野蔷薇组培快繁和高效瞬时表达体系的建立

doi:10.11983/CBB24061

植物学报 ›› 2025, Vol. 60 ›› Issue (2): 235-245.DOI: 10.11983/CBB24061 cstr: 32102.14.CBB24061

野蔷薇组培快繁和高效瞬时表达体系的建立

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^,^*()

¹华中农业大学园艺林学学院, 果蔬作物种质创新与利用全国重点实验室, 武汉 430070
²唐山师范学院生命科学系, 河北省植物生物技术研究与应用重点实验室, 唐山 063000
³华中农业大学花卉研究所, 武汉 430070

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:
国家自然科学基金(32072617);河北省自然科学基金(C2021105002);河北省现代农业产业技术体系建设专项(HBCT2024200405)

Tissue Culture, Rapid Propagation and Efficient Transient Expression Systems of Rosa multiflora

Xuemin Cao¹^,³^,^†, Ying Bao²^,^†, Yuexin Zhang¹^,³, Ruijie Li¹^,³, Jianxin Su¹^,³, Wei Zhang¹^,³^,^*()

¹National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture & Forestry Sciences of Huazhong Agricultural University, Wuhan 430070, China
²Hebei Key Laboratory of Plant Biotechnology Research and Application, Department of Life Science, Tangshan Normal University, Tangshan 063000, China
³Institute of Flowers Research, Huazhong Agricultural University, Wuhan 430070, China

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/Abstract

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^-1GA₃, 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^-16-BA+0.1 mg∙L^-1NAA, 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。

关键词: 野蔷薇, 快繁, 无菌再生苗, 真空渗透, 瞬时表达

Abstract: A rapid propagation system via tissue culture for Rosa multiflora was established using the stem segments with buds of the current-year as the experimental material. The results showed that the best explants were stem segments with axillary buds. The best disinfection method was to soak the explants in 75% ethanol for 30 seconds, and then soak them in 10% sodium hypochlorite solution for 20 minutes. The survival rate can reach 96%. The optimal bud-induction medium was MS+1.0 mg∙L^-1 6-BA+0.01 mg∙L^-1 NAA+0.1 mg∙L^-1 GA₃. The budding rate can reach 98% after 30 days of cultivation. WPM was the best basal medium for the proliferation of sterile regenerated plantlets, and the proliferation coefficient was 2.87. The best medium for rooting was 1/2MS+1.0 mg∙L^-1 6-BA+0.1 mg∙L^-1 NAA, and the rooting rate can reach 93%. The transplanting survival rate of sterile regenerated plantlets was 98%. On this basis, the transient expression system of R. multiflora was established. The results showed that the optimal transformation conditions for transient expression were OD₆₀₀ of 0.8 for the bacterium culture medium, vacuum negative pressure of -0.10 MPa and vacuum suction twice for 15 minutes each time. The transient expression efficiency can reach 96%. The results of this study laid a foundation for the establishment of regeneration and genetic transformation system of R. multiflora, and also provided technical support for studying on the gene function of Rosa plants.

Key words: Rosa multiflora, rapid propagation, sterile regenerated plantlets, vacuum infiltration, transient expression

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.

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https://www.chinbullbotany.com/CN/Y2025/V60/I2/235

