HORTSCIENCE 56(3):313–317. 2021. https://doi.org/10.21273/HORTSCI15546-20 events and resume normal growth when wa- ter is available (VanBuren et al., 2018). The phenomenon that the dry and visually In Vitro Propagation of Resurrection ‘‘dead’’ plants come alive after rewatering is fascinating to plant biologists and the lay Plant Selaginella pulvinata Using Frond public (Xiao et al., 2015), thus making res- urrection plants a special group of ornamen- Tips as Explants tal plants. Interestingly, the nuclear genomes of Selaginella are some of the smallest Rongpei Yu among green plants (Baniaga et al., 2016; School of Life Sciences, School of Ecology and Environmental Sciences, Little et al., 2007; Obermayer et al., 2002). Institute of Ecology and Geobotany, Yunnan University, Kunming, Yunnan Therefore, the resurrection species of Selag- inella are good candidates for exploring the 650091, China; Flower Research Institute, Yunnan Academy of Agricultural mechanisms of desiccation tolerance with Sciences, National Engineering Research Center for Ornamental genomic-based approaches (VanBuren et al., Horticulture, Kunming, Yunnan 650205, China; and Yuxi Yunxing Biotech 2018). Co., Ltd., Yuxi, Yunnan 653100, China Selaginella pulvinata (Hook. & Grev.) Maxim, a typical resurrection plant mainly Ying Cheng and Yanfei Pu distributed in exposed limestone areas, has School of Agriculture, Yunnan University, Kunming, Yunnan 650504, China varied usefulness in China (Zhang and Zhang, 2004). It is renowned as an ornamen- Fan Li tal plant because of its magical resurrection Flower Research Institute, Yunnan Academy of Agricultural Sciences, ability, and it is an important medicinal plant National Engineering Research Center for Ornamental Horticulture, listed in Chinese Pharmacopoeia (Chinese Pharmacopoeia Committee, 2015). S. pulvi- Kunming, Yunnan 650205, China; and Yuxi Yunxing Biotech Co., Ltd., nata is used in traditional Chinese medicine Yuxi, Yunnan 653100, China for the treatment of traumatic injury and asthma (Cao et al., 2010). Pharmacological Shugang Lu investigations revealed its various biological School of Life Sciences, School of Ecology and Environmental Sciences, activities, such as anti-cancer (Wang et al., Institute of Ecology and Geobotany, Yunnan University, Kunming, Yunnan 2016) and anti-inflammatory effects (Huang 650091, China et al., 2017). Unfortunately, because of its overexploitation, populations of S. pulvinata Additional index words. cytokinin, in vitro propagation, resurrection plants, Selaginella have decreased sharply. Therefore, there is an pulvinata urgent need to establish the in vitro propagation of S. pulvinata to produce high-quality plantlets Abstract. The resurrection plant Selaginella pulvinata (Hook. & Grev.) Maxim is used as for horticultural and medicinal use without an ornamental and medicinal plant. It is also a good candidate for exploring the resorting to harvesting wild populations. desiccation tolerance of resurrection plants. However, there is not an efficient propaga- Many fern species have been successfully tion method for S. pulvinata. In the present study, we evaluated the establishment of in established in an in vitro propagation system vitro propagation of S. pulvinata using frond tips as explants. The original shoot via spores (Barnicoat et al., 2011) and induction, adventitious shoot proliferation and plantlet growth media, and substrate shoot organogenesis from juvenile leaves type of plantlet acclimatization were investigated. The highest induction rate of original (Camloha et al., 1994), rhizomes (Winarto shoots (61.77 ± 5.17%) was obtained on half-strength (1/2) MS medium supplemented L L and Teixeira da Silva, 2012), callus (Hegde with 0.1 mg·L 1 N6-benzylaminopurine (BAP). The 1/2 MS with 1.0 mg·L 1 BAP was the et al., 2006), somatic embryos (Miku1a et al., most effective medium for the adventitious shoot proliferation. The quarter-strength (1/ 2015a, 2015b), and green globular bodies 4) MS containing 0.1% (w/v) active charcoal (AC) was optimum for plantlets proliferated (GGBs) (Amaki and Higuchi, 1991). Among from adventitious shoots and plantlet growth. Approximately 98 plantlets could be them, the GGB system is regarded as a obtained from one single original shoot via one-time shoot proliferation cultivation and remarkably efficient method (Higuchi et al., plantlet cultivation. The acclimated plants on a 5:1 (v/v) mixture of peat and perlite had 1987). However, studies of in vitro propaga- the highest survival rate (92.13 ± 1.67%). The acclimated plants maintained excellent tion of fern-ally Selaginella are limited. Park resurrection ability. et al. (2020) reported in vitro regeneration of nonresurrection species S. martensii by using Selaginella P. Beauv. (Selaginellaceae), estimated 700 to 800 species that exploit a shoot-tips as explants. Because of the differ- an ancient and distinctive group, is the largest diverse array of the arctic, temperate, tropi- ent germination times of megaspores and genus of seed-free vascular