An Efficient Method for In Vitro Shoot-Tip Culture and Sporophyte Production Using Selaginella martensii Spring Sporophyte

Abstract

Selaginella martensii, an evergreen perennial fern that is native to South America and New Zealand, is named “frosty fern” because of its beautiful white-colored leaves and it is used as an ornamental plant. Efficient propagation methods for this species have not been developed. We aimed to develop an efficient propagation method for S. martensii through in vitro culture. We investigated culture conditions that are suitable for shoot-tip proliferation and growth. The optimum shoot-tip culture conditions were determined while using Murashige and Skoog (MS) medium (quarter, half, full, or double strength) and macronutrients (sucrose and two nitrogen sources) at various concentrations. In MS medium, the shoot tips formed a maximum of 6.77 nodes per explant, and each node formed two new shoot tips (i.e., 26 or 64 shoot tips). When using branching segments containing an angle meristem, the shoot-to-rhizophore formation ratio could be controlled by medium supplementation with plant-growth regulators. Sporophytes that were grown from shoot tips in vitro were acclimated in ex vitro soil conditions and successfully survived in the greenhouse. Numerous shoot tips could be obtained from in vitro-grown sporophytes and be proliferated ex vitro to produce a large number of plants. This method provides a way of shortening the time that is required for producing a large stock of S. martensii planting material.

In Vitro Propagation of Resurrection Plant Selaginella pulvinata Using Frond Tips as Explants

“…Similarly, the addition of 0.1% (w/v) AC improved the root number and length of Magnolia 'Ann' (Parris et al, 2012), and 1/4 MS medium with 1.0% (w/v) AC was the recorded optimum for the root development of fern plant Matteuccia struthiopteris (Thakur et al, 1998). In contrast, MS without AC was the most efficient for root development of plantlets in nonresurrection species Selaginella martensii (Park et al, 2020).…”

“…The same type of substrate was also suitable for the plantlet acclimatization of Cibotium barometz (Yu et al, 2017b). Nevertheless, a 3:1 mixture of horticultural substrate and decomposed granite favored the growth of acclimated plantlets in Selaginella martensii (Park et al, 2020).…”

“…However, studies of in vitro propagation of fern-ally Selaginella are limited. Park et al (2020) reported in vitro regeneration of nonresurrection species S. martensii by using shoot-tips as explants. Because of the different germination times of megaspores and microspores in resurrection species S. eremophila, S. rupincola, and S. arizonica, a twostep in vitro propagation method was used: surface-sterilized megaspores were cultured alone for 3 weeks, followed by the addition of surface-sterilized microspores to the germinated megaspore cultures for co-culture; however, the final fertilization rate was only 12% in S. eremophila and S. rupincola, and no fertilization was observed in S. arizonica (Yu et al, 2017a).…”

Effective Propagation of Selaginella tamariscina through Optimized Medium Composition

“…Jha [32] produced plants in medium containing plant-growth regulators for the propagation of Selaginella microphylla, whereas in the present study, we showed that S. tamariscina regeneration was possible in medium without plant-growth regulators. In addition, sporophyte explants from Selaginella martensii do not form new sporophytes; instead, they merely grow [21]. In contrast, S. tamariscina sporophyte explants regenerate several sporophytes and thus ensure effective propagation.…”

“…In vitro culture can produce homogeneous plants throughout the year in a controlled environment and promote their rapid propagation [20,21]. Additionally, this method can be used for species conservation and for propagating plants that are difficult to reproduce naturally [22].…”