Chinese Bulletin of Botany ›› 2025, Vol. 60 ›› Issue (2): 235-245.DOI: 10.11983/CBB24061 cstr: 32102.14.CBB24061
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Xuemin Cao1,3,†, Ying Bao2,†, Yuexin Zhang1,3, Ruijie Li1,3, Jianxin Su1,3, Wei Zhang1,3,*(
)
HomeReceived:2024-04-24
Accepted:2024-08-20
Online:2025-03-10
Published:2024-08-22
Contact:
Wei Zhang
About author:First author contact:†These authors contributed equally to this paper
Xuemin Cao, Ying Bao, Yuexin Zhang, Ruijie Li, Jianxin Su, Wei Zhang. Tissue Culture, Rapid Propagation and Efficient Transient Expression Systems of Rosa multiflora[J]. Chinese Bulletin of Botany, 2025, 60(2): 235-245.
| Reagents | Required solvent dosage |
|---|---|
| 0.2 mol∙L-1 NaH2PO4 | 38 mL |
| 0.2 mol∙L-1 Na2HPO4 | 62 mL |
| 100 mmol∙L-1 K3Fe(CN)6 | 1 mL |
| 100 mmol∙L-1 K4Fe(CN)6 | 1 mL |
| 0.5 mol∙L-1 NaEDTA | 4 mL |
| 0.1% Triton X-100 | 200 μL |
| 20% methanol | 20 mL |
| X-Gluc | 200 mg |
Table 1 GUS staining solution formulation
| Reagents | Required solvent dosage |
|---|---|
| 0.2 mol∙L-1 NaH2PO4 | 38 mL |
| 0.2 mol∙L-1 Na2HPO4 | 62 mL |
| 100 mmol∙L-1 K3Fe(CN)6 | 1 mL |
| 100 mmol∙L-1 K4Fe(CN)6 | 1 mL |
| 0.5 mol∙L-1 NaEDTA | 4 mL |
| 0.1% Triton X-100 | 200 μL |
| 20% methanol | 20 mL |
| X-Gluc | 200 mg |
| Treatments | 75% ethanol immersion time (s) | NaClO (%) | NaClO immersion time (min) |
|---|---|---|---|
| A | 30 | 5 | 10 |
| B | 30 | 5 | 15 |
| C | 30 | 5 | 20 |
| D | 30 | 10 | 10 |
| E | 30 | 10 | 15 |
| F | 30 | 10 | 20 |
| G | 30 | 15 | 10 |
| H | 30 | 15 | 15 |
| I | 30 | 15 | 20 |
Table 2 Explant disinfection scheme
| Treatments | 75% ethanol immersion time (s) | NaClO (%) | NaClO immersion time (min) |
|---|---|---|---|
| A | 30 | 5 | 10 |
| B | 30 | 5 | 15 |
| C | 30 | 5 | 20 |
| D | 30 | 10 | 10 |
| E | 30 | 10 | 15 |
| F | 30 | 10 | 20 |
| G | 30 | 15 | 10 |
| H | 30 | 15 | 15 |
| I | 30 | 15 | 20 |
| Treatments | Basic medium | 6-BA (mg∙L-1) | NAA (mg∙L-1) | GA3 (mg∙L-1) |
|---|---|---|---|---|
| A1 | MS | 0.5 | 0.01 | 0 |
| A2 | MS | 1.0 | 0.01 | 0 |
| A3 | MS | 0.5 | 0.1 | 0 |
| A4 | MS | 1.0 | 0.1 | 0 |
| A5 | MS | 0.5 | 0.01 | 0.1 |
| A6 | MS | 1.0 | 0.01 | 0.1 |
| A7 | MS | 0.5 | 0.1 | 0.1 |
| A8 | MS | 1.0 | 0.1 | 0.1 |
Table 3 Treatment schemes for the initiation culture of Rosa multiflora
| Treatments | Basic medium | 6-BA (mg∙L-1) | NAA (mg∙L-1) | GA3 (mg∙L-1) |
|---|---|---|---|---|
| A1 | MS | 0.5 | 0.01 | 0 |
| A2 | MS | 1.0 | 0.01 | 0 |
| A3 | MS | 0.5 | 0.1 | 0 |
| A4 | MS | 1.0 | 0.1 | 0 |
| A5 | MS | 0.5 | 0.01 | 0.1 |
| A6 | MS | 1.0 | 0.01 | 0.1 |
| A7 | MS | 0.5 | 0.1 | 0.1 |
| A8 | MS | 1.0 | 0.1 | 0.1 |
| Treatments | Basic medium | Sucrose (%) | Agar (%) |
|---|---|---|---|
| B1 | MS | 3 | 0.75 |
| B2 | 1/2MS | 3 | 0.75 |
| B3 | QL | 3 | 0.75 |
| B4 | 1/2QL | 3 | 0.75 |
| B5 | WPM | 3 | 0.75 |
| B6 | B5 | 3 | 0.75 |
