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
:1. Introduction
2. Results
2.1. Cryoprotection and Regrowth of Pogostemon yatabeanus Shoot Tips: Effect of Ammonium
2.1.1. Effect of Ammonium at Different Protocol Stages (Experiment 1)
2.1.2. Substitution of Ammonium and Modification of Regrowth Conditions (Experiment 2)
2.1.3. Impact of Vitrification Solution Treatment (Experiment 3)
2.1.4. Impact of Individual Protocol Steps (Experiment 3, Continued)
2.2. Cryopreservation of Pogostemon yatabeanus Shoot Tips
2.2.1. Impact of Individual Protocol Steps and Vitrification Solution Treatment after Cryopreservation (Experiment 3, Continued)
2.2.2. Effect of Growth Regulators in Regrowth Medium (Experiment 4)
2.2.3. Combination of Growth Regulators and Ammonium-Free Regrowth Medium at Three Regrowth Steps (Experiment 5)
3. Discussion
4. Materials and Methods
4.1. Plant Material
4.2. Shoot Tip Cryopreservation by Droplet-Vitrification
4.3. Recovery Assessment and Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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No. | Protocol Steps | Survival (%) | Regeneration (%) | ||
---|---|---|---|---|---|
Preculture + Osmoprotection * | Cryoprotection + Unloading * | Regrowth (Step 1) ** | |||
1 | − | − | − (5 days) | 91.3 ± 8.4 a | 70.1 ± 5.4 c |
2 | − | − | + (5 days) | 87.5 ± 4.8 a | 71.6 ± 4.8 c |
3 | + | − | − (5 days) | 96.4 ± 3.1 a | 82.6 ± 2.9 b |
4 | + | + | − (5 days) | 97.7 ± 3.1 a | 95.7 ± 4.1 a |
5 | + | + | − (3 days) | 95.0 ± 2.7 a | 87.8 ± 7.5 ab |
6 | + | + | − (1 day) | 94.3 ± 5.4 a | 87.2 ± 7.9 ab |
7 | + | + | + (5 days) | 97.1 ± 3.2 a | 63.3 ± 6.2 c |
No. | Nitrogen Source in Cryoprotectant Medium * | Regrowth Media, Duration and Light/Dark Conditions ** | Survival (%) | Regeneration (%) |
---|---|---|---|---|
1 | NH4NO3 | RM2, 5 d → RM2, 23 d → MSF, 14 d | 90.6 ± 2.5 a | 70.0 ± 3.0 e |
2 | NH4NO3 | RM1, 5 d → RM2, 23 d → MSF, 14 d | 97.5 ± 4.0 a | 96.2 ± 4.5 a |
3 | NH4NO3 | RM1, 5 d → RM2, 7 d → RM2, 7 d → RM2, 7 d → MSF, 14 d | 93.3 ± 5.4 a | 94.6 ± 6.1 ab |
4 | NH4NO3 | NaNO3 in RM1, 5 d → RM2, 23 d → MSF, 14 d | 90.4 ± 6.1 a | 80.1 ± 5.6 cd |
5 | NaNO3 | NaNO3 in RM1, 5 d → RM2, 23 d → MSF, 14 d | 93.4 ± 4.1 a | 87.7 ± 4.8 abc |
6 | NH4NO3 | (10 mM NH4+30 mM NO3) in RM, 5 d → RM2, 23 d→ MSF, 14 d | 93.2 ± 7.3 a | 80.5 ± 1.6 cd |
7 | NH4NO3 | RM2+AC, 5 d → RM2, 23 d → MSF, 14 d | 89.2 ± 3.7 a | 73.3 ± 9.0 de |
8 | NH4NO3 | RM1+AC, 5 d → RM2, 23 d → MSF, 14 d | 92.5 ± 1.4 a | 87.7 ± 4.2 abc |
9 | NH4NO3 | RM2, L1, 5 d → RM2, 23 d → MSF, 14 d | 90.2 ± 2.8 a | 75.6 ± 6.9 de |
10 | NH4NO3 | RM1, L1, 5 d → RM2, 23 d→ MSF, 14 d | 92.9 ± 4.0 a | 85.8 ± 4.2 bc |
No. | Regrowth Steps * | LNC | LN | |||||
---|---|---|---|---|---|---|---|---|
Step 1 5 Days | Step 2 23 Days | Step 3 14 Days | Survival (%) | Regeneration (%) | Survival (%) | Regeneration (%) | ||
