毛华菊3种瓣型株系再生体系的建立

doi:10.11983/CBB23084

植物学报 ›› 2024, Vol. 59 ›› Issue (2): 245-256.DOI: 10.11983/CBB23084 cstr: 32102.14.CBB23084

毛华菊3种瓣型株系再生体系的建立

武晓云, 廖敏凌, 李雪茹, 舒梓淳, 辛佳潼, 张伯晗, 戴思兰^*()

北京林业大学园林学院, 城乡生态环境北京实验室, 花卉种质创新与分子育种北京市重点实验室, 国家花卉工程技术研究中心, 北京 100083

收稿日期:2023-06-25 接受日期:2023-09-19 出版日期:2024-03-10 发布日期:2024-03-10
通讯作者: * 戴思兰, 北京林业大学园林学院教授、博士生导师, 享受国务院政府特殊津贴。现任国家林业和草原局菊花产业国家创新联盟理事长, 中国风景园林学会菊花分会副理事长, 《植物学报》责任编委。曾获首届全国林业教学名师、宝钢优秀教师奖、中国观赏园艺特别荣誉奖、教育部自然科学二等奖等荣誉奖励。先后主持30余项国家和省部级科研项目, 发表学术论文380余篇, 出版著作2部。其研究团队以菊花为主要研究材料, 从菊花的历史文化、品种资源收集、整理和评价到花色、花型、开花期和抗逆性等观赏品质形成的遗传调控机理, 以及菊花优异新种质创制、产业化栽培技术等进行全面研究, 取得了一系列重要突破性进展和研究成果。E-mail: silandai@sina.com
基金资助:
国家自然科学基金(32371948);国家自然科学基金(31530064);国家重点研发计划(2018YFD1000405)

Establishment of Regeneration System of Chrysanthemum vestitum with Three Floret Forms

Xiaoyun Wu, Minling Liao, Xueru Li, Zichun Shu, Jiatong Xin, Bohan Zhang, Silan Dai^*()

National Engineering Research Center for Floriculture, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing Laboratory of Urban and Rural Ecological Environment, School of Landscape Architecture, Beijing Forestry University, Beijing 100083, China

Received:2023-06-25 Accepted:2023-09-19 Online:2024-03-10 Published:2024-03-10
Contact: * E-mail: silandai@sina.com

摘要/Abstract

摘要： 毛华菊(Chrysanthemum vestitum)是栽培菊花(C. × morifolium)的近缘六倍体野生种之一, 其自然群体中的舌状花与栽培菊花一样具有典型的平瓣型、管瓣型和混合瓣型变异, 是研究菊属植物瓣型变异的理想材料。其舌状花发育过程受生长素和花器官发育关键差异基因的影响, 但目前缺乏稳定高效的不同瓣型毛华菊株系的再生体系, 制约了毛华菊瓣型相关基因的研究。利用在河南省伏牛山收集的3种瓣型毛华菊株系, 以叶片和茎间薄层为外植体建立再生体系。结果表明, 以平瓣型叶片为外植体, 其愈伤组织诱导和不定芽分化最佳培养基为MS+1 mg∙L^-1NAA+2 mg∙L^-16-BA, 接种20天愈伤组织诱导率为100%, 不定芽分化率达100%; 最佳生根培养基为1/2MS+0.2 mg∙L^-1NAA, 生根率达100%。平瓣型毛华菊的叶片最佳再生体系也适用于管瓣型和混合瓣型株系, 不定芽分化率分别为83.46%和91.67%, 生根率均为100%。移栽后对开花植株进行观察, 发现利用叶片再生体系获得的3种不同瓣型再生植株花型稳定, 为后续利用不同瓣型株系解析舌状花形态变异机理提供了技术方法。

关键词: 毛华菊, 不同瓣型, 叶片, 再生体系, 茎间薄层

Abstract: Chrysanthemum vestitum, a closely related hexaploid wild species of cultivated C. × morifolium has the same and typical flat, tubular and mixed floret forms in its natural population as cultivated chrysanthemum and is an ideal material for studying the petal variation in chrysanthemum. The development progress of this ray floret is affected by key differential genes such as auxin and floral organ development, but the lack of stable and efficient regeneration systems for different floret forms has restricted the study of petal type-related genes in C. vestitum. In this paper, the authors used three floret forms of C. vestitum collected from Funiu Mountain in Henan Province to establish regeneration systems using leaves and transverse thin cell layers (tTCLs) as explants. The results showed that the optimal media for callus induction and adventitious bud differentiation were MS+1 mg∙L^-1NAA+2 mg∙L^-16-BA with flat floret leaves as explants, and the callus induction rate was 100% at 20 days after inoculation. The differentiation rate of adventitious buds was 100%. The optimal rooting medium was 1/2MS+0.2 mg∙L^-1NAA, and the rooting rate of regenerated plants was 100%. The optimal leaf regeneration system for the flat floret strain was also suitable for determining both the tubular and mixed floret strains. The differentiation rates of adventitious buds were 83.46% and 91.67%, respectively, and the rooting rates were 100%. The flowering plants were observed after transplanting, and it was found that the flower types of the regenerated plants with three different floret forms obtained by the leaf regeneration system were stable, which provided a technical method for the subsequent analysis of the morphological variation mechanism of ray florets by using different floret forms of C. vestitum.

