Vigorous Growing of Donor Plantlets by Liquid Overlay in Subcultures Is the Key to Cryopreservation of Endangered Species Pogostemon yatabeanus

Abstract

Cryopreservation is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. However, the wide application of cryopreservation for the protection of wild flora is hampered by some reasons: limits of source material available, difficulties in in vitro propagation, needs to re-optimize protocol steps for new species, etc. In this study, using an endemic and endangered Korean species, Pogostemon yatabeanus, we investigated subculture medium and supplements on in vitro growth of donor plants: medium strength, gelling agents, liquid overlay, plant hormones, and activated charcoal. Subculture conditions of each cycle tested significantly impacted on height and dry weight of subcultured donor plantlets. Among the treatments tested, the overlay of the liquid medium on top of gellan gum-gelled medium significantly increased the growth of shoots and roots. In the droplet-vitrification procedure, the survival and regeneration of cryopreserved shoot tips were critically impacted by the dry weight of donor plantlets (CORELL = 0.85~0.95) which was affected by the following subculture conditions. Moreover, every subsequent subculture cycle before cryopreservation positively or negatively impacted post-cryopreservation regeneration. This study highlights the vigor of donor plantlets for post-cryopreservation regeneration and provides practices for the revitalization of donor plants during subcultures.

Optimizing the Droplet-Vitrification Procedure by Balancing the Cryoprotection and Cytotoxicity of Alternative Plant Vitrification Solutions Based on the Nature of Donor Plant Vigor

“…Moreover, the subculture conditions critically determined LN regeneration (18.3~89.7%); the more vigorous the growth, the higher the LN regeneration. The dry weight of donor plants' shoots and roots and subsequent LN regeneration were highly correlated (r = 0.85-0.98) among the treatments tested [78]. The shoot tips subcultured with a liquid overlay on the gelled medium improved the growth of the donor plants (height 3.43 cm → 7.0 cm, dry weight 1.3 mg → 4.8 mg) (Figure 1A(G + LO)) and subsequently, caused higher LN regeneration of node-induced shoot tips (35.8% → 89.7%) (Figure 1B(G + LO)) than the no-liquid overlay (Figure 1A(G + LX),B(G + LX)) [78].…”

“…Rapid growth and, eventually, high dry weight with no or little growth hormones may indicate a higher DPV, since the explants from these donor plantlets can tolerate the cytotoxicity of PVSs, resulting in higher LN regeneration than the lower DPV [78]. Cold hardening of donor plants is often employed to improve cryotolerance, increasing membrane lipids' unsaturation ratio [79,80].…”

“…But, most species studied showed no less than some osmotic and chemical sensitivity. In this case, there are several approaches to adapting the plant material to these stresses during the procedure, i.e., vigorous growing of donor plants (subcultures), two-step preculture, balancing cryoprotection and cytotoxicity (pre-LN), rapid C/W and proper unloading (LN, warming), and regrowth initially with ammonium-free medium (regrowth) [7,15,36,74,78].…”

“…In this case, inoculating the apical section instead of the node and adding a liquid medium on the Gelrite-gelled medium has been applied spontaneously for two decades in the lab to rejuvenate the donor (stock, mother) plantlets. It has been proven to significantly improve the growing velocity and dry weight of subcultured donor plants and subsequent LN regeneration in P. yatabeanus [78], P. chinense [48], and D. grandiflourum [7]. Liquid overlay was used as a standard subculture in A. altaicus [70].…”

“…In vitro growth (height, dry weight) of P. yatabeana plantlets was affected by diverse factors of the subculture medium and culture conditions (overlay of liquid medium, gelling agent, medium strength, growth hormones, activated charcoal, and subculture duration) [78]. Moreover, the subculture conditions critically determined LN regeneration (18.3~89.7%); the more vigorous the growth, the higher the LN regeneration.…”