NH4NO3 | GA3 + BA | GA3 + BA | GA3 + BA | |||||
1 | − | + | + | − | 95.0 ± 7.1 a | 100.0 ± 0.0 a | 94.9 ± 6.8 a | 91.6 ± 1.0 a |
2 | − | + | − | − | 95.0 ± 7.1 a | 45.0 ± 7.1 d | 81.9 ± 10.3 ab | 32.2 ± 1.9 d |
3 | − | − | + | − | 90.0 ± 0.0 a | 75.0 ± 7.1 c | 77.6 ± 7.3 ab | 56.7 ± 17.9 bc |
4 | − | − | − | − | 95.0 ± 7.1 a | 88.8 ± 1.8 ab | 71.2 ± 5.2 bc | 35.4 ± 6.5 cd |
5 | + | + | + | − | 95.0 ± 7.1 a | 53.6 ± 5.1 d | 59.5 ± 18.1 cd | 50.0 ± 14.1 bcd |
6 | + | − | − | − | 77.9 ± 11.1 a | 53.6 ± 5.1 d | 53.0 ± 13.0 d | 7.9 ± 2.9 e |
7 | − | + | + | + | 95.0 ± 7.1 a | 85.0 ± 7.1 bc | 93.4 ± 5.9 a | 69.8 ± 7.6 b |
Pre-LN Step | Treatments * | Regrowth Medium ** | Code | LNC | LN |
---|---|---|---|---|---|
Preculture | No preculture | RM1 | noPC | + | + |
RM2 | + | ||||
10% sucrose 31 h → 25% sucrose 17 h | RM1 | S-25% | + | + | |
RM2 | + | ||||
10% sucrose 31 h | RM1 | standard | + | + | |
RM2 | + | ||||
10% sucrose 31 h → 17.5% sucrose 17 h | RM1 | S-17.5% | + | ||
Osmoprotection | No osmoprotection | RM1 | noOP | + | + |
RM2 | + | ||||
C4-35% 40 min | RM1 | standard | + | + | |
Cryoprotection | No vitrification solution treatment | RM1 | noVS | + | + |
RM2 | + | ||||
A3-80% ice 60 min | RM1 | A3-80% ice 60 standard | + | + | |
RM2 | + | ||||
A3-80% ice 30 min | RM1 | A3-80% ice 30 | + | ||
RM2 | + | ||||
A3-80% ice 90 min | RM1 | A3-80% ice 90 | + | + | |
RM2 | + | ||||
A3-80% RT 20 min | RM1 | A3-80% RT 20 | + | ||
RM2 | + | ||||
B5-80% RT 60 min | RM1 | B5-80% RT 60 | + | + | |
RM2 | + | ||||
B1-100% RT 60 min | RM1 | B1-100% RT 60 | + | ||
RM2 | + | ||||
Container | Cryovial, 2 mL | RM1 | Vial | − | + |
Solution | Composition (%, w/v) * | Total Concentration of Cryoprotectants (%, w/v) |
---|---|---|
S-10% | S 10.0 | 10.0 |
S-35% | S 35.0 | 35.0 |
C4-35% | G 17.5 + S 17.5 | 35.0 |
A1-73.7% (PVS2) | G 30.0 + DMSO 15.0 + EG 15.0 + S 13.7 | 73.7 |
A3-90% | G 37.5 + DMSO 15.0 + EG 15.0 + S 22.5 | 90.0 |
A3-80% | G 33.3 + DMSO 13.3 + EG 13.3 + S 20.1 | 80.0 |
A3-70% | G 29.2 + DMSO 11.7 + EG 11.7 + S 17.4 | 70.0 |
B1-100% (PVS3) | G 50.0 + S 50.0 | 100.0 |
B5-80% | G 40.0 + S 40.0 | 80.0 |
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Lee, H.-E.; Popova, E.; Park, H.-N.; Park, S.-U.; Kim, H.-H. Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators. Plants 2021, 10, 2018. https://doi.org/10.3390/plants10102018
Lee H-E, Popova E, Park H-N, Park S-U, Kim H-H. Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators. Plants. 2021; 10(10):2018. https://doi.org/10.3390/plants10102018
Chicago/Turabian StyleLee, Hyo-Eun, Elena Popova, Ha-Na Park, Sang-Un Park, and Haeng-Hoon Kim. 2021. "Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators" Plants 10, no. 10: 2018. https://doi.org/10.3390/plants10102018