Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant and Oomycete Material
2.2. In Vitro Culture Establishment
2.3. Shoot Stabilization and Proliferation
2.4. Shoot Rooting
2.5. Cold Storage of Shoot Cultures
2.6. Somatic Embryogenesis
2.7. Dual Cultures
2.8. Total Phenols Determinations
2.9. Gene Expression Analyses
2.10. Statistical Analysis
3. Results
3.1. Micropropagation by Axillary Budding
3.1.1. Establishment, Stabilization, and Proliferation of Axillary Shoot Cultures
3.1.2. Rooting of Axillary Shoots
3.1.3. Medium-Term Conservation of Axillary Shoots
3.2. Somatic Embryogenesis
3.3. Dual Cultures
3.4. Total Phenols Content of In Vitro Grown Holm Oak Shoots
3.5. Gene Expression in Leaves of In Vitro Grown Holm Oak Shoots
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
BA | benzyladenine |
Bs | basal part of the tree |
C | crown of the tree |
CAD | NADPH-dependent cinnamyl alcohol dehydrogenase |
ChS | chalcone synthase |
CS | chorismate synthase |
EF | elongation factor |
GD | Gresshoff and Doy medium |
IAA | indole-3-acetic acid |
IBA | indol-3-butyric acid |
MS | Murashige and Skoog medium |
NAA | α-naphthalene acetic acid |
NSs | nodular embryogenic structures |
PAL | phenylalanineammonia-lyase |
Pc | Phytophthora cinnamomic |
PDA | Potato Dextrose Agar medium |
PGRs | plant growth regulators |
PPA | Plant Propagation Agar |
qPCR | quantitative PCR |
SE | somatic embryogenesis |
SH | Schenk and Hildebrandt |
STS | silver thiosulphate |
WPM | Woody Plant Medium |
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Gene | Forward Primer | Reverse Primer |
---|---|---|
CAD | CAGATGATAAGCCATTTGCG | AGGAACTTCAGGGTGCTAC |
ChS | TGAGATCACAGCAGTTAC | CAAGTTGAAACAGTGGAC |
CS | TGGATTGATTGGAAACAGATTAC | CAAGGAAGCAGCACACAG |
PAL | ATTAGCAGGGATTGATGG | CAAGTGGTCTGTAAATTCG |
EF | TTGTGCCGTCCTCATTATTGACT | TCACGGGTCTGACCATCCTT |
Clones | Flushing Capacity of Branch Segments (4 w) 1 | Response to In Vitro Establishment (8 w) 2 | ||||||
---|---|---|---|---|---|---|---|---|
Branch Segment Number | Branch with Sprouting (%) | Total Number Shoots | Length of Shoots (mm) | Number of Initial Explants | Contamination Rate (%) | Response Rate (%) 3 | Shoot Establishment | |
Mediterranean | ||||||||
H1 | 30 | 86.7 | 48 | 16.2 ± 5.8 | 66 | 18.3 | 21.8 | No |
H5 | 22 | 68.2 | 58 | 20.1 ± 5.8 | 94 | 27.7 | 26.5 | No |
