芳香堆心菊离体再生体系的建立

doi:10.11983/CBB19211

摘要/Abstract

摘要： 芳香堆心菊(Helenium aromaticum)全株具芳香气味, 且头状花序仅含管状花, 是研究菊科植物花香和花型的良好材料, 但目前尚缺乏对其转基因技术体系的研究。为建立高效的芳香堆心菊离体再生体系, 以叶片、茎段和下胚轴为外植体, 进行25组不同激素及不同浓度配比的不定芽诱导研究。结果表明, 以芳香堆心菊叶片为外植体, 培养基为MS+ 0.2 mg·L^-1 NAA+1 mg·L^-1 6-BA+0.2 mg·L^-1 TDZ, 培养20天后愈伤组织诱导率高达100%, 丛生芽的诱导率为62.10%; 将不定芽接种于1/2MS培养基中进行生根培养, 16天即可生根, 且生根率为63.33%; 生根后继续培养14天现蕾, 开花率达93.33%。此外, 研究表明芳香堆心菊的再生受外植体来源、激素种类和浓度的影响。2,4-D不利于芳香堆心菊不定芽的诱导, 适宜浓度的6-BA和TDZ组合能有效促进芳香堆心菊不定芽的形成。研究初步建立了芳香堆心菊组织培养条件下的离体再生体系, 为建立其遗传转化体系奠定了坚实的基础。研究结果还可用于后续有关菊科植物花香和花型的研究。

关键词: 芳香堆心菊, 再生体系, 组织培养

Abstract: Helenium aromaticum has a fragrant odor and its capitulum contains only disk flowers. H. aromaticum is a good model for studying development of floral patterns in Compositae. However, methods for genetic transformation of H. aromaticum is currently not available. In this study, we report the establishment of an efficient in vitro regeneration system of the H. aromaticum. The induction of adventitious buds was tested with various combinations and concentrations of 25 different phytohormones by using leaves, stem segments, and hypocotyls as explants. We found that when leaf explants were cultured on MS medium supplemented with 0.2 mg·L^-1 NAA, 1 mg·L^-1 6-BA, and 0.2 mg·L^-1 TDZ for 20 days, the induction of callus was 100% and the induction rate of adventitious buds was 62.10%. The newly-formed adventitious buds were then cultured on 1/2MS medium for 16 days and the rooting rate reached 63.33%. After culturing for an additional 14 days, floral buds initiated and eventually flowering with a rate of 93.33%. In addition, we also found that the regeneration of H. aromaticum was affected by the nature of explants, the types and concentrations of phytohormones. Whereas 2,4-D is incapable of inducing adventitious buds, the combination of appropriate concentrations of 6-BA and TDZ effectively promotes the formation of adventitious buds of H. aromaticum. This study has established an in vitro regeneration system of H. aromaticum, which is a key perquisite for the subsequent establishment of its genetic transformation system. Moreover, this method will also be an important reference for studies on the floral development patterns in Compositae.

Key words: Helenium aromaticum, regeneration system, tissue culture

罗虹, 温小蕙, 周圆圆, 戴思兰. 芳香堆心菊离体再生体系的建立. 植物学报, 2020, 55(3): 318-328.

Hong Luo, Xiaohui Wen, Yuanyuan Zhou, Silan Dai. Establishment of In Vitro Regeneration System of Helenium aromaticum. Chinese Bulletin of Botany, 2020, 55(3): 318-328.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19211

https://www.chinbullbotany.com/CN/Y2020/V55/I3/318

图/表 10

表1 芳香堆心菊愈伤组织诱导及不定芽分化培养基

Table 1 Medium for callus induction and adventitious buds differentiation of Helenium aromaticum

Treatments	Plant growth regulators (mg·L^-1)
Treatments	NAA	6-BA	2,4-D	TDZ
A1	0.5	1	0	0
A2	0.5	2	1	0
A3	0.5	3	2	0
A4	1	1	1	0
A5	1	2	2	0
A6	1	3	0	0
A7	2	1	2	0
A8	2	2	0	0
A9	2	3	1	0
B1	0.2	1	0	0
B2	1	1	0	0
B3	0.2	2	0	0
B4	0.5	2	0	0
B5	1	2	0	0
B6	0.2	3	0	0
B7	0.5	3	0	0
C1	0.1	0.5	0	0.2
C2	0.2	1	0	0.2
C3	0.5	1.5	0	0.2
C4	0.2	0.5	0	0.5
C5	0.5	1	0	0.5
C6	0.1	1.5	0	0.5
C7	0.5	0.5	0	1
C8	0.1	1	0	1
C9	0.2	1.5	0	1

