Herbicide-resistant weeds result from 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. To develop such gene editing systems, 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 culture conditions necessary to maintain protoplast health before, during, and after introduction of genetic material (transformation), to facilitate gene editing. We also propose to continue similar research in Palmer amaranth by producing cultures from which protoplasts can be produced.

A) Evaluate waterhemp protoplast stress under various culture conditions.
B) Establish laboratory-grown cell suspension cultures of Palmer amaranth.

A) Ability to perform waterhemp genetic research in a laboratory where escape of seeds and pollen is not a concern.
B) Production of Palmer amaranth cultures for laboratory-based genetic research.

Updated January 5, 2024:
Research Overview and Objectives:
Waterhemp and Palmer amaranth are problematic weeds that negatively impact corn production, especially when control is reduced due to herbicide resistance. Emerging genetic biocontrol technologies hold promise to help combat herbicide resistance in these weeds, but research to develop these strategies requires safety considerations to ensure that any organisms carrying genetic changes are not allowed to reproduce and escape containment. 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.

We have previously developed a tissue culture system for waterhemp and have been successful in producing waterhemp protoplasts (cells without cell walls), in preparation for polyethylene glycol (PEG)-mediated transformation. However, we have not been successful in obtaining subsequent recovery and growth of these cells. This is an indication that something is affecting the health of protoplasts within our current protocols.

Oxidative stress is a condition where reactive oxygen is generated by a cell under stress, leading to breakdown of membranes and eventual death of the cell. Oxidative stress is a negative factor in the maintenance of protoplast health, and we have identified oxidative stress among waterhemp protoplasts after isolation. Our current research investigates strategies to minimize oxidative stress during protoplast isolation.

In preparation for similar research on Palmer amaranth, we are also establishing cell suspension cultures in liquid media using callus cultures (clumps of cells), grown on solid media. This will allow future laboratory-based research on Palmer amaranth, with goals similar to those of waterhemp.

Specific objectives for this research project are:
Objective 1: Evaluate waterhemp protoplast stress under various culture conditions.
Objective 2: Establish laboratory-grown cell suspension cultures of Palmer amaranth.

Completed work:
Oxidative stress of waterhemp protoplasts was evaluated with 2',7'-dichlorofluorescein diacetate (DCFH-DA), a fluorescent stain that is activated by the presence of reactive oxygen species (ROS) within living cells. Previous research evaluated DCFH-DA fluorescence as an indicator of oxidative stress using a fluorescence microsope, but this method was limited in the number of protoplasts that could be evaluated (typically about 200 protoplasts per sample). Microscopic evaluation also did not provide quantitative measurements on the intensity of fluorescence (indicating the level of oxidative stress) and was also subject to human error.

To more efficiently evaluate waterhemp protoplast oxidative stress, we have established a protocol using a flow cytometer to evaluate DCFH-DA fluorescence among thousands of protoplasts per sample. The flow cytometer exposes protoplast samples to various wavelengths of light, analyzing how the light scatters and detecting resulting wavelengths to distinguish between protoplasts, cells with intact cell walls, and cellular debris. Protoplast size and fluorescence is also determined. This entire process can be carried out at the rate of thousands of cells per second, providing greater confidence and efficiency than traditional fluorescence microscopy can provide.

As DCFH-DA requires intracellular ROS to fluoresce, changes to oxidative stress levels are reflected by changes in fluorescence intensity among DCFH-DA stained cells. By comparing fluorescence intensity to that of the closely related stain fluorescein diacetate (FDA), which is activated in all living protoplasts, we are able to identify the level of oxidative stress among living waterhemp protoplasts. In this method, decreased relative fluorescence for cells stained with DCFH-DA indicates decreased oxidative stress. This has allowed us to more accurately identify protoplast isolation and culture conditions that keep oxidative stress to a minimum.

Four Palmer amaranth cell suspension cultures have also been established. These can be used in future research investigating genetic biocontrol strategies for this weed.

Progress of Work and Results to Date:
The protoplast generation process relies upon enzymes, such as cellulase and macerozyme, to degrade the cell walls that encapsulate and connect individual plant cells. The optimum concentrations of these enzymes are known to vary widely between different plant species and even between different cell lines within the same species. To further complicate the issue, the quality and efficacy of enzymes from different commercial sources has been found to vary.