图/表 17

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

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

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

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

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

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

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

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

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

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

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

参考文献 28

[1]	丁萌 (2012). 野蔷薇再生体系的建立及其遗传转化的研究. 硕士论文. 武汉: 华中农业大学. pp. 14-38.
[2]	龚维红 (2022). 大马士革玫瑰组培与快繁技术研究. 安徽农学通报 28, 19-21.
[3]	李刚, 宋平丽, 王翔, 马青翠, 张海霞, 张玉星, 许建锋, 亓宝秀 (2021). 农杆菌介导的杜梨叶片瞬时转化方法的建立. 果树学报 38, 2006-2013.
[4]	李惠玲, 罗玉兰, 章漳, 尹丽娟, 李圃锦, 张冬梅 (2020). 细梗蔷薇种子发芽及组织培养技术研究. 中国农学通报 36 (13), 89-93. DOI
[5]	李佳慧, 叶维雁, 朱鹏锦, 庞新华, 张继, 唐毓玮, 韦俏宇 (2022). 猫须草无菌短枝组织培养与快速繁殖体系的建立. 热带作物学报 43, 2063-2070. DOI
[6]	李静, 陈敏, 刘现伟, 沈法富, 王鹏 (2006). 莴苣高效瞬时表达体系的建立. 园艺学报 33, 405-407.
[7]	李晓亮, 张军云, 张钟, 董春富, 杨世先, 王文智, 张建康, 张翠萍 (2017). 滇红食用玫瑰生根培养基的试验筛选研究. 西南农业学报 30, 656-663.
[8]	李心悦, 张克闯, 张道远, 李进, 王玉成 (2018). 新疆野苹果瞬时遗传转化方法建立及初步验证. 分子植物育种 16, 7315-7321.
[9]	李增武, 钟玲, 赵梁军 (2011). 野蔷薇种子休眠和萌发整齐度研究. 种子 30(10), 42-44.
[10]	刘雪梅, 王蕾, 文添龙, 何鹏, 俞嘉宁 (2014). 农杆菌介导的棉花子叶瞬时表达系统的建立. 植物学报 49, 587-594. DOI
[11]	刘亚娟, 杨小艳, 谢树章, 吴红, 高立均 (2018). 芳纯月季组培快繁技术研究. 西南师范大学学报(自然科学版) 43(8),52-56.
[12]	鲁艺, 史文君, 黄淑丹, 肖明 (2022). 苦水玫瑰组培快繁技术研究. 青海大学学报 40(3), 41-46.
[13]	穆建鑫 (2021). ‘中梨一号’梨再生体系建立及秋子梨遗传转化体系探索. 硕士论文. 南京: 南京农业大学. pp. 13-24.
[14]	彭奎莉, 孟繁博, 周志达, 郑关任, 张金柱, 杨涛, 车代弟 (2018). 不同基因型丰花月季快繁体系差异的研究. 见:张启翔. 中国观赏园艺研究进展2018. 北京: 中国林业出版社. pp. 480-486.
[15]	强泽宇 (2015). 中华金叶榆繁殖技术研究. 硕士论文. 保定: 河北农业大学. pp. 6-12.
[16]	任菲宏, 王姣, 李晓松, 石丽姝, 任红艳, 吴茂宏 (2023). 外植体取样时间和消毒方式对沙子空心李诱导培养的影响. 现代农业科技 (12), 77-79.
[17]	宋常美, 文晓鹏 (2015). 4种贵州樱桃的高效离体再生. 西南大学学报(自然科学版) 37(9),19-24.
[18]	王悦, 吴艳菊, 孟大伟, 郎晨婧, 赖薪宇, 刘佳, 葛禹凡, 杨丽萍 (2020). 植物瞬时表达体系真空侵染法的优化. 分子植物育种 18, 6743-6748.
[19]	徐立军, 李志斌, 蒋淑磊, 李振勤 (2015). 大马士革玫瑰组织培养技术研究. 河北林业科技 (2), 19-21.
[20]	闫海霞, 蒋月喜, 黄昌艳, 邓杰玲, 何荆洲, 王晓国, 卜朝阳 (2016). 月季‘卡罗拉’的组培快繁技术. 热带作物学报 37, 1741-1746.
[21]	闫允青, 姜桦韬, 谷超, 吴俊 (2017). ‘雪花梨’扩繁和叶片再生体系的建立. 南京农业大学学报 40, 68-75.
[22]	杨丽萍, 许亚男, 刘雨晴, 孟大伟, 金太成 (2016). 利用根部真空侵染法在烟草中瞬时表达外源蛋白. 分子植物育种 14, 3385-3389.
[23]	张燕红, 黄乐平, 周小云, 王冬梅 (2008). 农杆菌真空渗透法转化棉花花粉的初步研究. 棉花学报 20, 354-358. DOI
[24]	赵月玲, 王汉海, 程贯召, 杜延飞 (2001). 枣组织培养中外植体取材部位的比较研究. 潍坊学院学报 1(2), 53-54, 43.
[25]	周玉洁, 韦雪芬, 申长青, 李焜钊, 孙朝辉, 黄久香 (2019). 濒危植物四药门花的组培快繁. 植物生理学报 55, 635-641.
[26]	Lu J, Bai MJ, Ren HR, Liu JY, Wang CQ (2017). An efficient transient expression system for gene function analysis in rose. Plant Methods 13, 116. DOI PMID
[27]	Nakamura N, Hirakawa H, Sato S, Otagaki S, Matsumoto S, Tabata S, Tanaka Y (2018). Genome structure of Rosa multiflora, a wild ancestor of cultivated roses. DNA Res 25, 113-121. DOI PMID
[28]	Zhang Y, Zhao MJ, Zhu W, Shi CM, Bao MZ, Zhang W (2021). Nonglandular prickle formation is associated with development and secondary metabolism-related genes in Rosa multiflora. Physiol Plant 173, 1147-1162. DOI PMID

野蔷薇组培快繁和高效瞬时表达体系的建立

Tissue Culture, Rapid Propagation and Efficient Transient Expression Systems of Rosa multiflora

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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	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

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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	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	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	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