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 system to generate protoplasts (cells without cell walls) from cultured waterhemp cells, and more recently confirmed successful recovery and cell division of these protoplasts. The current proposal will adapt these waterhemp protocols to the production of healthy protoplasts from existing Palmer amaranth cell cultures. Successful production of Palmer amaranth protoplasts will facilitate gene editing experiments to develop genetic biocontrol methods for control of herbicide-resistant Palmer amaranth.