Cell wall degrading enzymes from Yakult Pharmaceutical Industry Co. (Tokyo, JP), are recommended for the production of protoplasts from various plant species. To our knowledge, a comparison of ROS levels and oxidative stress after protoplast isolation from waterhemp cell suspension cultures using Yakult vs. other enzymes has not previously been completed. To begin this evaluation, we began with strained waterhemp cell suspension cultures grown in modified Murashige and Skoog media. Oxidative stress levels of waterhemp protoplasts prepared with Yakult enzymes were then compared to protoplasts prepared from domestically sourced commercial enzymes. Comparing fluorescence of FDA to DCFH-DA-stained samples, domestic enzymes produced 36.38 ± 3.08% relative fluorescence, while the Yakult enzymes produced 12.47 ± 1.92% relative fluorescence. These preliminary results indicated that waterhemp protoplasts produced by Yakult enzymes have 65.72% lower oxidative stress levels than those produced by alternatives. These preliminary results suggest that modification of the enzymes used in our waterhemp protoplast isolation procedures may help to promote protoplast health, facilitating the goal of protoplast recovery and growth. However, these experiments still need to be repeated, followed by additional statistical analyses, to be confident in the results.

Work to be Completed:
In addition to implementing a more accurate method for analyzing oxidative stress and determining the ideal source of enzymes for waterhemp protoplast isolation, we have begun work identifying compounds that may be added to the cell wall degradation solution during protoplast isolation to further reduce oxidative stress. Initial results have found that the addition of ascorbic acid, an antioxidant, may also reduce levels of oxidative stress. Work to confirm these preliminary results is ongoing.

Palmer amaranth cell suspension cultures are growing more slowly than our waterhemp cultures. Hormone levels in the culture media are currently being modified to improve Palmer amaranth growth.

Summary:
Tissue culture is a promising laboratory technique for research into emerging genetic biocontrol methods for waterhemp and Palmer amaranth. Protoplasts amenable to genetic transformation can be generated from cultured cells by removing cell walls using enzymes. However, protoplasts generated using current protocols are under oxidative stress and do not recover and divide. This project has identified different enzymes that have not generated as much oxidative stress in preliminary trials. In addition, use of the antioxidant ascorbic acid is promising but needs to be confirmed. Cultures of Palmer amaranth have also been established for future work in this area.

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Updated July 1, 2024:
Research Overview and Objectives:
Waterhemp and Palmer amaranth are problematic weeds that negatively impact soybean production, an issue that is worsened when control is reduced due to herbicide resistance. Emerging genetic biocontrol technologies hold promise to help combat herbicide resistance in these weeds, but research to develop these strategies requires safety considerations to ensure that any organisms carrying genetic changes are not allowed to reproduce and escape containment. 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.

We previously developed a tissue culture system for waterhemp and have been successful in producing waterhemp protoplasts (plant cells without cell walls), in preparation for polyethylene glycol (PEG)-mediated transformation. However, subsequent recovery and growth of these cells had not been achieved. This was an indication that something within the protoplast isolation protocol was affecting the health of the protoplasts.

Oxidative stress is a condition where reactive oxygen is generated by a cell under stress, leading to breakdown of membranes and eventual death of the cell. Oxidative stress is a negative factor in the maintenance of protoplast health, and we have identified oxidative stress among waterhemp protoplasts after isolation. Our research investigated strategies to minimize oxidative stress during protoplast isolation for the purpose of facilitating protoplast recovery and cell division.

In preparation for similar research on Palmer amaranth, we are also establishing cell suspension cultures in liquid media using callus cultures (clumps of cells), grown on solid media. This will allow future laboratory-based research on Palmer amaranth, with goals similar to those of waterhemp.

Specific objectives for this research project are:
Objective 1: Evaluate waterhemp protoplast stress under various culture conditions.
Objective 2: Establish laboratory-grown cell suspension cultures of Palmer amaranth.