VA5 | 32 | 96.9 | 109 | 30.1 ± 10.5 | 206 | 2.4 | 30.9 | Yes |
VA11-Bs 4 | 21 | 100.0 | 131 | 43.7 ± 20.2 | 225 | 15.6 | 15.8 | Yes |
VA11-C 4 | 12 | 75.0 | 33 | 18.4 ± 7.2 | 51 | 2.0 | 42.0 | Yes |
South-West | ||||||||
PL-T1 | 26 | 30.8 | 36 | 14.7 ± 5.2 | 41 | 61.0 | 100.0 | Yes |
PL-T2 | 31 | 70.9 | 57 | 15.4 ± 7.7 | 96 | 52.1 | 15.2 | Yes |
PL-T3 | 23 | 65.2 | 40 | 13.9 ± 6.4 | 59 | 25.4 | 11.4 | No |
PL-T4 | 32 | 31.3 | 19 | 12.9 ± 4.8 | 26 | 34.6 | 11.8 | No |
PL-T5 | 12 | 33.3 | 6 | 12.5 ± 3.7 | 6 | 33.3 | 0.0 | - |
PL-T6 | 32 | 0.0 | 0 | - | - | - | - | - |
Clone | Responsive Explants (%) | Total Shoots (Nº) | Shoots 0.5–1.0 (Nº) | Shoots ≥ 1 cm (Nº) | Longest Shoot Length (mm) |
---|---|---|---|---|---|
VA11-Bs | 100 ± 0.0 | 7.82 ± 0.38 | 2.53 ± 0.27 | 5.29 ± 0.21 | 19.33 ± 0.48 |
VA11-C | 100 ± 0.0 | 8.30 ± 0.17 | 4.96 ± 0.15 | 3.34 ± 0.12 | 15.57 ± 0.39 |
VA5 | 100 ± 0.0 | 17.76 ± 0.56 | 10.33 ± 0.52 | 7.43 ± 0.31 | 22.40 ± 0.83 |
PL-T2 | 100 ± 0.0 | 5.34 ± 0.17 | 1.96 ± 0.18 | 3.38 ± 0.16 | 13.30 ± 0.52 |
ANOVA I | 1.0 ns | 0.001 *** | 0.001 *** | 0.001 *** | 0.001 *** |
Clone | Rooting (%) | Root Number | Root Length (mm) |
---|---|---|---|
VA5 | 36.0 ± 9.3 | 4.4 ± 0.7 | 47.5 ± 5.7 |
VA11-Bs | 12.0 ± 3.1 | 1.2 ± 0.1 | 17.3 ± 2.1 |
VA11-C | 8.0 ± 3.1 | 1.0 ± 0.0 | 40.0 ± 7.8 |
ANOVA I | 0.012 * | 0.004 ** | 0.028 * |
CLONE | Storage Period (Months) | Recovery (%) 1 | Total Shoots (Nº) | Shoots 0.5–1.0 cm (Nº) | Shoots ≥1 cm (Nº) | Longest Shoot Length (mm) |
---|---|---|---|---|---|---|
Q10 | 0 | 100 ± 0.0 | 3.2 ± 0.2 | 1.3 ± 0.1 | 1.9 ± 0.2 | 16.2 ± 0.5 |
6 | 100 ± 0.0 | 4.8 ± 0.3 | 1.8 ± 0.2 | 3.0 ± 0.2 | 14.7 ± 1.4 | |
9 | 97.1 ± 2.5 | 4.3 ± 0.3 | 1.5 ± 0.2 | 2.8 ± 0.2 | 12.8 ± 1.1 | |
12 | 100 ± 0.0 | 3.7 ± 0.2 | 2.0 ± 0.2 | 1.7 ± 0.2 | 15.7 ± 0.6 | |
ANOVA I | 0.418 ns | 0.008 ** | 0.045 * | 0.003 ** | 0.81 ns | |
Q3 | 0 | 100 ± 0.0 | 2.7 ± 0.1 | 1.7 ± 0.2 | 1.0 ± 0.1 | 16.2 ± 0.5 |
6 | 78.6 ± 4.0 | 2.5 ± 0.4 | 1.4 ± 0.2 | 1.1 ± 0.2 | 14.5 ± 0.3 | |
9 | 64.3 ± 6.3 | 2.8 ± 0.1 | 1.6 ± 0.2 | 1.2 ± 0.1 | 11.2 ± 0.5 | |
12 | 100 ± 0.0 | 2.3 ± 0.3 | 1.2 ± 0.2 | 1.1 ± 0.2 | 11.2 ± 2.4 | |
ANOVA I | 0.001 *** | 0.523 ns | 0.386 ns | 0.818 ns | 0.62 ns | |
E00 | 0 | 100 ± 0.0 | 4.4 ± 0.3 | 1.8 ± 0.1 | 2.6 ± 0.1 | 23.3 ± 1.0 |