Key words: Chrysanthemum vestitum, different floret forms, leaf, regeneration system, transverse thin cell layer

武晓云, 廖敏凌, 李雪茹, 舒梓淳, 辛佳潼, 张伯晗, 戴思兰. 毛华菊3种瓣型株系再生体系的建立. 植物学报, 2024, 59(2): 245-256.

Xiaoyun Wu, Minling Liao, Xueru Li, Zichun Shu, Jiatong Xin, Bohan Zhang, Silan Dai. Establishment of Regeneration System of Chrysanthemum vestitum with Three Floret Forms. Chinese Bulletin of Botany, 2024, 59(2): 245-256.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB23084

https://www.chinbullbotany.com/CN/Y2024/V59/I2/245

图/表 13

图1 毛华菊3种瓣型野生型株系的头状花序 (A) 平瓣型头状花序; (B) 管瓣型头状花序; (C) 混合瓣型头状花序。Bars=1 cm

Figure 1 Capitula of wild-type strains of Chrysanthemum vestitum with three floret forms (A) Capitulum with a flat floret form; (B) Capitulum with a tubular floret form; (C) Capitulum with a mixed floret form. Bars=1 cm

表1 毛华菊平瓣型株系叶片愈伤组织诱导和不定芽分化培养基配方

Table 1 Culture medium settings for callus induction and adventitious buds differentiation of leaves of Chrysanthemum vestitum with flat floret form

No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
A1	0.5	1
A2	0.5	2
A3	0.5	3
A4	1	1
A5	1	2
A6	1	3
A7	2	1
A8	2	2
A9	2	3

表2 毛华菊平瓣型株系茎间薄层愈伤组织诱导和不定芽分化培养基配方

Table 2 Culture medium settings for callus induction and adventitious buds differentiation in the transverse thin cell layer of Chrysanthemum vestitum with flat floret form

No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2

表3 生根培养基配方

Table 3 Rooting medium settings

No.	Basal medium	NAA (mg∙L^-1)
R1	1/2MS	0
R2	1/2MS	0.2
R3	1/2MS	0.5

表4 不同培养基配方对毛华菊平瓣型株系叶片愈伤组织诱导和不定芽分化的影响

Table 4 Effects of different medium settings on callus induction and adventitious bud differentiation of leaves of Chrysanthemum vestitum with flat floret form

Treatments	Callus formation rate (%)	Differentiation rate (%)	Average reproduction coefficient
A1	100.00±0 a	62.50±1.29 b	3.24±0.94 b
A2	100.00±0 a	72.92±1.67 ab	3.82±1.29 a
A3	100.00±0 a	91.37±2.31 a	2.05±1.67 cd
A4	100.00±0 a	89.58±2.05 ab	4.08±1.26 a
A5	100.00±0 a	91.67±2.87 a	4.34±2.34 a
A6	100.00±0 a	75.00±0.96 ab	2.40±2.16 bc
A7	97.92±0.37 a	87.50±1.12 ab	2.31±1.53 d
A8	97.92±0.37 a	77.08±2.11 ab	3.04±1.21 cd
A9	100.00±0 a	89.58±1.77 ab	3.61±1.46 bc

表5 不同培养基配方对毛华菊平瓣型株系茎间薄层愈伤组织诱导和不定芽分化的影响

Table 5 Effects of different medium settings on callus induction and adventitious bud differentiation in transverse thin cell layers of Chrysanthemum vestitum with flat floret form

Treatments	Callus formation rate (%)	Differentiation rate (%)
M1	80.00±7.12 b	0.28±0.06 b
M2	100.00±0.00 a	0.49±0.08 a
M3	87.33±2.62 b	0.31±0.70 b
M4	83.76±2.47 b	0.15±0.40 bd
M5	64.80±1.47 bc	0.09±0.70 bc
M6	77.93±3.93 b	0.20±0.08 b
M7	72.67±9.53 bc	0.19±0.02 bc
M8	81.76±2.68 b	0.21±0.09 b
M9	70.53±3.45 bc	0.27±0.05 bc
M10	78.76±2.78 b	0.27±0.05 b
M11	78.76±3.21 b	0.32±0.17 b
M12	90.60±2.30 ab	0.37±0.13 b
M13	53.06±9.69 bcd	0.02±0.03 bcd
M14	73.87±5.53 b	0.03±0.05 bcd
M15	50.33±5.44 bcd	0.07±0.10 bcd
M16	75.77±5.86 b	0.03±0.05 bc

图2 毛华菊平瓣型株系叶片再生过程从左至右分别为刚接入分化培养基的叶片(0天)、切口边缘膨大的叶片(7天)、叶片诱导的愈伤组织(15天)、不定芽芽点(30天)、长至0.5 cm的不定芽(45天)及接入生根培养基10天的不定芽。Bar=1 cm

