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. Initial studies investigating the application of new genetic technologies to 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 such capacity, yet still maintain most of the physiological processes that are targeted by herbicides. This proposal seeks to continue previous research by using existing cultured waterhemp cells in liquid media (suspension cultures), to produce cells capable of efficiently accepting the chemical components necessary for future gene editing research. These cells, called protoplasts because they lack a cell wall, will also be tested to see if new cultures can be derived from single protoplast cells. Because current gene editing technology is imperfect, establishing cultures from single edited cells is necessary to produce cell cultures that are genetically uniform and homogeneous.

1) Successfully generate and maintain healthy protoplasts (cells after removal of cell walls), from a waterhemp cell suspension culture (plant tissue grown in liquid media in a laboratory).
2) Establish micro-colonies (small clumps of cells), from single waterhemp protoplasts.

1) The ability to produce and study genetic alterations in waterhemp without risking unintentional release into the environment.
2) The ability to safely assess emerging gene drive technology and explore its potential for waterhemp management in North Dakota.

Updated December 1, 2020:
Completed work:
The conditions necessary to remove cell walls from waterhemp cell suspension cultures, in order to generate protoplasts, were investigated. In the first experiment, 5 ml of a 1-month-old waterhemp cell suspension culture was centrifuged to pellet the cells, and the supernatant was replaced with 5 ml of the following filter-sterilized enzyme solution: 0.55 M sorbitol, 0.6% cellulase R-10, 0.2% hemicellulase, and 0.2% pectinase, pH 5.8. Cells were incubated at room temperature for 5 hours with gentle agitation, filtered, and washed 6-7 times with resuspension in 0.55 M sorbitol. The second experiment was a repeat using a younger 3-day-old waterhemp cell suspension culture with incubation at room temperature for 7 hours using the same enzyme solution.
In the third experiment, a different enzyme solution was prepared by dissolving 0.2 g driselase in 10 ml of 4 M mannitol, pH 5.2. Because driselase is poorly soluble in aqueous liquids, the solution was centrifuged to remove undissolved enzyme and the supernatant was collected. Waterhemp cells from a 5-day-old cell suspension culture were then incubated in this driselase solution at 30 C for 90 minutes with gentle agitation.
A fourth experiment used an enzyme solution of sorbitol, cellulase R-10, hemicellulose, and pectinase similar to the first two experiments, but with increased cellulase R-10 (0.8%). A 10-day-old cell suspension culture was incubated in this enzyme solution for 4 hours at 30 C.

Preliminary results:
Digestion of cells walls and the generation of protoplasts was observed by microscopy. In all cases, cell walls were not successfully removed. While the driselase incubation (third experiment) was primarily performed to ensure that the enzyme would remain in solution during incubation, the other experiments used published protocols from other plant species. However, the enzymes that successfully generate protoplasts are known to vary by plant species due to different cell wall compositions. Our preliminary results indicate that testing additional cell wall digestion enzymes is warranted. Culture age and incubation temperature did not affect our results.

Work to be completed:
Experiments to test the enzymes pectolyase Y23 and cellulase Onozuka RS are in process. Upon completion of Objective 1, i.e., the successful generation of protoplasts from waterhemp cell suspension cultures, research to generate micro-colonies from protoplasts (Objective 2) will begin.

Updated June 30, 2021:

View uploaded report

Research Conducted
Cell suspension cultures, which are living cells suspended in a liquid nutrient solution, are often used in laboratory research. We tested six different solutions for their ability to generate waterhemp protoplasts (cells without cell walls). One solution successfully generated protoplasts, the majority of which were confirmed to be alive and viable. Subsequent cell wall regeneration and cell division, however, was not observed under our tested conditions.

Why the research is important to ND soybean growers
Herbicide-resistant waterhemp represents an important problem in North Dakota soybean production, and new tools are needed to study emerging methods for controlling this weed. When growing herbicide-resistant weeds for research, it is important to minimize the risk of seeds escaping and spreading into fields. This is especially important when researching new genetic methods of weed control, where weed genes may be altered. The cells that make up cell suspension cultures need to be maintained in a laboratory and cannot grow or produce new plants in the field. This makes waterhemp suspension cultures very valuable herbicide resistance research tools. The ability to remove cell walls from these cultured cells, generating protoplasts, adds value to the cultures because of their increased ability to take up genetic material for experimentation compared to intact cells, due to the lack of a cell wall barrier in protoplasts.

Final findings of the research
We found that while most tested solutions did not generate protoplasts from waterhemp cells, a solution containing the enzyme ‘macerozyme R-10’ successfully produced viable waterhemp protoplasts. However, under our tested conditions, waterhemp protoplasts were not observed to subsequently regenerate cell walls or start dividing. This will be an area of future research.

Benefits/Recommendations to North Dakota soybean farmers and industry
Waterhemp plant material in the form of cell suspension cultures are now available for further studies investigating herbicide resistance. Researchers also now have a protocol to generate protoplasts from these cell suspension cultures, facilitating genetic research to investigate new ways for waterhemp control without risk of weed escape into North Dakota fields.

Development of new herbicide modes of action has greatly declined in recent decades, while 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 is progresses, it’s 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, 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.