Stevia has become one of the most commercially important natural sugar substitutes in the world, prized by the pharmaceutical and food industries because its sweetening compounds deliver intense sweetness with essentially no calories. The sweetness comes from steviol glycosides, a family of diterpene-derived molecules accumulated in the plant’s tissues, with stevioside and rebaudioside A among the most valuable members. Extracting these compounds traditionally requires cultivating vast acreage of the perennial plant, harvesting its leaves, and processing them through multi-step purification. Plant cell suspension culture offers an alternative: cells grown in liquid medium can, in principle, produce the same metabolites year-round under controlled conditions. The catch has always been yield, since undifferentiated cells in culture typically produce far less of these specialized metabolites than intact leaves do.

That is where elicitation comes in. Plants in nature defend themselves against microbial attack by ramping up their chemical arsenals, flooding their tissues with defensive secondary metabolites. Fungal molecules, including components of fungal cell walls and secreted metabolites, act as signaling molecules that mimic an attack and trigger this defensive chemistry. Researchers exploit this ancient alarm system by adding carefully measured doses of fungal preparations to plant cultures, essentially tricking the cells into thinking they are under siege so they manufacture more of the valuable compounds. The Punjab team applied this strategy using the Fo47 endophytic strain of Fusarium oxysporum, an ascomycete fungus whose metabolites served as the elicitor in their experiments.

The experimental design was systematic and thorough. The researchers established callus cultures—masses of undifferentiated plant cells growing on solid medium—and cell suspension cultures, where cells float and multiply in agitated liquid medium. Both culture types were treated with varying concentrations of the crude fungal extract: 0, 1, 2, 3, 4 and 5 milligrams per liter. Calli were exposed to the treatments for thirty days, while cell suspension cultures received twenty days of treatment. After these periods, the team measured a battery of parameters including morphological growth characteristics, total chlorophyll content, antioxidant enzyme activities, soluble sugars, and, most importantly, the concentrations of stevioside and rebaudioside A in the biomass.

The growth results revealed a clear dose-response pattern with an optimum at 4 milligrams per liter. At this concentration, calli achieved their maximum fresh weight of 3.74 grams and dry weight of 0.35 grams, while cell suspension cultures reached 3.94 grams fresh weight and 0.39 grams dry weight. These figures indicate that moderate elicitor doses did not merely stress the cells into defensive overdrive at the expense of growth; instead, the treatment appeared to support robust biomass accumulation. This balance matters enormously for industrial application, because the total yield of a valuable metabolite depends on both the concentration of the compound in the cells and the amount of biomass produced. An elicitor that boosts compound concentration while collapsing growth would offer little commercial advantage.

Physiological measurements reinforced the picture of a culture thriving under the right elicitor dose. Calli treated with 4 milligrams per liter of the fungal extract showed maximum total chlorophyll content at 0.68 milligrams per gram of fresh weight, along with peak catalase activity of 9.09 units per milliliter of enzyme, peroxidase activity of 2.77 milligrams per gram, and soluble sugar content of 17.63 milligrams per gram. Interestingly, the cell suspension cultures peaked slightly differently: maximum catalase activity of 9.19 units per milliliter, peroxidase activity of 2.87 milligrams per gram, and soluble sugars of 18.21 grams were recorded at 3 milligrams per liter of extract. The elevated antioxidant enzyme activities suggest that the elicitor treatment activates the cells’ oxidative stress response machinery, a biochemical cascade known to be intertwined with secondary metabolite production in plants.

The headline result concerns the sweet compounds themselves. In cell suspension cultures, the maximum steviol glycoside yields were recorded at 4 milligrams per liter of crude extract, reaching 13.95 milligrams of stevioside per gram of dry weight and 33.76 milligrams of rebaudioside A per gram of dry weight. Callus cultures treated at the same concentration produced 11.13 milligrams of stevioside and 31.39 milligrams of rebaudioside A per gram of dry weight. Rebaudioside A is particularly significant commercially because it is considered to have a cleaner, more sugar-like taste profile than stevioside, with less of the bitter aftertaste that has historically limited stevia’s acceptance in beverages and foods. Achieving high rebaudioside A levels in suspension culture therefore represents a meaningful step toward producing premium sweetener material outside the field.

The biochemical logic behind these results connects several threads of plant physiology. When fungal elicitor molecules are perceived by plant cells, they typically initiate a signaling cascade involving reactive oxygen species, calcium fluxes, and defense-related enzymes such as catalase and peroxidase. The observed rise in antioxidant enzyme activity in the treated cultures is consistent with this pathway being activated. Because steviol glycosides are secondary metabolites whose biosynthesis shares precursors and regulatory networks with defense chemistry, stimulating the stress response plausibly channels more metabolic flux toward glycoside production. The soluble sugar measurements add another dimension, since sugars serve both as energy sources and as signaling molecules that can influence specialized metabolism in cultured cells.

What makes this study practically interesting is the simplicity of the elicitor. Rather than purifying specific fungal cell wall fragments or buying expensive commercial elicitor preparations, the team used a crude extract from the Fo47 strain of Fusarium oxysporum, an endophytic isolate known for its relatively benign relationship with plant hosts. Crude extracts contain a complex mixture of fungal metabolites and cell wall components, and this complexity may actually contribute to the strong response by activating multiple signaling routes simultaneously. For a process intended to scale to industrial bioreactors, using an inexpensive, easily prepared elicitor could substantially reduce production costs compared with synthetic alternatives.

The authors suggest that their findings provide useful information for the industrial production of natural sweeteners from Stevia rebaudiana cell cultures using fungal extract. The work joins a growing body of research demonstrating that elicitation strategies can make plant cell culture commercially viable for high-value natural products. Challenges remain before such processes reach factory scale, including optimizing elicitor timing, scaling culture conditions from flasks to large bioreactors, and ensuring consistent product quality across batches. But the demonstration that a simple fungal extract can push stevioside and rebaudioside A yields to their highest recorded levels in these cultures—while simultaneously maintaining healthy biomass growth—offers a compelling proof of concept. As demand for natural, calorie-free sweeteners continues to climb globally, bioreactor-grown stevia cells, prodded into productivity by their fungal allies, may one day sweeten products around the world without a single stevia leaf ever being harvested.

Subject of Research: Elicitation of steviol glycoside biosynthesis in Stevia rebaudiana cell cultures using fungal extract

Article Title: Fungal elicitor-based improvement of steviol glycoside production in Stevia rebaudiana Bertoni cell suspension cultures

Article References: Saddiqa, A., Shahzadi, A., & Sajid, Z. A. (2026). Fungal elicitor-based improvement of steviol glycoside production in Stevia rebaudiana Bertoni cell suspension cultures. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10034-6

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10034-6

Keywords: Stevia rebaudiana, steviol glycosides, fungal elicitor, Fusarium oxysporum, cell suspension culture, plant biotechnology, secondary metabolites, stevioside, rebaudioside A, natural sweeteners, antioxidant enzymes, elicitation