Materials and Methods:
For Objective 1, waterhemp protoplasts were generated using 1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10. These are enzymes that digest plant cell walls to produce protoplasts. Following the isolation of living waterhemp protoplasts using these enzymes, experiments to assess the expression of foreign genetic material introduced via PEG-mediated transformation failed to yield any successful results. Further assessment of the waterhemp protoplasts indicated that they were alive, but failed to regenerate cell walls or undergo cellular division. Staining for reactive oxygen species (ROS), molecules that cause oxidative stress and are known to inhibit protoplast recovery, revealed high levels of oxidative stress in all waterhemp protoplasts that were produced.

To begin assessing approaches for reducing oxidative stress in waterhemp protoplasts, a flow cytometer (Figure 1) was used to quantify oxidative stress after staining with dichlorodihydrofluorescein diacetate (DCFH-DA), a dye that fluoresces when oxidized in living cells (fluorescence intensity increases with increased oxidative stress). This was achieved by separating protoplast samples into two equal subfractions and staining with 10 µM DCFH-DA or with 5 µg/ml fluorescein diacetate (FDA), a dye that fluoresces in living cells. The fluorescence of individual protoplasts is measured by the flow cytometer, allowing for the analysis of thousands of protoplasts from each sample.

Flow cytometry was utilized to assess the effects of ascorbic acid on oxidative stress levels in waterhemp protoplasts. This was performed by comparing protoplasts prepared with modified Murashige & Skoog media containing 6.5% mannitol and 0.1% CaCl2 and either 0, 114, or 285 µM ascorbic acid. The samples were then stained with either 10 µM DCFH-DA or 5 µg/ml FDA.

Flow cytometry was utilized to assess the effects of different commercial sources of cell wall degrading enzymes (Cellulase Onozuka R-10 and Macerozyme R-10) on oxidative stress levels in waterhemp protoplasts. This was performed by comparing protoplasts prepared with 1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10 from one of four different commercial sources prepared in modified Murashige & Skoog media containing 6.5% mannitol and 0.1% CaCl2. The samples were then stained with either 10 µM DCFH-DA or 5 µg/ml FDA.

We attempted to utilize activated charcoal in a similar manner to reduce oxidative stress, but it was found that activated charcoal prevented the staining of protoplasts with fluorescent dyes. As we would be unable to quantify oxidative stress without the dyes, we did not perform additional experiments with activated charcoal.

As the goal of assessing and reducing oxidative stress was to enable protoplast recovery, and after conversations with Adrian Monthony (personal communicaion), we investigated 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL). By inhibiting PAL, AIP prevents the synthesis of phytochemicals known for producing oxidative stress in response to cell wall degradation. Protoplasts derived from cell suspension treated with either 0 or 10 µM AIP were suspended in a droplet of low-gelling-temperature agarose submerged in KM 5/5 media with 10 µM AIP and incubated in darkness at 25 C. These protoplasts were monitored for recovery and cell division using microscopy, but were not analyzed for oxidative stress.

For Objective 2, Palmer amaranth calli (clumps of undifferentiated cells) were used to start cell suspension cultures in liquid media. Four sets of callus cultures from the previous year’s research were transferred to fresh media to initiate these cultures, each representing calli grown from a single Palmer amaranth seedling hypocotyl.

Research Results/Outcomes:
For Objective 1, the initial results for the treatment of protoplasts with ascorbic acid were promising. Dosing with a low level of ascorbic acid demonstrated a significant improvement in oxidative stress levels in protoplasts. However, assessments of higher concentrations of ascorbic acid indicated that there was a negative effect on waterhemp protoplasts.

Initial results comparing commercial sources of cell wall degrading enzymes for different levels of oxidative stress were promising, but additional experiments that controlled for the length of time between treatment and analysis did not show significance among the four sources of enzymes (p = 0.438) (results not shown).

