植物学报 ›› 2025, Vol. 60 ›› Issue (2): 235-245.DOI: 10.11983/CBB24061 cstr: 32102.14.CBB24061
曹雪敏1,3,†, 包颖2,†, 张悦新1,3, 李瑞杰1,3, 苏健馨1,3, 张蔚1,3,*()
收稿日期:
2024-04-24
接受日期:
2024-08-20
出版日期:
2025-03-10
发布日期:
2024-08-22
通讯作者:
张蔚
作者简介:
第一联系人:†共同第一作者
基金资助:
Xuemin Cao1,3,†, Ying Bao2,†, Yuexin Zhang1,3, Ruijie Li1,3, Jianxin Su1,3, Wei Zhang1,3,*()
Received:
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
摘要: 以野蔷薇(Rosa multiflora)当年生带芽茎段为试材, 建立了其组培快繁体系。结果表明, 最佳外植体是带腋芽茎段, 外植体最佳消毒方法是先用75%乙醇浸泡30秒, 再用10%次氯酸钠溶液浸泡20分钟, 成活率可达96%; 带芽茎段萌芽最佳诱导培养基为MS+1.0 mg∙L-1 6-BA+0.01 mg∙L-1 NAA+0.1 mg∙L-1 GA3, 培养30天, 萌芽率可达98%; 无菌再生苗增殖最佳基础培养基为WPM, 增殖系数为2.87; 无菌再生苗生根最佳培养基为1/2MS+1.0 mg∙L-1 6-BA+0.1 mg∙L-1 NAA, 生根率可达93%; 无菌再生苗移栽成活率达98%。在此基础上, 以野蔷薇无菌再生苗为受体, 建立了野蔷薇瞬时表达体系。结果表明, 瞬时表达最佳转化条件是菌液OD600为0.8, 负压为-0.10 MPa, 真空抽吸2次, 每次15分钟, 瞬时表达效率可达96%。研究结果为建立野蔷薇再生及遗传转化体系奠定了基础, 并为蔷薇属植物基因功能研究提供技术支持。
曹雪敏, 包颖, 张悦新, 李瑞杰, 苏健馨, 张蔚. 野蔷薇组培快繁和高效瞬时表达体系的建立. 植物学报, 2025, 60(2): 235-245.
Xuemin Cao, Ying Bao, Yuexin Zhang, Ruijie Li, Jianxin Su, Wei Zhang. Tissue Culture, Rapid Propagation and Efficient Transient Expression Systems of Rosa multiflora. 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 |
表1 GUS染色液配方
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 |
表2 外植体消毒方案
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 |
表3 野蔷薇启动培养的处理方案
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 |
表4 野蔷薇增殖培养处理方案
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 |
表5 野蔷薇生根培养处理方案
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 |
表6 野蔷薇无菌再生苗瞬时表达实验设计
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 |
表7 不同消毒方法对野蔷薇带顶芽茎段消毒效果的影响
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 |
表8 不同消毒方法对野蔷薇带腋芽茎段消毒效果的影响
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 |
图1 野蔷薇在不同启动培养基上的生长状态 A1-A8同表3。每处理接种10瓶, 每瓶接种3个外植体, 每处理重复3次。Bars=1.5 cm
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 |
表9 不同植物生长调节剂类型及浓度对野蔷薇萌发效果的影响
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 |
图2 基础培养基类型对野蔷薇增殖系数的影响 B1-B6同表4。不同小写字母表示差异显著(P<0.05)。
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).
图3 野蔷薇在不同增殖培养基上的生长状态 B1-B6同表4。每处理接种5瓶, 每瓶接种3个外植体, 每处理重复3次。Bars=1 cm
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
图4 野蔷薇在不同生根培养基上的根部状态 C1-C8同表5。每处理接种5瓶, 每瓶接种3个外植体, 每处理重复3次。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 |
表10 不同培养基配方对野蔷薇生根的影响
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 |
图5 野蔷薇生根苗炼苗移栽过程 (A) 生根的无菌再生苗; (B) 移栽当天的植株; (C) 移栽30天后的植株。每个重复移栽40株生根苗, 重复3次。Bars=1 cm
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 |
表11 不同处理对野蔷薇瞬时表达效率的影响
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 |
图6 不同处理下野蔷薇无菌再生苗GUS染色效果 D1-D9同表6。每处理接种3瓶, 每瓶接种3个外植体, 每处理重复3次。Bars=1 cm
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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