6 | 100 ± 0.0 | 3.1 ± 0.1 | 1.5 ± 0.1 | 1.6 ± 0.2 | 15.5 ± 0.2 | |
9 | 71.4 ± 13.3 | 2.5 ± 0.5 | 1.4 ± 0.1 | 1.1 ± 0.4 | 12.7 ± 2.9 | |
12 | 68.6 ± 8.4 | 1.8 ± 0.1 | 1.3 ± 0.1 | 0.5 ± 0.1 | 10.3 ± 0.9 | |
ANOVA I | 0.012 * | 0.001 *** | 0.082 ns | 0.001 *** | 0.001 *** | |
PL-T2 | 0 | 100 ± 0.0 | 5.5 ± 0.3 | 2.1 ± 0.3 | 3.4 ± 0.3 | 13.4 ± 0.9 |
6 | 84.0 ± 3.4 | 4.0 ± 0.1 | 1.7 ± 0.1 | 2.3 ± 0.1 | 10.0 ± 0.8 | |
9 | 54.3 ± 15.8 | 1.9 ± 0.2 | 1.4 ± 0.3 | 0.5 ± 0.1 | 7.4 ± 0.9 | |
12 | - | - | - | - | - | |
ANOVA I | 0.018 * | 0.001 *** | 0.207 ns | 0.001 *** | 0.006 ** | |
VA11-C | 0 | 100 ± 0.0 | 8.4 ± 0.2 | 4.9 ± 0.1 | 3.5 ± 0.1 | 16.4 ± 0.3 |
6 | 100 ± 0.0 | 9.2 ± 0.5 | 5.8 ± 0.6 | 3.4 ± 0.2 | 19.7 ± 0.6 | |
9 | 100 ± 0.0 | 5.8 ± 0.3 | 3.1 ± 0.3 | 2.7 ± 0.2 | 18.4 ± 0.7 | |
12 | 100 ± 0.0 | 6.7 ± 0.2 | 4.0 ± 0.2 | 2.7 ± 0.2 | 18.7 ± 0.8 | |
ANOVA I | 1.0 ns | 0.001 *** | 0.002 ** | 0.007 ** | 0.026 * | |
VA11-Bs | 0 | 100 ± 0.0 | 8.3 ± 0.4 | 2.9 ± 0.3 | 5.4 ± 0.1 | 19.3 ± 0.28 |
6 | 100 ± 0.0 | 9.6 ± 0.5 | 5.7 ± 0.4 | 3.9 ± 0.2 | 19.7 ± 0.6 | |
9 | 100 ± 0.0 | 7.1 ± 0.2 | 4.4 ± 0.1 | 2.7 ± 0.2 | 17.9 ± 0.6 | |
12 | 100 ± 0.0 | 6.0 ± 0.5 | 3.1 ± 0.3 | 2.9 ± 0.3 | 19.2 ± 0.9 | |
ANOVA I | 1.0 ns | 0.001 *** | 0.001 *** | 0.001 *** | 0.378 ns | |
VA5 | 0 | 100 ± 0.0 | 18.5 ± 0.6 | 10.5 ± 0.6 | 8.0 ± 0.3 | 20.2 ± 0.6 |
6 | 100 ± 0.0 | 15.3 ± 0.8 | 9.2 ± 0.3 | 6.1 ± 0.6 | 24.3 ± 0.9 | |
9 | 100 ± 0.0 | 10.2 ± 1.4 | 3.4 ± 0.6 | 6.8 ± 0.9 | 23.5 ± 0.7 | |
12 | 97.1 ± 2.5 | 5.2 ± 0.8 | 4.6 ± 0.7 | 0.6 ± 0.2 | 10.3 ± 0.7 | |
ANOVA I | 0.418 ns | 0.001 *** | 0.001 *** | 0.001 *** | 0.001 *** |
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Martínez, M.T.; Arrillaga, I.; Sales, E.; Pérez-Oliver, M.A.; González-Mas, M.d.C.; Corredoira, E. Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees. Forests 2021, 12, 1634. https://doi.org/10.3390/f12121634
Martínez MT, Arrillaga I, Sales E, Pérez-Oliver MA, González-Mas MdC, Corredoira E. Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees. Forests. 2021; 12(12):1634. https://doi.org/10.3390/f12121634
Chicago/Turabian StyleMartínez, Mª Teresa, Isabel Arrillaga, Ester Sales, María Amparo Pérez-Oliver, Mª del Carmen González-Mas, and Elena Corredoira. 2021. "Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees" Forests 12, no. 12: 1634. https://doi.org/10.3390/f12121634