Construction of a Genetic Transformation System for Populus wulianensis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Source of Materials
2.1.1. Plant Materials of P. wulianensis
2.1.2. Strain and Plasmid
2.2. Methods
2.2.1. Leaf Adventitious Bud Induction
2.2.2. Antibiotic and Bacteriostatic Agent Concentration Screening Experiment
2.2.3. Genetic Transformation of P. wulianensis Leaf Discs
2.2.4. Evaluation of Transformation Factors on Genetic Efficiency
2.2.5. PCR and RT-PCR Analyses
2.2.6. Observation of EGFP Expression
2.2.7. Statistical Analysis
3. Results
3.1. Effects of Medium Components on the Induction of Adventitious Buds of P. wulianensis Leaves
3.2. Screening of Critical Antibiotic Concentrations
3.2.1. Determination of Critical Screening Pressure for Timentin
3.2.2. Determination of Kan Critical Screening Pressure
3.3. Optimization of a Genetic Transformation System for P. wulianensis
3.4. Verification of a Genetic Transformation System for P. wulianensis
4. Discussion
4.1. Comparison of Adventitious Bud Regeneration System of Poplar
4.2. Comparison of Poplar Genetic Transformation Systems
4.3. Analysis of Factors Affecting Conversion Efficiency
4.4. False Positive Exclusion Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
MS | Murashige and Skoog medium |
NAA | Naphthalene acetic acid |
6-BA | 6-Benzylaminopurine |
EGFP | Enhanced green fluorescent protein |
DAPI | 4′,6-Diamidino-2-phenylindole |
PCR | Polymerase chain reaction |
β-AS | Acetylsyringone |
WT | Wild type |
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Level | Basal Medium (A) | NAA (mg/L) (B) | 6-BA (mg/L) (C) | Dark Culture Time (d) (D) |
---|---|---|---|---|
1 | (1/2NH4NO3) MS | 0.01 | 0.1 | 0 |
2 | 1/2MS | 0.05 | 0.5 | 4 |
3 | MS | 0.10 | 1.0 | 8 |
Code | A | B | C | D | Average Number of Regenerated Buds |
---|---|---|---|---|---|
1 | 3(MS) | 2 (0.05) | 3 (1.0) | 1 (0 d) | 1.67 ± 0.72 e |
2 | 3(MS) | 3 (0.10) | 1 (0.1) | 2 (4 d) | 17.33 ± 1.70 c |
3 | 2(1/2MS) | 1 (0.01) | 3 (1.0) | 2 (4 d) | 10.00 ± 0.82 d |
4 | 2(1/2MS) | 3 (0.10) | 2 (0.5) | 1 (0 d) | 3.33 ± 2.05 e |
5 | 2(1/2MS) | 2 (0.05) | 1 (0.1) | 3 (8 d) | 15.00 ± 3.27 c |
6 | 1(1/2(NH4NO3) MS) | 3 (0.10) | 3 (1.0) | 3 (8 d) | 22.33 ± 2.05 b |
7 | 1(1/2(NH4NO3) MS) | 1 (0.01) | 1 (0.1) | 1 (0 d) | 19.00 ± 2.94 c |
8 | 3(MS) | 1 (0.01) | 2 (0.5) | 3 (8 d) | 17.67 ± 1.70 c |
9 | 1(1/2(NH4NO3) MS) | 2 (0.05) | 2 (0.5) | 2 (4 d) | 34.67 ± 3.30 a |
Code | Concentration mg/L | Middle of the Leaf | Top of the Leaf | Bottom of the Leaf |
---|---|---|---|---|
A | 0 | 32.30 ± 1.15 cd | 16.00 ± 0.66 bc | 14.70 ± 0.18 e |
B | 50 | 37.70 ± 0.35 bc | / | / |
C | 100 | 39.30 ± 0.98 bc | 17.70 ± 0.98 bc | 15.30 ± 1.12 e |
D | 150 | 41.30 ± 1.52 ab | / | / |
E | 200 | 42.30 ± 1.34 ab | 20.001.25 ab | 24.00 ± 1.75 cd |
F | 250 | 49.30 ± 0.33 a | / | / |
G | 300 | 50.30 ± 1.71 a | 24.30 ± 1.26 a | 35.70 ± 1.27 b |
H | 350 | 40.00 ± 1.11 bc | / | / |
I | 400 | 34.00 ± 0.68 bc | 20.70 ± 1.31 ab | 44.00 ± 2.05 a |
J | 450 | 27.30 ± 1.28 d | / | / |
K | 500 | 16.00 ± 0.53 e | 13.00 ± 1.09 cd | 26.00 ±1.02 c |
L | 550 | 14.70 ± 1.14 e | / | / |
M | 600 | 11.70 ± 0.39 ef | 7.70 ± 0.88 d | 17.00 ± 0.72 de |
N | 650 | 8.00 ± 0.27 ef | / | / |