表2 芳香堆心菊生根培养基

Table 2 Medium for root culture of Helenium aromaticum

Treatments	Plant growth regulators (mg·L^-1)
Treatments	NAA
R0	0
R1	0.1
R2	0.2
R3	0.3

图1 芳香堆心菊整株及花器官形态 (A) 整株(Bar=2 cm); (B) 头状花序(Bar=2 mm); (C) 单个小花(Bar=1 mm)。PE: 花瓣; OV: 子房; PI: 雌蕊; GT: 腺毛

Figure 1 The whole plant and flower morphology of Helenium aromaticum (A) The whole plant (Bar=2 cm); (B) The capitulum (Bar=2 mm); (C) Single flower (Bar=1 mm). PE: Petal; OV: Ovary; PI: Pistil; GT: Glandular trichomes

图2 芳香堆心菊叶片愈伤组织诱导及不定芽分化的阶段(C2) (A) 叶片外植体; (B) 愈伤组织(20天, 箭头所示); (C) 不定芽(30天, 箭头所示)。Bars=1 cm。C2同表1。

Figure 2 Several stages of callus induction and adventitious bud differentiation of Helenium aromaticum leaves (C2) (A) Leaf explants; (B) Calli (20 d, shown by arrow); (C) Adventitious buds (30 d, shown by arrow). Bars=1 cm. C2 is the same as Table 1.

图3 芳香堆心菊茎段愈伤组织诱导及不定芽分化的阶段(B1) (A) 茎段外植体; (B) 愈伤组织(17天, 箭头所示); (C) 不定芽(65天, 箭头所示)。Bars=1 cm。B1同表1。

Figure 3 Several stages of callus induction and adventitious bud differentiation of Helenium aromaticum stem fragments (B1) (A) Stem segment explants; (B) Calli (17 d, shown by arrow); (C) Adventitious buds (65 d, shown by arrow). Bars=1 cm. B1 is the same as Table 1.

图4 芳香堆心菊下胚轴愈伤组织诱导的阶段(C4) (A) 下胚轴外植体; (B) 愈伤组织(20天)。Bars=1 cm。C4同表1。

Figure 4 Several stages of callus induction of Helenium aromaticum hypocotyls (C4) (A) Hypocotyl explants; (B) Calli (20 d). Bars=1 cm. C4 is the same as Table 1.

表3 芳香堆心菊不同外植体再生体系

Table 3 Different explant regeneration systems of Helenium aromaticum

Explant types	Callus induction medium	Adventitious bud differentiation medium	Regeneration rate (%)	Rooting medium
Leaf	MS+0.2 mg·L^-1 NAA+1 mg·L^-1 6-BA+0.2 mg·L^-1 TDZ		62.1	1/2MS
Stem segment	MS+0.2 mg·L^-1 NAA+1 mg·L^-1 6-BA		9.75	1/2MS
Hypocotyl	MS+0.2 mg·L^-1 NAA+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 TDZ	-	-	-

表4 芳香堆心菊不定芽生根情况

Table 4 Rooting results of adventitious buds of Helenium aromaticum

Treatments	Time of rooting (d)	The situation of root and plant	Rate of rooting (%)	Budding time (d)	Rate of flowering (%)
R0	16	Root system is sparse and slender; the plant is robust with normal stems and leaves, flowering normally	63.33	30	93.33
R1	10	The root system is dense and crude, some of which are deformed roots; the plants are dwarf with large leaves	45.46	34	86.36
R2	13	The root system is few and short, most of which are deformed roots; the plants dwarf can’t grow with normal leaves	12.50	-	0
R3	18	Root deformity and plants hardly grow taller and leaves weaker	14.71	-	0

图5 芳香堆心菊不定芽生根及植株状态(从左至右分别为R0、R1、R2和R3) (A1) 生根情况(17天); (A2) 植株状态(17天); (B1) 生根情况(32天); (B2) 植株状态(32天)。Bars=1 cm。R0-R3同表2。

Figure 5 Rooting and plant status of Helenium aromaticum adventitious buds (from left to right are R0, R1, R2, and R3, respectively) (A1) Rooting condition (17 d); (A2) Plant status (17 d); (B1) Rooting condition (32 d); (B2) Plant status (32 d). Bars=1 cm. R0-R3 are the same as Table 2.