Figure 2 The flat floret regeneration phase for leaves in Chrysanthemum vestitum From left to right are leaves that had just been added to the differentiation medium (0 d), leaf blades with swollen edges (7 d), leaf-induced calli (15 d), adventitious points (30 d), adventitious buds up to 0.5 cm (45 d), and adventitious buds inserted into the rooting medium for 10 days, respectively. Bar=1 cm

图3 毛华菊平瓣型株系茎间薄层与叶片的再生状况 (A)-(D) 茎间薄层在分化培养基中培养7、15、20和35天的分化情况; (E)-(G) 叶片在分化培养基中培养7、15和20天的分化情况; (H) 移入生根培养基15天后叶片再生芽的情况。Bars=1 cm

Figure 3 Regeneration of transverse thin cell layers (tTCLs) and leaves of Chrysanthemum vestitum with a flat floret (A)-(D) Regeneration system of tTCLs in differentiation media for 7, 15, 20, and 35 days; (E)-(G) Regeneration system of leaves in differentiation media for 7, 15, and 20 days; (H) Plant status of regeneration buds from leaves after 15 days transplanting into a rooting medium. Bars=1 cm

表6 毛华菊平瓣型株系在不同生根培养基中的生根和植株生长情况

Table 6 Rooting and plant status of Chrysanthemum vestitum with a flat floret in different rooting medium

No.	Rooting rate (%)	Number of roots	Average root length (cm)	Average plant height (cm)
R1	79.17	2.21±1.15 a	4.11±2.16 b	3.05±0.72 a
R2	100.00	1.96±1.54 a	7.65±3.24 a	3.16±1.01 a
R3	95.83	2.04±2.26 a	4.37±1.80 b	2.60±1.26 b

图4 毛华菊平瓣型株系再生苗在R1 (A)、R2 (B)和R3 (C)培养基中培养15天的生根情况 R1-R3同表3。Bars=1 cm

Figure 4 Rooting status of regenerated seedlings from Chrysanthemum vestitum with a flat floret in R1 (A), R2 (B), and R3 (C) media for 15 days R1-R3 are the same as shown in Table 3. Bars=1 cm

表7 毛华菊3种瓣型株系叶片再生体系

Table 7 Leaf regeneration systems of Chrysanthemum vesititum with three floret forms

Floret form	Callus formation rate (%)	Differentiation rate (%)	Average reproduction coefficient	Rooting rate (%)
Flat	100.00±0.00	100.00±2.05	3.59±2.46	100.00
Tubular	100.00±0.00	83.46±6.59	1.75±0.25	100.00
Mix	100.00±0.00	91.67±2.87	4.34±2.34	100.00

图5 毛华菊管瓣型和混合瓣型株系叶片再生过程 (A) 管瓣型株系叶片再生过程; (B) 混合瓣型株系叶片再生过程。从左至右分别为刚接入分化培养基的叶片(0天)、切口边缘膨大的叶片(7天)、叶片诱导的愈伤组织(15天)、不定芽芽点(30天)、长至0.5 cm的不定芽(45天)和接入生根培养基10天的不定芽。Bars=1 cm

Figure 5 The regeneration phase of leaf of Chrysanthemum vesititum in the presence of tubular and mixed floret forms (A) The regeneration phase of leaves by tubular floret forms; (B) The regeneration phase of leaves by mixed floret forms. From left to right are leaves that had just been added to the differentiation medium (0 d), leaf blades with swollen edges (7 d), leaf-induced calli (15 d), adventitious points (30 d), adventitious buds up to 0.5 cm (45 d), and adventitious buds inserted into the rooting medium for 10 days, respectively. Bars=1 cm

图6 毛华菊3种瓣型野生型株系与再生植株的头状花序 (A1)-(A3) 平瓣型、管瓣型和混合瓣型野生型株系的花序; (B1)-(B3) 平瓣型、管瓣型和混合瓣型再生植株的花序。Bars=1 cm

Figure 6 Capitula of wild-type plants and regenerated plants of Chrysanthemum vestitum with three floret forms (A1)-(A3) Capitula of wild-type strains with flat, tubular and mixed floret forms; (B1)-(B3) Capitula of regenerated plants with flat, tubular and mixed floret forms. Bars=1 cm

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毛华菊3种瓣型株系再生体系的建立

Establishment of Regeneration System of Chrysanthemum vestitum with Three Floret Forms

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No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2

No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2

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No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2

No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2

No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2

No.	NAA (mg∙L^-1)	6-BA (mg∙L^-1)
M1	0.1	0.1
M2	0.5	0.1
M3	1	0.1
M4	2	0.1
M5	0.1	0.5
M6	0.5	0.5
M7	1	0.5
M8	2	0.5
M9	0.1	1
M10	0.5	1
M11	1	1
M12	2	1
M13	0.1	2
M14	0.5	2
M15	1	2
M16	2	2