O | 700 | 7.70 ± 1.02 ef | 5.70 ± 0.42 e | 12.70 ± 0.93 e |
P | 750 | 7.00 ± 0.83 ef | / | / |
Q | 800 | 2.70 ± 0.57 f | 0.00 ± 0.00 | 3.70 ± 0.14 f |
R | 850 | 1.70 ± 0.24 f | / | / |
S | 900 | / | 0.00 ± 0.00 | 1.70 ± 0.70 f |
Kan Concentration(mg/L) | Indeterminate Number of Buds | Leaf-Induced Growth Status | Rooting Rate (%) | Adventitious Buds Induce Rooting |
---|---|---|---|---|
0 | 33.30 ± 1.28 a | All three parts of the leaf blade were dark green and had the highest number of differentiated buds. | 100.00 | All rooted, green plants, healthy growth. |
25 | 15.70 ± 1.09 b | All three parts of the leaf blade were green, and 50% of the B part of the leaf produced adventitious buds. | 50.00 | Half rooted, and the other leaves were light green. |
50 | 5.00 ± 0.27 c | Leaf A was slightly yellow; the rest was green, and 30% of leaf B produced adventitious buds. | 42.67 | Some were rooted, whereas others were yellowed. |
75 | 3.70 ± 0.33 c | Only the C part of the leaf produced adventitious buds, whereas the other parts of the leaf did not produce adventitious buds. | 23.08 | A small part of the leaves took root, and a small part of the leaves appeared vitrified. |
100 | 2.00 ± 0.18 c | Only a small part of the leaf C part produced adventitious buds, and the leaf B part appeared yellow. | 0.00 | The base was swollen; none of them took root, and the adventitious buds grew slowly. |
125 | 1.70 ± 0.15 c | Very few leaves had adventitious bud differentiation in the leaf C part, and no adventitious buds were produced in the others. | 0.00 | A few had swollen bases; none of them took root, and adventitious buds had stagnated growth. |
150 | 0.00 ± 0.00 | Dehydration began to appear in all three parts of the leaves, and there was no redifferentiation or adventitious buds. | 0.00 | The base was not enlarged and did not take root, and the adventitious bud leaves were partially yellowed. |
175 | 0.00 ± 0.00 | The three parts of the leaf showed different degrees of whitening, with no redifferentiation or adventitious buds. | 0.00 | The base was not swollen and did not take root, and the adventitious bud leaves were curled and yellowed. |
200 | 0.00 ± 0.00 | The three parts of the leaves were dehydrated and whitish, and there were no adventitious buds. | 0.00 | The base was not swollen and did not take root, and the leaves of adventitious buds were partially dry. |
OD600 | Number of Resistant Buds | Growth State |
---|---|---|
0.60 | 1.44 ± 0.51 b | The leaves gradually turned pale green and eventually withered, often displaying white plaques. |
0.30 | 5.11 ± 0.92 a | A few leaf edges showed signs of fading with faintly visible colonies. |
0.15 | 3.89 ± 0.87 ab | Several leaves exhibited fading with faintly visible colonies. |
Infection Time/min | Number of Resistant Buds | Growth State |
---|---|---|
5 | 3.36 ± 0.45 b | Leaf disks remained green, with no colonies visible. |
15 | 6.38 ± 1.28 a | Leaf blade margins displayed reddish brown, whereas wounds exhibited multiple buds and pale mycorrhizae. |
20 | 2.92 ± 0.31 b | Leaf disks appeared dry with visible marginal mycorrhiza. |
β-AS Concentration /μmol·L−1 | Number Of Resistant Buds | Growth State |
---|---|---|