Table 4 Treatment schemes for the propagation culture of Rosa multiflora
| Treatments | Basic medium | Sucrose (%) | Agar (%) |
|---|---|---|---|
| B1 | MS | 3 | 0.75 |
| B2 | 1/2MS | 3 | 0.75 |
| B3 | QL | 3 | 0.75 |
| B4 | 1/2QL | 3 | 0.75 |
| B5 | WPM | 3 | 0.75 |
| B6 | B5 | 3 | 0.75 |
| Treatments | Basic medium | NAA (mg∙L-1) | IBA (mg∙L-1) |
|---|---|---|---|
| C1 | MS | 0.05 | 0 |
| C2 | MS | 0.1 | 0 |
| C3 | 1/2MS | 0.05 | 0 |
| C4 | 1/2MS | 0.1 | 0 |
| C5 | MS | 0 | 0.5 |
| C6 | MS | 0 | 1.0 |
| C7 | 1/2MS | 0 | 0.5 |
| C8 | 1/2MS | 0 | 1.0 |
Table 5 Treatment schemes for the rooting culture of Rosa multiflora
| Treatments | Basic medium | NAA (mg∙L-1) | IBA (mg∙L-1) |
|---|---|---|---|
| C1 | MS | 0.05 | 0 |
| C2 | MS | 0.1 | 0 |
| C3 | 1/2MS | 0.05 | 0 |
| C4 | 1/2MS | 0.1 | 0 |
| C5 | MS | 0 | 0.5 |
| C6 | MS | 0 | 1.0 |
| C7 | 1/2MS | 0 | 0.5 |
| C8 | 1/2MS | 0 | 1.0 |
| Treatments | Bacterium OD600 | Vacuum pressure (MPa) | Vacuum processing time (min) |
|---|---|---|---|
| D1 | 0.6 | -0.06 | 5 |
| D2 | 0.8 | -0.08 | 5 |
| D3 | 1.0 | -0.10 | 5 |
| D4 | 1.0 | -0.08 | 10 |
| D5 | 0.8 | -0.06 | 10 |
| D6 | 0.6 | -0.10 | 10 |
| D7 | 0.6 | -0.08 | 15 |
| D8 | 0.8 | -0.10 | 15 |
| D9 | 1.0 | -0.06 | 15 |
Table 6 Experimental design for transient expression of sterile re-generated plantlets of Rosa multiflora
| Treatments | Bacterium OD600 | Vacuum pressure (MPa) | Vacuum processing time (min) |
|---|---|---|---|
| D1 | 0.6 | -0.06 | 5 |
| D2 | 0.8 | -0.08 | 5 |
| D3 | 1.0 | -0.10 | 5 |
| D4 | 1.0 | -0.08 | 10 |
| D5 | 0.8 | -0.06 | 10 |
| D6 | 0.6 | -0.10 | 10 |
| D7 | 0.6 | -0.08 | 15 |
| D8 | 0.8 | -0.10 | 15 |
| D9 | 1.0 | -0.06 | 15 |
| Treatments | Contamination rate (%) | Browning rate (%) | Survival rate (%) |
|---|---|---|---|
| A | 11.11±0.00 a | 59.26±6.42 b | 29.60±6.42 a |
| B | 0.00±0.00 b | 92.59±6.42 a | 7.41±6.42 b |
| C | 7.41±6.42 ab | 100.00±0.00 a | 0.00±0.00 b |
| D | 0.00±0.00 b | 92.59±6.42 a | 7.41±6.42 b |
| E | 3.70±6.42 b | 100.00±0.00 a | 0.00±0.00 b |
| F | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| G | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| H | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| I | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
Table 7 Effects of different disinfection methods on the stem segments with apical buds of Rosa multiflora
| Treatments | Contamination rate (%) | Browning rate (%) | Survival rate (%) |
|---|---|---|---|
| A | 11.11±0.00 a | 59.26±6.42 b | 29.60±6.42 a |
| B | 0.00±0.00 b | 92.59±6.42 a | 7.41±6.42 b |
| C | 7.41±6.42 ab | 100.00±0.00 a | 0.00±0.00 b |
| D | 0.00±0.00 b | 92.59±6.42 a | 7.41±6.42 b |
| E | 3.70±6.42 b | 100.00±0.00 a | 0.00±0.00 b |
| F | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| G | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| H | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| I | 0.00±0.00 b | 100.00±0.00 a | 0.00±0.00 b |