Protoplasts pre-treated with 10 µM AIP during cell suspension (prior to protoplast isolation), and protoplasts not pre-treated with AIP, were suspended in agarose droplets and immersed in KM 5/5 with 10 µM AIP. Light microscopy of protoplasts prepared in this manner found evidence of cellular division, both with and without pre-treatment with AIP (Figure 2, Table 1). This represents a significant acheivement compared to previous attempts to attain waterhemp protopast recovery and cell division without the use of AIP. While cell division was observed with and without AIP pre-treatment, protoplasts derived from cell suspensions pre-treated with AIP have a larger and healthier appearance. However, cells with and without AIP pre-treatment appear to stop dividing after one or two divisions, indicating that additional refinement of the protoplast culture conditions are necessary.

For Objective 2, two of four Palmer amaranth cell suspension cultures were successfully initiated and are being maintained for future research.

Discussion:
For Objective 1, initial results indicated that ascorbic acid is capable of reducing oxidative stress in waterhemp protoplasts. However, there appears to be a negative effect on protoplasts at higher dosages of ascorbic acid. Due to this, ascorbic acid might have some usage as a supplement to protoplast media in order to reduce oxidative stress, but it is unlikely to be a viable solution for being the primary agent of oxidative stress reduction.

Initial results into the effects of the commercial source of Cellulase Onozuka R-10 and Macerozyme R-10 were promising. However, the effects of sample variation in the the length of time between staining with stress-indicating dye and analysis with flow cytometry appears to be high enough to hide any statistical significances among commercial sources, if any. This issue might be remedied in the future by modifications to the fluorescent staining protocol for protoplasts. If there is a further need to reduce oxidative stress within protoplasts, this would remain a possible area for investigation.

We now have a procedure for protoplast culturing that allows protoplasts to undergo cell division (Figure 2). Further experiments will be directed at improving cell culture conditions for these dividing protoplasts and assessing their capacity for transgene expression after PEG-mediated transformation.

For Objective 2, we currently have two Palmer amaranth cell suspension cultures that are being established (Figure 3). These cultures will be used for future research, including protoplast transformation.

Conclusion/Benefits to the North Dakota Soybean Farmers and the Industry:
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. Protoplast transformation is an important tool for genetic research, as it allow for introduction of transgenes and gene editing components into individuals cells. Recovery and division of transformed protoplasts is then necessary to produce tissue cultures that serve as perpetual sources of homogenous and uniform plant tissue for additional research. Cell division of waterhemp protoplasts obtained in the current research is a critical step toward this goal, and will facilitate similar research in Palmer amaranth as well. 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.

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Herbicide-resistant waterhemp and Palmer amaranth represent serious concerns for North Dakota soybean growers. Conducting research on emerging 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.

We previously used waterhemp cell suspension cultures to generate protoplasts (cells without cell walls), to facilitate genetic transformation. However, these protoplasts did not express introduced genes and did not recover and divide. Further investigation identified significant oxidative stress among the protoplasts, and the current research project explored methods to reduce oxidative stress and stimulate waterhemp protoplast cell division.

Ascorbic acid at low concentrations was found to have potential in lowering oxidative stress. This benefit was reduced at higher concentrations, however, perhaps due to acidity or other negative effects. Cell wall digesting enzymes from four different commercial sources were also compared, and contrary to our previous research, significant differences in waterhemp protoplast oxidative stress among the enzyme sources was not observed.

Inhibition of phenylalanine ammonia lyase (PAL) was also tested for reduction of oxidative stress and stimulation of cell division in waterhemp protoplasts. Incorporation of the PAL inhibitor 2-aminoindane-2-phosphonic acid (AIP) into culture media successfully produced cell division among waterhemp protoplasts, albeit at a low rate. After 10 days of culture with AIP, 5.5-6.0% of protoplasts were observed to be dividing. Additional pretreatment of cell suspension cultures with AIP for 10 days before protoplast isolation produced similar rates of division, although cells appeared to be fuller and healthier than without AIP pretreatment.

While obtaining cell division of waterhemp protoplasts is a significant advance, growth continued for only a few divisions and more research needs to be done to obtain continued division and growth of waterhemp protoplasts. In addition, two cell suspension cultures were produced from Palmer amaranth and will be used to produce protoplasts for research in a manner similar to waterhemp.

This research improved current protocols for genetic research in waterhemp by obtaining cell division of protoplasts. 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 such as waterhemp and Palmer amaranth 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. 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. The proposed research is a necessary step toward these goals.