Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum
Abstract
:1. Introduction
2. Results and Discussion
2.1. Role of Various Growth Regulators in Shoot Regeneration
2.2. Effect of Various Antibiotics on Shoot Regeneration and Rooting
2.3. Molecular and Histochemical Analyses of Transgenic Plants
2.4. T-DNA Insertion Locus of pG10-2
3. Materials and Methods
3.1. Materials
3.2. Assessment of Growth Regulators of the Untransformed Plants
3.3. Assessment of Antibiotic Sensitivity of the Untransformed Plants
3.4. Agrobacterium-Mediated Transformation
3.5. PCR Detection
3.6. GUS Staining
3.7. Identification of the T-DNA Insertion Locus Using High-Throughput Sequencing
3.8. Data Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Types of Antibiotics | Concentration of Antibiotic (mg·L−1) | Callus Induction Rate (%) | Budding Leaf Disc Rate (%) | Budding Rate (%) |
---|---|---|---|---|
Kanamycin | 0 | 100 ± 0 a | 77.33 ± 5.81 b | 114.67 ± 8.11 a |
4.0 | 96.00 ± 2.31 ab | 22.67 ± 7.06 c | 36.00 ± 6.11 b | |
6.0 | 60.00 ± 2.31 d | 13.33 ± 7.06 cd | 16.00 ± 9.24 de | |
8.0 | 25.33 ± 3.53 f | 4.00 ± 4.00 de | 5.33 ± 5.33 de | |
10.0 | 8.00 ± 2.31 gh | 0 ± 0 | 0 ± 0 | |
Hygromycin | 0 | 100 ± 0 a | 78.67 ± 3.53 ab | 128.00 ± 8.33 a |
1.0 | 87.67 ± 2.91 b | 9.43 ± 4.28 de | 12.62 ± 5.91 c | |
1.5 | 72.00 ± 5.29 c | 7.35 ± 2.15 de | 9.87 ± 3.34 de | |
2.0 | 42.67 ± 8.11 e | 3.51 ± 2.15 de | 3.51 ± 2.15 de | |
2.5 | 13.33 ± 2.40 g | 0 ± 0 | 0 ± 0 | |
Glufosinate-ammonium | 0 | 100 ± 0 a | 89.33 ± 3.53 a | 118.67 ± 7.06 a |
0.3 | 85.33 ± 5.33 b | 24.00 ± 2.31 c | 33.33 ± 4.81 bc | |
0.4 | 54.67 ± 5.81 d | 13.33 ± 3.53 cd | 18.67 ± 4.81 cd | |
0.5 | 28.00 ± 4.62 f | 5.33 ± 3.53 de | 5.33 ± 3.53 de | |
0.6 | 0 ± 0 | 0 ± 0 | 0 ± 0 |
Types of Antibiotics | Concentration of Antibiotic (mg·L−1) | Frequency of Rooting (%) |
---|---|---|
Kanamycin | 0.00 | 100 ± 0 a |
6.00 | 33.3 ± 6.67 c | |
8.00 | 26.7 ± 6.67 c | |
10.00 | 14.4 ± 6.67 c | |
12.00 | 0 ± 0 | |
Hygromycin | 0.00 | 100 ± 0 a |
1.50 | 13.3 ± 6.67 c | |
2.00 | 0 ± 0 | |
2.50 | 0 ± 0 | |
3.00 | 0 ± 0 | |
Glufosinate-ammonium | 0.00 | 100 ± 0 a |
0.20 | 80 ± 11.55 b | |
0.30 | 60 ± 0 b | |
0.40 | 33.3 ± 6.67 c | |
0.50 | 0 ± 0 |
Code | 6-BA (mg·L−1) | NAA (mg·L−1) | Budding Leaf Disc Rate (%) | Budding Rate (%) |
---|---|---|---|---|
M1 | 1 | 0.2 | 96.00 ± 2.31 a | 162.67 ± 16.38 a |
M2 | 1 | 0.5 | 70.67 ± 9.33 bc | 92.00 ± 10.58 bc |
M3 | 1 | 0.8 | 13.33 ± 3.53 f | 16.00 ± 4.00 e |
M4 | 2 | 0.2 | 61.33 ± 8.74 bcd | 97.33 ± 20.95 bc |
M5 | 2 | 0.5 | 78.67 ± 8.74 ab | 124.00 ± 6.93 b |
M6 | 2 | 0.8 | 52.00 ± 2.31 cd | 90.67 ± 4.81 bc |
M7 | 3 | 0.2 | 14.67 ± 1.33 f | 16.00 ± 0 e |
M8 | 3 | 0.5 | 29.33 ± 9.61 ef | 41.33 ± 8.11 de |
M9 | 3 | 0.8 | 42.67 ± 9.33 de | 61.33 ± 15.03 cd |
Primer Name | Sequence (5′–3′) | Amplicon Length | Application |
---|---|---|---|
GUS/NPTII-F | GGGCAACAAGCCGAAAGA | 252 bp | Evaluate transgenic plants of Gojo-0 |
GUS/NPTII-R | TGAGCGTCGCAGAACATTACAT | ||
pG10-Backbone-F | GCCTTCTTGACGAGTTCTTCTGA | 505 bp | Evaluate pG10-RUBY vector backbone |
pG10-Backbone-R | CATGCTACCCTCCGCGAGA | ||
Cse2.0_LG3-F | AGGGTGGTGGTCATTTAGCCT | ~700 bp | Evaluate insertion site of line 2 |
pG10-LB-R | TGGGCTGACCGCTTCCTC |
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Zhang, J.; Zhang, J.; Li, P.; Gao, Y.; Yu, Q.; Sun, D.; Zhang, L.; Wang, S.; Tian, J.; Wang, Z.; et al. Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum. Int. J. Mol. Sci. 2022, 23, 11426. https://doi.org/10.3390/ijms231911426
Zhang J, Zhang J, Li P, Gao Y, Yu Q, Sun D, Zhang L, Wang S, Tian J, Wang Z, et al. Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum. International Journal of Molecular Sciences. 2022; 23(19):11426. https://doi.org/10.3390/ijms231911426
Chicago/Turabian StyleZhang, Jiali, Jing Zhang, Peiling Li, Yuan Gao, Qi Yu, Daojin Sun, Lingling Zhang, Siqi Wang, Jing Tian, Zhenxing Wang, and et al. 2022. "Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum" International Journal of Molecular Sciences 23, no. 19: 11426. https://doi.org/10.3390/ijms231911426