| Treatments | Contamination rate (%) | Browning rate (%) | Survival rate (%) |
|---|---|---|---|
| A | 3.70±6.42 a | 18.52±6.42 ab | 77.78±11.11 a |
| B | 7.41±6.42 a | 25.93±6.42 a | 66.67±0.00 a |
| C | 3.70±6.42 a | 14.81±6.42 ab | 81.48±6.42 a |
| D | 7.41±6.42 a | 11.11±0.00 b | 81.48±6.42 a |
| E | 14.81±16.97 a | 14.81±6.42 ab | 70.37±16.97 a |
| F | 3.70±6.42 a | 0.00±0.00 c | 96.30±6.42 a |
| G | 11.11±11.11 a | 14.81±6.42 ab | 74.07±16.97 a |
| H | 0.00±0.00 a | 25.93±6.42 a | 74.07±6.42 a |
| I | 18.52±12.83 a | 11.11±0.00 b | 70.37±12.83 a |
Table 8 Effects of different disinfection methods on the stem segments with axillary buds of Rosa multiflora
| Treatments | Contamination rate (%) | Browning rate (%) | Survival rate (%) |
|---|---|---|---|
| A | 3.70±6.42 a | 18.52±6.42 ab | 77.78±11.11 a |
| B | 7.41±6.42 a | 25.93±6.42 a | 66.67±0.00 a |
| C | 3.70±6.42 a | 14.81±6.42 ab | 81.48±6.42 a |
| D | 7.41±6.42 a | 11.11±0.00 b | 81.48±6.42 a |
| E | 14.81±16.97 a | 14.81±6.42 ab | 70.37±16.97 a |
| F | 3.70±6.42 a | 0.00±0.00 c | 96.30±6.42 a |
| G | 11.11±11.11 a | 14.81±6.42 ab | 74.07±16.97 a |
| H | 0.00±0.00 a | 25.93±6.42 a | 74.07±6.42 a |
| I | 18.52±12.83 a | 11.11±0.00 b | 70.37±12.83 a |
Figure 1 Growth condition of Rosa multiflora on different initiation media A1-A8 are the same as shown in Table 3. Each treatment is inoculated with 10 vials, each vial is inoculated with 3 explants, and each treatment is repeated 3 times. Bars=1.5 cm
| Treatments | Germination rate (%) | Plant height (cm) | Number of compound leaves (slice) |
|---|---|---|---|
| A1 | 92.22±7.70 a | 1.30±0.50 bc | 4.37±1.39 cd |
| A2 | 97.78±1.92 a | 1.52±0.59 a | 4.94±1.72 abc |
| A3 | 96.67±3.34 a | 1.20±0.59 bc | 4.45±1.46 cd |
| A4 | 98.89±1.92 a | 1.25±0.50 bc | 5.17±1.41 a |
| A5 | 98.89±1.92 a | 1.11±0.39 c | 4.74±1.64 abcd |
| A6 | 98.89±1.92 a | 1.35±0.45 b | 5.07±1.61 ab |
| A7 | 97.78±1.92 a | 1.15±0.48 bc | 4.17±1.71 d |
| A8 | 96.67±5.77 a | 1.25±0.41 bc | 4.69±1.77 abcd |
Table 9 Effects of different plant growth regulator types and concentrations on the germination of Rosa multiflora
| Treatments | Germination rate (%) | Plant height (cm) | Number of compound leaves (slice) |
|---|---|---|---|
| A1 | 92.22±7.70 a | 1.30±0.50 bc | 4.37±1.39 cd |
| A2 | 97.78±1.92 a | 1.52±0.59 a | 4.94±1.72 abc |
| A3 | 96.67±3.34 a | 1.20±0.59 bc | 4.45±1.46 cd |
| A4 | 98.89±1.92 a | 1.25±0.50 bc | 5.17±1.41 a |
| A5 | 98.89±1.92 a | 1.11±0.39 c | 4.74±1.64 abcd |
| A6 | 98.89±1.92 a | 1.35±0.45 b | 5.07±1.61 ab |
| A7 | 97.78±1.92 a | 1.15±0.48 bc | 4.17±1.71 d |
| A8 | 96.67±5.77 a | 1.25±0.41 bc | 4.69±1.77 abcd |
Figure 2 Effects of basal medium types on the proliferation coefficient of Rosa multiflora B1-B6 are the same as shown in Table 4. Different lowercase letters indicate significant differences (P<0.05).
