Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators

Abstract

Cryopreservation provides a secure long-term conservation option for rare and endangered plant species with non-orthodox or limitedly available seeds. Wide application of cryopreservation to biobank wild flora is hampered by the need to re-optimize nearly all protocol steps for every new species. We applied a systematic approach to simplify optimization of a multi-stage droplet-vitrification method for the endangered wetland Korean species, Pogostemon yatabeanus. This approach consisted of a standard procedure pre-selected based on material type and size, which was complemented with 11 additional treatments to reveal the most impactful conditions. Effect of ammonium nitrate at various protocol steps was also tested. The highest shoot tip survival (92%) and plant regeneration (90%) after cryopreservation were achieved using preculture with 10% sucrose followed by 40 min osmoprotection and 60 min treatment with vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose) on ice. A three-step regrowth procedure starting with ammonium-free medium with 1 mg/L GA₃ and 1 mg/L BA followed by ammonium-containing medium with and without growth regulators was essential for the development of healthy plants from cryopreserved shoot tips. This approach enables fast optimization of the cryopreservation procedure for new osmotic stress-sensitive plant species.

Keywords: alternative vitrification solutions; ammonium stress; droplet-vitrification; endangered species conservation; osmoprotection; plant growth regulators; regrowth medium.

Figure 1

Effect of vitrification solution treatment conditions and initial (first 5 days) regrowth on ammonium-free (RM1) or ammonium–containing (RM2) medium on survival and regeneration of Pogostemon yatabeanus shoot tips in the droplet-vitrification procedure. Shoot tips were cryoprotected but not cryopreserved. Treatment conditions are listed in the table in Section 4.2. (LNC).

Figure 2

Effect of individual pre-LN protocol steps (preculture, osmoprotection, cryoprotection) and initial (first 5 days) regrowth on ammonium-free (RM1) or ammonium–containing (RM2) medium on survival and regeneration of Pogostemon yatabeanus shoot tips in the droplet-vitrification procedure. Shoot tips were cryoprotected but not cryopreserved. Treatment conditions are listed in the table in Section 4.2. (LNC).

Figure 3

Effect of preculture, osmoprotection, cryoprotection and container for cooling/rewarming on survival (surv) and regeneration (rege) of cryopreserved Pogostemon yatabeanus shoot tips in the droplet-vitrification procedure. Treatment S-10% represents a standard protocol: preculture with 10% sucrose for 31 h, osmoprotection with C4-35% for 40 min, and cryoprotection with A3-80% on ice for 60 min, followed by cooling and warming using aluminum foil strips. Treatments are listed in the table in Section 4.2. (LN).

Figure 4

Effect of growth regulators in regrowth medium (steps 1 and 2 of the three-step regrowth procedure) on survival (surv) and regeneration (rege) of cryoprotected (LNC) and cryopreserved (LN) Pogostemon yatabeanus shoot tips in the droplet-vitrification procedure. G0, G1-0 and 1 mg/L gibberellic acid-3; B0, B0.5, B0.7 and B1-0, 0.5, 0.7 and 1 mg/L benzyl adenine; Z1-1 mg/L zeatin; I0.3 and I0.5-0.3 and 0.5 mg/L indolyl acetic acid; Free—medium without growth regulators.