progresses, it is also important that it is performed in a manner that does not risk negative impacts on North Dakota agriculture through unintentional weed escapes. Establishing methods for genetic biocontrol research in waterhemp and Palmer amaranth, using laboratory-contained tissue cultures, supports the goal of bringing additional weed control options to North Dakota soybean growers.

Herbicide-resistant waterhemp and Palmer amaranth represent serious concerns for North Dakota soybean growers. Emerging genetic biocontrol technologies to supplement herbicidal weed control or reverse herbicide resistance need to be explored. Conducting research on genetic biocontrol strategies requires safety considerations to ensure that any plants carrying genetic changes are not allowed to reproduce and escape containment. Plants grown in tissue culture as undifferentiated cells do not have capacity to propagate outside the laboratory, yet they still maintain most of the physiological processes that are targeted by herbicides.

To advance potential genetic biocontrol technologies for weed control, we previously developed a tissue culture system where callus tissue (clumps of undifferentiated cells), are grown from seedling hypotocols, followed by establishment of cell suspension cultures in liquid media. We then used these waterhemp cultures to generate protoplasts (cells without cell walls), to facilitate genetic transformation. However, these protoplasts did not express introduced genes. Further investigation identified significant oxidative stress among the protoplasts, and it is likely that this stress was inhibiting transgene expression. The current research project explored methods to reduce oxidative stress during waterhemp protoplast production.

Generation of waterhemp protoplasts was done using two cell wall digesting enzymes, and we sourced each enzyme from at least two different suppliers in order to detect any differences in the production of oxidative stress during protoplast generation. Oxidative stress was significantly higher, by a factor of 1.5-fold, using enzymes from one source compared to the other. This confirmed that the enzyme supplier is important in the production of waterhemp protoplasts. Further studies using lowered concentrations of enzymes did not lower oxidative stress.

To begin similar tissue culture research in Palmer amaranth, we successfully established 6 callus tissue cultures of this weed. These calluses will be used to establish cell suspension cultures in liquid media. Another 12 cultures of Palmer amaranth were also generated, but these produced undesired roots in addition to undifferentiated cells.

This research improved current protocols for genetic research in waterhemp by lowering oxidative stress during protoplast generation. Palmer amaranth cultures were also established, and these cultures will be valuable in future genetic research in this important weed. Both results will facilitate research investigating emerging genetic biocontrol methods for control of herbicide-resistant weeds.

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.