Presentation: 2025 ND EPSCoR Annual conference 

October 21, 2025, NDSU Memorial Union, Fargo, North Dakota

Improving Protoplast Survival and Division for Single-Cell Plant Biology

Herbicide-resistant waterhemp (Amaranthus tuberculatus) is a growing challenge for Midwestern agriculture and calls for expansion of research tools. We work with plant protoplasts—cells stripped of their cell walls—to build a platform for single-cell studies. The most common challenge for protoplast research in any plant species is cell death during or shortly after the protoplast isolation process. To our knowledge, we have developed the first protocol for the isolation of waterhemp protoplasts that recover and divide after isolation. Further, we show that a pretreatment with 100 µM 2-aminoindane-2-phosphonic acid (AIP), a phenylalanine ammonia-lyase inhibitor, improves early recovery and division. In cell suspensions, AIP pretreatment roughly doubled the rate of cell divisions one day after isolation (5.90% vs 2.95%; p = 2.04 × 10⁻⁵). Protoplast yield after enzymatic cell-wall digestion and sucrose-cushion purification increased four-fold with AIP pretreatment (p = 2.6 × 10⁻⁴). Using a flow cytometer, we found that the AIP-treated group contained a higher proportion of intact cells after isolation (61% vs 53%; p = 0.016). We also identified two subpopulations of cells with different rates of cell wall regeneration for both treatment groups. AIP shifted distributions toward the subpopulation with lower immediate cell wall regeneration and reduced oxidative stress in this subpopulation by 48% (p = 0.01). Together, these findings show that AIP pretreatment increases intact-cell yield and early divisions while reducing oxidative stress. Practically, this opens a new avenue of research for studying and addressing the problem of herbicide-resistant waterhemp and other weedy plant species.