图6 芳香堆心菊叶片离体再生体系的建立 (A) 叶片; (B) 叶片膨大(15天); (C) 叶片的愈伤组织(20天); (D) 愈伤组织分化的不定芽(50天); (E) 不定芽的生根诱导(60天); (F) 生根诱导16天(76天)。Bars=2 cm

Figure 6 Establishment of in vitro regeneration system of Helenium aromaticum leaves (A) The leaf; (B) The leaf swelling (15 d); (C) Calli were induced from leaves (20 d); (D) Adventitious buds could be regenerated from calli (50 d); (E) Inducing rooting (60 d); (F) The adventitious roots induced after 16 days (76 d). Bars=2 cm

参考文献 46

[1]	包晗, 张芮, 张美玲, 张虹, 陈任 (2018). 甜叶菊叶片外植体再生体系的建立. 北方园艺 42(6), 16-22.
[2]	陈华, 李平, 刘晶, 李银心 (2005). 药蒲公英再生体系的建立和优化. 生物工程学报 21, 244-249.
[3]	陈雪, 张金柱, 潘兵兵, 桑成瑾, 马雪, 杨涛, 车代弟 (2011). 月季愈伤组织的诱导及植株再生. 植物学报 46, 569-574.
[4]	程密密 (2015). 翠菊(Callistephus chinensis (L.) Nees)高频再生体系的建立及多倍体诱导. 硕士论文. 重庆: 西南大学. pp. 17.
[5]	程越 (2014). 菊花脑再生体系建立的研究. 硕士论文. 南京: 南京农业大学. pp. 4.
[6]	丁晓霞 (2018). 菊花组培再生体系研究进展. 辽宁林业科技(4), 64-65, 68.
[7]	冯欢, 易姝利, 谢佳恒, 雷梦琦, 黄萱 (2014). 微型月季愈伤组织诱导及植株再生. 植物学报 49, 595-602.
[8]	付建新, 张超, 王翊, 戴思兰 (2012). 甘菊下胚轴离体再生体系的建立. 北京林业大学学报 34(3), 91-96.
[9]	高亚曼, 李妹芳, 冀芦沙 (2016). 珍珠半夏(Pinellia ternate)高效再生体系的建立及其遗传稳定性检测. 分子植物育种 14, 980-985.
[10]	郝志华, 顾偌铖, 黄洁兰, 武彦博, 田铃 (2017). 常见多肉植物繁育技术的研究进展. 广东蚕业 51(4), 13-16.
[11]	荆甜蕊, 徐思雅, 倪亦飞, 张勇娟, 郭斌, 尉亚辉 (2016). 天山雪莲生物技术研究进展. 基因组学与应用生物学 35, 2201-2210.
[12]	李海萍, 张鲁刚, 张静, 茹磊, 刘学成, 孙希禄 (2011). 萝卜带柄子叶高频再生体系的建立. 植物学报 46, 331-337.
[13]	李金童, 吴雅妮, 丁兵, 齐学军, 张旸, 解莉楠 (2016). 四种露地菊再生体系的建立. 北方园艺 40(19), 119-124.
[14]	刘晨旭, 马欣, 董凤丽, 周蕴薇 (2015). 地被菊‘紫妍’和‘纽9722’的再生体系建立. 草业科学 32, 188-195.
[15]	刘娟旭, 刘玲, 王静, 余义勋 (2008). 大叶相思下胚轴离体培养再生植株的研究. 林业科学研究 21, 403-406.
[16]	刘军, 赵兰勇, 丰震, 张美蓉, 吴银凤 (2004). 菊花叶盘片转基因再生体系的优化选择. 植物学通报 21, 556-558.
[17]	刘萌萌 (2017). 盆栽小菊高频再生体系建立与试管开花研究. 硕士论文. 银川: 宁夏大学. pp. 1.
[18]	罗虹, 温小蕙, 刘影, 蒲娅, 戴思兰 (2018). 芳香堆心菊试管开花及释香机制初探. 见: 张启翔主编. 中国观赏园艺研究进展(2018). 北京: 中国林业出版社. pp. 448-460.
