Improving Ex Vitro Rooting and Acclimatization Techniques for Micropropagated American Chestnut1

Abstract

Limited rooting and acclimatization success when micropropagating certain hardwood tree species may hinder conservation efforts of certain threatened and endangered species. Restoration efforts for such trees, such as the American chestnut [Castanea dentata (Marsh.) Borkh.], require a massive number of plantlets to be produced by micropropagation for testing, initial distribution, and orchard establishment. Therefore, increasing the number and quality of lab-produced plantlets is a key research focus. After previously determining that an ex vitro rooting system produced significantly more robust plantlets, we examined extending the time in elongation medium, rooting substrates, exogenous auxin applications, root-promoting substrate soaks, submerging the cut site, and light intensity. The most effective methods included seven weeks in elongation medium, using Jiffy peat pellets soaked in water as the rooting substrate, cutting off callus while submerged, then dipping in 0.31% IBA rooting gel, and placing plantlets in low light of 60 μmol·m-2·s-1 after rooting. By increasing the number of roots and improving acclimatization success, we can ensure that many more blight-tolerant American chestnuts will be available for field studies and eventual public distribution. Demonstrating the ecological safety and blight survival of these trees will help restore this foundational tree species and assist future restoration efforts for other threatened species. Index words: Rooting, ex vitro, American chestnut, Castanea dentata, IBA, substrate. Species used in this study: American chestnut, [Castanea dentata (Marsh.) Borkh.]. Chemicals used in this study: IBA (indole-3-butyric acid).

Acclimation and hardening of a slow‐growing woody species emblematic to western North America from in vitro plantlets

“…The 16-week acclimation and hardening of in vitro-grown sagebrush plantlets (taking place in a laboratory setting) resulted in low to medium survival rates reflecting genotypes (Table 1, Appendix S1). These results agree with previous findings, where a strong genotype effect was observed on rooting and growth rates of in vitro individual lines (Barron et al, 2020), and are comparable to other studies, especially in woody species exhibiting faster growth rates (e.g., 40-80% depending on treatments in American chestnut; Oakes et al, 2020). The outplanting of acclimated and hardened plantlets in the greenhouse in preparation for field G×E experiments resulted in low (30%) to high (100%) survival rates, again reflecting genotypes (Table 2).…”

“…Therefore, in vitro plantlets must go through an acclimation and hardening process to develop functional leaves and rooting systems (Fila et al, 1998; Hazarika et al, 2006; Chandra et al, 2010). Such morphogenesis has been more easily applied to herbaceous, fast‐growing, annual plants (e.g., tomato, sunflower; Cruz‐Mendívil et al, 2011; Nowakowska et al, 2020), but is challenging with woody, slow‐growing, perennial species (Oakes et al, 2020). Indeed, most of these species share similar anatomical characteristics (e.g., amphistomatous leaves, clonal splitting; Downs and Black, 1999; Schenk et al, 2008; Drake et al, 2019) preventing the application of classical biotechnological protocols.…”

“…Although approaches to acclimation are similar across studies, soil mixes and duration of plantlets' exposure to high humidity differ (Hailu et al, 2013 ; Zayova et al, 2018 ), but these differences do not seem to impact survival. On the other hand, a lack of consistency in slow‐growing, woody species has been shown to have repercussions on survival (Oakes et al, 2020 ), necessitating standardized protocols for these taxa. Developing and validating such protocols to slow‐growing, woody Artemisia species has not been achieved yet, but an in vitro propagation method is available for A. tridentata (Barron et al, 2020 ), which provides the starting material for the protocol presented here.…”

Micropropagation of Duboisia Species via Shoot Tip Meristem

“…Rooting in vitro regenerated shoots in substrates is a common practice for many plant species, including some woody plants such as cherry [58], green ash [59], chestnut [60] and wolfberry [61]. In addition, substrate-based rooting reportedly accelerates the propagation process [62], which is critical for commercial production.…”