0 | 2.94 ± 0.73 b | Dry spots appeared with yellowing of the leaf margins. |
100 | 5.95 ± 1.12 a | Leaf disks remained green, with many buds forming around the wounds. |
200 | 6.45 ± 1.34 a | Brown spots developed, with more buds forming around the wound. |
Co-Culture Time/d | Number of Resistant Buds | Growth State |
---|---|---|
1 | 0.00 ± 0.00 c | Leaf disks appeared greenish but dry without any visible adventitious buds. |
3 | 6.11 ± 1.43 a | Yellowing was observed along the margins of the leaf disks, with green adventitious buds but no visible mycorrhizae. |
6 | 2.95 ± 0.22 b | Late leaf discs appeared dry and exhibited white fungal spots. |
Populus Species/Hybrid | Explants | Adventitious Buds | Reference |
---|---|---|---|
P. tomentosa (clone GM107) | Leaf | 20.00 | Huang et al., 2023 [31] |
P. alba × P. glandulosa | Leaf | 20.00 | Li et al., 2007 [32] |
P. adenopoda × poplar84K | Leaf | 14.80 | Zhang et al., 2010 [33] |
P. dabidiana × P. bolleana | Leaf | 12.10 | Wang et al., 2010 [34] |
P. hopeiensis | Leaf | 11.40 | Wu et al., 2021 [35] |
P. tremuloides | Leaf | 10.00 | Huang et al., 2011 [28] |
P. Leucopyramidalis | Leaf | 8.20 | Li et al., 2008 [32] |
P. tomentosa (male diploid) | Leaf | 6.63 | Yu et al., 2022 [13] |
P. tremula × P. alba | Leaf | 4.27 | Wang et al., 2009 [30] |
P. tomentosa | Leaf | 3.40 | Li et al., 2016 [27] |
P. tremula × P. tremuloides | Leaf | 2.73 | Wang et al., 2009 [30] |
Populus Species/Hybrid | Positive Rate | Selective Agent and Its Concentration(mg/L) | Bacterial Inhibitor and Its Concentration (mg/L) | Reference |
---|---|---|---|---|
P. dabidiana × P. bolleana | 9.30% | 25–50, Kan | 500, Cef | Wang et al., 2011 [2] |
P. tomentosa | 9.38% | 20–50, Kan | 150–250, Cef | Long et al., 2010 [36] |
Male diploid P. tomentosa | 16.28% | 30, Kan | 200 Cef + 200 timentin | Yu et al., 2022 [13] |
P. alba var. pyramidalis | 23.60% | 90, Hygromycin | 400, Cef | Ma et al., 2019 [15] |
P. dabidiana × P. bolleana | 30.00% | 50, Kan | Unmarked | Han et al., 2013 [4] |
P. tomentosa | 36.32% | 30–50, Kan | 300, Cef | Hu et al., 2005 [37] |
P. alba × P. glandulosa cv. 84K | 51.54% | 50–60, Kan | 300, Cef | Zhang et al., 2005 [40] |
P. dabidiana × P. bolleana | 93.33% | 30–60, Kan | Unmarked | Zang et al., 2008 [38] |
P. canescens × P. grandidentata | 93.33% | 60–80, Kan | 250 Cef + 500 Car | Dai et al., 2003 [41] |
P. tremuloides (clone 271) | 94.17% | 40–100, Kan | 300, Cef | Tsai et al., 1994 [39] |
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Share and Cite
Wang, Y.; Song, C.; Han, Y.; Wang, R.; Guan, L.; Mu, Y.; Sun, T.; Xie, X.; Zhao, Y.; Xu, J.; et al. Construction of a Genetic Transformation System for Populus wulianensis. Forests 2024, 15, 1474. https://doi.org/10.3390/f15081474
Wang Y, Song C, Han Y, Wang R, Guan L, Mu Y, Sun T, Xie X, Zhao Y, Xu J, et al. Construction of a Genetic Transformation System for Populus wulianensis. Forests. 2024; 15(8):1474. https://doi.org/10.3390/f15081474
Chicago/Turabian StyleWang, Yan, Chenxia Song, Yi Han, Ruilong Wang, Lingshan Guan, Yanjuan Mu, Tao Sun, Xiaoman Xie, Yunchao Zhao, Jichen Xu, and et al. 2024. "Construction of a Genetic Transformation System for Populus wulianensis" Forests 15, no. 8: 1474. https://doi.org/10.3390/f15081474