Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus
Abstract
:1. Introduction
2. Results
2.1. Influence of Basal Media, Growth Regulators and Gelling Agents on Regeneration Efficiency of MBs
2.2. Hansen 536 MBs Transformation Efficiency and the Role of GFP and Kanamycin as Selectable Markers
2.3. Molecular Analysis of Putatively Transgenic Callus Lines
3. Discussion
4. Material and Methods
4.1. Plant Material and Establishment of In Vitro Culture
4.2. Influence of Different Factors on the Regeneration Efficiency of Hansen 536 MBs
4.2.1. Effect of Different Basal Media and Plant Growth Regulator Combinations
4.2.2. Effect of Different Gelling Agents on Shoot Regeneration Efficiency and Shoot Hyperhydricity
4.3. Genetic Transformation
4.3.1. Gene Construct and Agrobacterium Tumefaciens Strain
4.3.2. Genetic Transformation Protocol and Selection of GFP Fluorescing Calli
4.4. Molecular Analysis of GFP Fluorescing Calli
4.5. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
References
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Regeneration Media | Frequency of Regeneration (%) ± SE * | Average Number of Regenerated Shoots/MB ± SE |
---|---|---|
QL1 | 61.1 ± 5.5 (abc) | 1.8 ± 0.5 (defg) |
QL2 | 83.3 ± 9.6 (a) | 6.4 ± 1.2 (ab) |
QL3 | 61.1 ± 11.1 (abc) | 3.8 ± 1 (bcde) |
QL4 | 50 ± 0 (abc) | 2.8 ± 1.9 (cdefg) |
QL5 | 66.7 ± 9.6 (abc) | 4.3 ± 1.2 (bcd) |
QL6 | 83.3 ± 16.7 (a) | 7.2 ± 1.3 (a) |
DKW1 | 38.9 ± 5.5 (abc) | 1.4 ± 0.6 (defg) |
DKW2 | 38.9 ± 22.2 (abc) | 1.3 ± 0.5 (defg) |
DKW3 | 27.8 ± 5.5 (bc) | 0.8 ± 0.3 (fg) |
DKW4 | 22.2 ± 11.1 (c) | 0.4 ± 0.2 (g) |
DKW5 | 27.8 ± 5.5 (bc) | 1.2 ± 0.5 (efg) |
DKW6 | 72.2 ± 14.7 (ab) | 3.5 ± 0.8 (bcdef) |
WPMm1 | 61.1 ± 22.2 (abc) | 2.6 ± 0.7 (cdefg) |
WPMm2 | 72.2 ± 11.1 (ab) | 5.5 ± 1.4 (abc) |
WPMm3 | 38.9 ± 5.5 (abc) | 1.4 ± 0.5 (defg) |
WPMm4 | 72.2 ± 11.1 (ab) | 3.3 ± 0.8 (cdefg) |
WPMm5 | 77.8 ± 5.5 (a) | 4.2 ± 0.9 (bcde) |
WPMm6 | 50 ± 25.4 (abc) | 2.1 ± 0.7 (defg) |
Gelling Agent Concentration (g L−1) | Average Number of Regenerated Shoots/MB ± SE | Frequency of Vitrified Shoots (%) ± SE * | |||||
---|---|---|---|---|---|---|---|
QL2 | QL6 | WPMm5 | QL2 | QL6 | WPMm5 | ||
PA | 7 | 6.7 ± 0.5 (ab) | 7.4 ± 1.3 (ab) | 4.4 ± 0.7 (b) | 82.6 ± 5.7 (b) | 98.5 ± 8.9 (a) | 59.2 ± 5.9 (cd) |
B&V | 6 | 5.6 ± 1 (ab) | 8.9 ± 0.7 (a) | 7.8 ± 1.4 (a) | 53.7 ± 4.3 (d) | 64.3 ± 7.4 (c) | 22.8 ± 4.9 (f) |
Kan Concentration (μM) | Number of Weeks after Co-Culture | Mean Number of GFP Spot/Explant ± SE | GFP MB (%) ± SE * | Number of MB Slices with Lzs | GFP MB Lzs (%) ± SE ** |
---|---|---|---|---|---|
0 | 3 | 1.17 ± 0.37 (b) | 26.67 ± 5.75 (c) | 12 | 20 ± 5.21 (ns) |
12 | 0.82 ± 0.38 (A) | 6.67 ± 3.25 (A) | 2 | 3.33 ± 2.34 (NS) | |
17 | 3 | 2.10 ± 0.33 (ab) | 63.33 ± 6.5 (a) | 15 | 25 ± 5.64 (ns) |
12 | 0.78 ± 0.29 (A) | 13.33 ± 4.42 (A) | 4 | 6.67 ± 3.25 (NS) | |
42 | 3 | 3.17 ± 0.24 (a) | 48.33 ± 6.27 (b) | 22 | 36.67 ± 6.27 (ns) |
12 | 0.68 ± 0.36 (A) | 8.33 ± 3.60 (A) | 1 | 1.67 ± 1.67 (NS) |
Culture Media | Basal Salts | Growth Regulators (μM) | |||
---|---|---|---|---|---|
BAP | IBA | TDZ | NAA | ||
QL1 | QL | 4.4 | 0.5 | ||
QL2 | QL | 8.9 | 0.5 | ||
QL3 | QL | 4.4 | 0.2 | ||
QL4 | QL | 8.9 | 0.2 | ||
QL5 | QL | 4.4 | 0.1 | ||
QL6 | QL | 8.9 | 0.1 | ||
DKW1 | DKW | 4.4 | 0.5 | ||
DKW2 | DKW | 8.9 | 0.5 | ||
DKW3 | DKW | 4.4 | 0.2 | ||
DKW4 | DKW | 8.9 | 0.2 | ||
DKW5 | DKW | 4.4 | 0.1 | ||
DKW6 | DKW | 8.9 | 0.1 | ||
WPMm1 | WPMm | 4.4 | 0.5 | ||
WPMm2 | WPMm | 8.9 | 0.5 | ||
WPMm3 | WPMm | 4.4 | 0.2 | ||
WPMm4 | WPMm | 8.9 | 0.2 | ||
WPMm5 | WPMm | 4.4 | 0.1 | ||
WPMm6 | WPMm | 8.9 | 0.1 |
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Sabbadini, S.; Ricci, A.; Limera, C.; Baldoni, D.; Capriotti, L.; Mezzetti, B. Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus. Plants 2019, 8, 178. https://doi.org/10.3390/plants8060178
Sabbadini S, Ricci A, Limera C, Baldoni D, Capriotti L, Mezzetti B. Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus. Plants. 2019; 8(6):178. https://doi.org/10.3390/plants8060178
Chicago/Turabian StyleSabbadini, Silvia, Angela Ricci, Cecilia Limera, Dania Baldoni, Luca Capriotti, and Bruno Mezzetti. 2019. "Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus" Plants 8, no. 6: 178. https://doi.org/10.3390/plants8060178