In vitro and in vivo germination of Viola elatior Fries: implications for conservations

Viola elatior (Violaceae) is one of the most rare and endangered European species and occurs in Italy in small fragmented populations situated at the SW margin of its range. It is a river corridor plant, typical of humid grasslands, banks, field margins and woodlands, growing in places where the water table shows notable annual level variations (flooding in winter and strong summer aridity). From late 1800 onwards, it underwent a strong decline due to drainage, urban sprawl, soil eutrophication, pollution: today it is Endangered at a national level (1). To cope with such a decline, which is still running, we studied the in vitro and in vivo propagation by seed, in order to obtain a number of individuals for population reinforcing. A first preliminary in vitro experiment was performed in 2015 on 25 seeds collected in late May of that year (chasmogamous flowering) from the plants maintained at the Botanic Garden of Modena. Seeds were sterilised for 14’ in 5% NaOCl, then posed on a 1/2 MS + 6 g/l agar cultivation terrain. After sowing, they were kept in the dark, for 11 days at 25 °C, then 35 days at 4 °C and then again at 25 °C for 4 days. In a second experiment, we took into account 3 populations of the province of Mantua, which are particularly notable for number of individuals and vegetative strength. Seeds were collected during spring and summer 2015, sampling separately the production of chasmogamous and cleistogamous flowering. Seeds were kept in the dark at 18 °C until the sowing (January 2016). Two tests were carried out: sowing in vitro after 30 days of chilling at 4 °C; sowing outdoor in pots without chilling, in an area near the collection places. Germination occurrence was recorded for 50 days after sowing. Totally, in the second experiment 1724 seeds were sowed. In the first experiment (in vitro) we obtained a germination rate by 96%. In the second one, the highest germination percentage was observed in vivo, with seeds coming from cleistogamous flowering (85.9%); the lowest percentage was recorded in vitro, for seeds taken from chasmogamous flowering (14%). Germination percentage was higher for the seeds coming from the cleistogamous flowers (56.4% in vivo, vs. 34.3% in vitro), in comparison with seeds coming from the chasmogamous flowers (50% and 23%, respectively). Such findings are conflicting with the literature data, taken from central European populations (2), which refer lower germination rates for seeds from cleistogamous flowers and practically no germination under controlled conditions (3). The in vivo germination values are quite similar to what is known for V. elatior (2); the in vitro germination values are instead always notably different both from literature data and from precedent in vitro experiments (4) performed on Viola pumila Chaix, which is ecologically and biologically similar to V. elatior (5). Results similar to those obtained from the populations of the province of Mantua were found also for Jacobaea paludosa (L.) G.Gaertn., B.Mey. & Scherb. subsp. angustifolia (Holub.) B.Nord. & Greuter (6), another river corridor species living in marshes and wetlands. Briefly, in conditions of peripheral rarity (Mantuan sites) our tests show a higher germination of the seeds coming from the cleistogamous flowering, both in vitro and in vivo; in more central areas of the distribution range, data from literature show a higher germination for the seeds from chasmogamous flowering. These results could be correlated to the climate of the Mantua province, which is warmer than in central-eastern Europe, and could be exacerbated by the fact that 2015 has been the warmest year since 1860. The nearly total germination obtained in vitro in the first experiment (seeds from the chasmogamous flowers) may have been influenced by mother plants cultivation in the botanic garden. At present, the ex situ protocols for V. elatior seed germination should be improved, individuating more properly the ecological conditions to which seeds are subjected in the central zones of the species’ distribution area, where a high germination occurs (2).