Figure 3 Growth condition of Rosa multiflora on different proliferation media B1-B6 are the same as shown in Table 4. Each treatment is inoculated with 5 vials, each vial is inoculated with 3 explants, and each treatment is repeated 3 times. Bars=1 cm
Figure 4 Root condition of Rosa multiflora on different rooting media C1-C8 are the same as shown in Table 5. Each treatment is inoculated with 5 vials, each vial is inoculated with 3 explants, and each treatment is repeated 3 times. Bars=1 cm
| Treatments | Rooting rate (%) | Average number of roots (root) | Root status |
|---|---|---|---|
| C1 | 88.33±2.89 ab | 8.22 | Short roots, weak growth |
| C2 | 88.33±2.89 ab | 9.30 | Strong roots, grow well |
| C3 | 93.33±7.64 ab | 9.78 | Short roots, weak growth |
| C4 | 93.33±2.89 a | 8.85 | Strong roots, grow well |
| C5 | 81.67±5.77 ab | 8.25 | Short roots, weak growth |
| C6 | 81.67±2.89 ab | 8.47 | Strong roots, grow well |
| C7 | 78.33±5.77 b | 6.68 | Short roots, weak growth |
| C8 | 86.67±5.77 ab | 7.18 | Short roots, weak growth |
Table 10 Effects of different media formulations on the rooting of Rosa multiflora
| Treatments | Rooting rate (%) | Average number of roots (root) | Root status |
|---|---|---|---|
| C1 | 88.33±2.89 ab | 8.22 | Short roots, weak growth |
| C2 | 88.33±2.89 ab | 9.30 | Strong roots, grow well |
| C3 | 93.33±7.64 ab | 9.78 | Short roots, weak growth |
| C4 | 93.33±2.89 a | 8.85 | Strong roots, grow well |
| C5 | 81.67±5.77 ab | 8.25 | Short roots, weak growth |
| C6 | 81.67±2.89 ab | 8.47 | Strong roots, grow well |
| C7 | 78.33±5.77 b | 6.68 | Short roots, weak growth |
| C8 | 86.67±5.77 ab | 7.18 | Short roots, weak growth |
Figure 5 The transplanting process of rooted plantlets of Rosa multiflora (A) Rooted sterile regenerated plantlets; (B) Plantlets on the first day of transplanting; (C) Plantlets transplanted 30 days later. Transplant 40 rooted plantlets per replicate and repeat 3 times. Bars=1 cm
| Treatments | Transient expression efficiency (%) | Staining effect |
|---|---|---|
| D1 | 14.07±12.24 d | Pale blue, flake area blue |
| D2 | 30.63±2.44 cd | Pale blue, flake area blue |
| D3 | 65.27±17.71 b | Blue, flake area blue |
| D4 | 52.38±10.82 bc | Pale blue, leaf blue |
| D5 | 15.87±5.72 d | Blue, flake area blue |
| D6 | 52.89±17.16 bc | Blue, leaf blue |
| D7 | 4.76±8.25 d | Blue, leaf blue |
| D8 | 96.30±6.42 a | Blue, leaf blue |
| D9 | 31.19±7.84 cd | Blue, flake area blue |
Table 11 Effects of different treatments on the transient expression efficiency of Rosa multiflora
| Treatments | Transient expression efficiency (%) | Staining effect |
|---|---|---|
| D1 | 14.07±12.24 d | Pale blue, flake area blue |
| D2 | 30.63±2.44 cd | Pale blue, flake area blue |
| D3 | 65.27±17.71 b | Blue, flake area blue |
| D4 | 52.38±10.82 bc | Pale blue, leaf blue |
| D5 | 15.87±5.72 d | Blue, flake area blue |
| D6 | 52.89±17.16 bc | Blue, leaf blue |
| D7 | 4.76±8.25 d | Blue, leaf blue |
| D8 | 96.30±6.42 a | Blue, leaf blue |
| D9 | 31.19±7.84 cd | Blue, flake area blue |
Figure 6 Effects of different treatments on GUS staining of sterile regenerated plantlets of Rosa multiflora D1-D9 are the same as shown in Table 6. Each treatment is inoculated with 3 vials, each vial is inoculated with 3 explants, and each treatment is repeated 3 times. Bars=1 cm
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