Sterilization and gas exchange on in vitro cultivation of Sinningia rupicola (Mart.) Wiehler: production of seedlings from an endangered species

Abstract The Ferruginous Rupestrian Grassland is an ecosystem that harbors endemic and endangered species, such as Sinningia rupicola . The low germination rate and restricted habitat of this species increase its risk of extinction, making the development of conservation strategies essential. Micropropagation is a recommended technique in such cases and, although effective for seedling production, in vitro cultivation requires careful measures such as strict contamination control during inoculation, as well as process optimizations that may induce morphophysiological changes favoring acclimatization. In this context, sterilization methods, the use of permeable membranes, and mini-incubator system emerge as promising strategies to ensure the production and survival of these plants, mitigating anthropogenic impacts and preserving biodiversity. This study aimed to establish in vitro seed culture according to exposure time to sodium hypochlorite, to evaluate the effect of gas exchange on the multiplication, elongation, and rooting of Sinningia rupicola , and to assess the effectiveness of the mini-incubator system in acclimatizing the seedlings. During the in vitro establishment stage, exposure to active chlorine for 15 min resulted in the lowest contamination percentage, while 5 and 10-min treatments yielded the highest germination rates (55.0%). In the multiplication stage, the treatment with three membranes (3Mem) showed the best vigor, with an average value of 1.04. Regarding the clump diameter variable, the treatments without membranes (0Mem) and with one membrane (1Mem) had the best results in both the multiplication and elongation stages. In the elongation stage, concerning shoot length, treatments 1Mem and 3Mem showed the best performances, with averages of 4.3 cm and 3.95 cm, respectively. The use of 3Mem also resulted in higher levels of photosynthetic pigments across all evaluated stages. A survival of 85.8% was achieved during acclimatization, and all seedlings transferred to the greenhouse survived after 45 d, demonstrating the effectiveness of the in vitro cultivation conditions and the mini-incubator system in producing micropropagated plants.