plants, with an cal, and semi-arid habitats (Arrigo et al., microspores in resurrection species S. eremo- phila, S. rupincola, and S. arizonica, a two- 2013; Banks, 2009; Jermy, 1986; Singh step in vitro propagation method was used: et al., 2019; Zhou et al., 2016). Some species surface-sterilized megaspores were cultured of Selaginella are resurrection plants, alone for 3 weeks, followed by the addition of Received for publication 3 Nov. 2020. Accepted such as S. tamariscina (Wang et al., 2010; for publication 11 Dec. 2020. surface-sterilized microspores to the germi- Published online 26 January 2021. Xu et al., 2018), S. lepidophylla (Pampurova nated megaspore cultures for co-culture; We thank the National Natural Science Founda- et al., 2014; Rafsanjani et al., 2015; Yobi however, the final fertilization rate was only tions of China (grant no. 31860569) and the Sci- et al., 2013), S. bryopteris (Deeba et al., 12% in S. eremophila and S. rupincola, and ence and Technology Talents and Platform 2016), S. arizonica, S. eremophila, and S. no fertilization was observed in S. arizonica Program of Yunnan Province - Rongpei Yu rupincola of the North American southwest- (Yu et al., 2017a). Additionally, there are no (2021-2026) for financial support. ern deserts (Yu et al., 2017a). With the S.L. is the corresponding author. E-mail: shuganglu@ reports of in vitro vegetative propagation of 163.com. remarkable vegetative desiccation tolerance, resurrection species in Selaginella. This is an open access article distributed under the the resurrection plants are able to survive We describe an in vitro vegetative prop- CC BY-NC-ND license (https://creativecommons. nearly complete anhydrobiosis (<10% rela- agation protocol for resurrection plant S. org/licenses/by-nc-nd/4.0/). tive water content) during prolonged drought pulvinata using frond tips as explants. We HORTSCIENCE VOL. 56(3) MARCH 2021 313 investigated the optimal media for original (0.1, 0.5, or1.0 mg·L–1) and Thidiazuron peat and a mixture of peat and perlite at 5:1 shoot induction, adventitious shoot prolifer- (TDZ) (0.1, 0.5, or 1.0 mg·L–1). Cultures and 8:1 (v/v)]. All substrate type treatments ation and plantlet growth, and the optimal were maintained in the dark at 25 ± 2 °C. included two groups: one group had 2.0 substrate type for plantlet acclimatization. After 6 weeks of culture, the induction rate kg/m3 granular slow-release fertilizer (nitro- This new protocol will be beneficial for of the original shoots was recorded. The gen, 14%; phosphorus, 14%; potassium, horticultural and medicinal applications of original shoots were the shoots derived from 14%; w/w) added and the other group did S. pulvinata and will be a critical tool for the apical and lateral bud primordium. The not. These plantlets were maintained in the researching the biology of desiccation toler- explant forming at least one original shoot shaded greenhouse (PPFD, 170 mmol·m–2·s–1 ance. was identified as successful induction. at 12:00 PM)at20±5°C. Spray irrigation was For the proliferation of adventitious performed every 7 to 9 d for 4 weeks. After- Materials and Methods shoots, the original shoots induced on 1/2 ward, the shadecloth of the greenhouse was MS with 0.1 mg·L–1 BAP were separated removed, and the PPFD was 1600 Plant material. S. pulvinata plants (plant from explants and subsequently cultivated on mmol·m–2·s–1 at 12:00 PM. Plantlets were irri- diameter, 5.0–8.0 cm) were collected from 1/2 MS media with various concentrations of gated with tap water every 5 to 6 d. The exposed limestone in the Xishan Mountains, cytokinin (described previously); then, they survival rate of plantlet acclimatization was Yunnan Province, China (lat. 24°57#6$ N, were maintained in a controlled environment recorded after 4 weeks of culture in the long. 102°38#22$ E). Plants were cultivated room at 25 ± 2 °C under light intensity of 40 unshaded greenhouse. in plastic pots containing a humus soil ob- mmol·m–2·s–1 (16 h light/8 h dark) provided Resurrection assessment of acclimated tained from the original habitat and irrigated by cool-white fluorescent lamps (Philips, plants. Hydrated plants cultivated in the un- with tap water every 5 d; they were main- Netherlands). After 8 weeks of culture, the shaded greenhouse for 8 months were used to tained in a greenhouse for 2 months with number of adventitious shoots proliferated assess the resurrection ability in a controlled natural light [photosynthetic photon flux den- from the single original shoot was assessed. environment room (as described previously). sity (PPFD) 1600 mmol·m–2·s–1 at 12:00 PM] Plantlet cultivation and acclimatization. Hydrated plants were not irrigated for more and relative humidity 50% to 60% at 25 ± To obtain plantlets from adventitious than 10 d to ensure complete dehydration. 5 °C. shoots, the single adventitious shoot The dehydrated plants were sequentially kept Induction of original shoots and proliferation (height $5.0 mm) from 1/2 MS supple- dry for 1 week and then rehydrated for 24 h.