Document Type : Research Article
Authors
1
plant breeding department, Ka.C, Islamic Azad University, Karaj, Iran
2
Faculty member of Islamic Azad University-Karaj Branch
10.22067/jhs.2026.98122.1502
Abstract
Introduction: Venus flytrap (Dionaea muscipula Ellis) is one of the most remarkable carnivorous plant species due to its unique trapping mechanism, ornamental value, medicinal importance, and commercial potential. This species contains several valuable secondary metabolites, including naphthoquinones, flavonoids, and phenolic compounds, widely used in pharmaceutical and medicinal studies. Despite its economic and scientific importance, natural populations of Venus flytrap are severely threatened by excessive harvesting, habitat destruction, environmental changes, and the species’ inherently low propagation rate under natural conditions. Conventional propagation methods are relatively slow and insufficient for large-scale production. Therefore, plant tissue culture techniques have become an important biotechnological approach for rapid propagation, conservation of genetic resources, and sustainable commercial exploitation of this endangered carnivorous plant. Successful micropropagation depends on several factors, including explant type, culture medium composition, plant growth regulators, mineral nutrient concentration, and acclimatization conditions. The present study was conducted to optimize a direct micropropagation protocol for Venus flytrap through rhizome explants and to evaluate the effects of different concentrations of plant growth regulators and culture media on regeneration induction, shoot regeneration, proliferation, rooting, and acclimatization of regenerated plantlets.
Materials and Methods: Healthy donor plants of Venus flytrap were selected as the source of rhizome explants. Explants were thoroughly washed and surface sterilized under aseptic conditions using ethanol, sodium hypochlorite, and mercuric chloride treatments followed by several rinses with sterile distilled water. Sterilized explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose and 7.5 g l⁻¹ agar. In the regeneration induction stage, the effects of different concentrations of NAA (0.5, 1, and 1.5 mg l⁻¹) combined with BAP concentrations of 0 and 0.5 mg l⁻¹ were evaluated. Explants showing regeneration responses were subsequently transferred to regeneration media containing different concentrations of BAP (0, 1.5, 2, and 2.5 mg l⁻¹). In the proliferation stage, regenerated shoots were cultured on full-strength MS, half-strength MS (1/2MS), and 70% MS media to investigate the effects of mineral nutrient concentration on growth characteristics, including shoot number, shoot length, and trap size. Rooting treatments were not independently designed because preliminary observations showed that explants were capable of spontaneous root formation during regeneration and proliferation stages. For acclimatization, regenerated plantlets were transferred to different substrates, including sphagnum moss, peat moss, and coco peat + peat moss mixture, under greenhouse conditions to evaluate adaptation and survival percentages.
Results and Discussion: The results demonstrated that plant growth regulators significantly affected regeneration induction and shoot regeneration of Venus flytrap explants. Among all tested treatments, MS medium supplemented with 1 mg l⁻¹ NAA without BAP produced the highest regeneration induction percentage (73.33%). This finding highlights the important role of auxins in stimulating cell division, organogenic competence, and initiation of regenerative responses in rhizome tissues. Auxins regulate cellular differentiation and organ initiation in plant tissue culture systems, and the results confirmed their essential role during the early stages of direct regeneration in Venus flytrap.
In the regeneration stage, MS medium supplemented with 1.5 mg l⁻¹ BAP produced the highest regeneration percentage (86.66%). Increasing the BAP concentration beyond this level significantly reduced regeneration efficiency. These findings indicate that balanced cytokinin concentration is crucial for successful shoot organogenesis. Cytokinins such as BAP stimulate cell division and shoot formation; however, excessive concentrations may disrupt hormonal balance and negatively affect morphogenesis and explant development. Similar observations have been reported in previous tissue culture studies of carnivorous and ornamental plant species, where moderate cytokinin concentrations promoted direct shoot regeneration while higher concentrations reduced regeneration capacity.
Significant differences were also observed among different culture media during the proliferation stage. Half-strength MS medium (1/2MS) produced the highest shoot number (60.33), shoot length (0.53 cm), and trap size (0.36 cm), whereas full-strength MS medium resulted in comparatively weaker growth responses. The superior performance of 1/2MS medium suggests that reduced mineral salt concentration provides more suitable nutritional conditions for this carnivorous species. Venus flytrap naturally grows in nutrient-poor and acidic environments and is highly sensitive to excessive mineral accumulation. Therefore, reduction of macronutrient concentration in the culture medium likely decreased osmotic stress and improved physiological growth conditions. Trap size and shoot development are important indicators of plantlet quality because they reflect normal morphological and physiological development of regenerated plants.
No separate rooting experiment was conducted because regenerated explants naturally formed roots during regeneration induction and proliferation stages. Although the roots were relatively short and few in number, they were capable of nutrient uptake and plantlet survival. The rooting response may be attributed to the residual effect of auxins used during induction stages as well as the natural rooting ability of Venus flytrap. Elimination of a separate rooting stage reduces both the duration and cost of the micropropagation process, making the protocol more suitable for commercial application.
Acclimatization results showed that different substrates significantly influenced survival percentages of regenerated plantlets. Sphagnum moss produced the highest acclimatization percentage (73.33%), followed by peat moss (46.66%), whereas the coco peat + peat moss mixture showed the lowest survival rate. The superiority of sphagnum moss may be associated with its high water-holding capacity, proper aeration, high porosity, and acidic properties, which resemble the natural habitat conditions of Venus flytrap. These characteristics improve root establishment and reduce transplant shock during adaptation to greenhouse conditions. Since in vitro plantlets generally possess weak cuticles, poorly functioning stomata, and limited root systems, selection of an appropriate acclimatization substrate is essential for improving survival under ex vitro conditions.
Conclusion: Overall, the results of this study provide an efficient, reliable, and low-cost protocol for direct micropropagation of Venus flytrap through rhizome explants. The optimized protocol includes regeneration induction on MS medium containing 1 mg l⁻¹ NAA, shoot regeneration on MS medium supplemented with 1.5 mg l⁻¹ BAP, proliferation on 1/2MS medium, natural rooting without a separate rooting stage, and acclimatization using sphagnum moss substrate. The complete micropropagation process required approximately 4–5 months to obtain acclimatized plantlets. The protocol developed in this study can be effectively used for large-scale propagation, conservation of genetic resources, and commercial production of this valuable carnivorous plant species.
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