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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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.