[19]	孙永莲, 戴晓港, 李小平, 陈赢男 (2019). 簸箕柳组培再生体系的建立. 南京林业大学学报(自然科学版) 43(2), 31-37.
[20]	王碧玉 (2017). 菊花再生及遗传转化体系的研究. 硕士论文. 沈阳: 沈阳农业大学. pp. 8.
[21]	王洪霞, 郭尚敬 (2012). TDZ对甜椒不定芽分化的影响. 北方园艺 36(3), 104-106.
[22]	王欢, 刘洋, 苑禹, 李旭, 杜凤国 (2015). 蚂蚱腿子离体快繁技术研究. 植物生理学报 51, 2275-2279.
[23]	王树芸, 吕波, 李坤, 吴佳洁, 付道林 (2012). “济麦”系列品种成熟胚再生体系的优化研究. 分子植物育种 10, 476-484.
[24]	王自布, 莫国秀, 罗会兰, 张德英 (2015). 菊花不同外植体组培快繁及其再生体系的研究. 北方园艺 39(18), 106-109.
[25]	吴志苹, 高亦珂, 范敏, 高耀辉 (2020). 菊花‘金不凋’再生及遗传转化体系的构建. 分子植物育种 18, 150-158.
[26]	咸宏康, 支秋娟, 李卉, 王长泉 (2019). 金樱子(Rosa laevigata Michx.)叶片直接再生不定芽体系的建立. 北方园艺 43(13), 93-100.
[27]	徐术菁 (2016). 东北蒲公英(Taraxacum ohwianum)叶片再生体系建立与优化. 硕士论文. 沈阳: 沈阳农业大学. pp. 24.
[28]	徐晓峰, 黄学林 (2003). TDZ: 一种有效的植物生长调节剂. 植物学通报 20, 227-237.
[29]	杨怡帆, 杨清江, 潘峰, 张新利 (2019). 基于主成分分析的不同浓度TDZ处理下‘红地球’葡萄膨大效果的综合评价. 农业科技通讯 (7), 165-168.
[30]	张俊华, 苏振华, 张泽鑫, 李妹芳, 郭尚敬, 曹雪松, 冀芦沙 (2019). 甜椒离体再生体系的构建. 分子植物育种 17, 8208-8214.
[31]	张旭红, 王頔, 梁振旭, 孙美玉, 张金政, 石雷 (2018). 欧洲百合愈伤组织诱导及植株再生体系的建立. 植物学报 53, 840-847.
[32]	张媛, 王康才, 汤兴利 (2008). 杭白菊子叶和下胚轴组织培养技术的研究. 中草药 39, 1721-1723.
[33]	赵喜亭, 蒋丽微, 王苗, 朱玉婷, 张文芳, 李明军 (2016). 怀黄菊间接体胚受体再生体系的建立及CmTGA1的遗传转化. 植物学报 51, 525-532.
[34]	中国科学院中国植物志编辑委员会 (1984). 中国植物志, Vol. 74. 北京: 科学出版社. pp. 1.
[35]	左静静, 闫贵云, 安晓宁, 霍利光, 刘少翔 (2017). 正交设计优化北苍术组织培养研究. 中国农学通报 33(11), 21-24.
[36]	Alamgir ANM (2017). Medicinal, non-medicinal, biopestici- des, color- and dye-yielding plants; secondary metabolites and drug principles; significance of medicinal plants; use of medicinal plants in the systems of traditional and complementary and alternative medicines (CAMs). In: Alamgir ANM, ed. Therapeutic Use of Medicinal Plants and Their Extracts. Cham Heidelberg: Springer. pp. 61-104.
[37]	Barkley TM, Brouillet L, Strother JL (2006). Asteraceae. In: Flora of North America, Vol. 19. New York: Oxford Uni- versity Press. pp. 3-69.
[38]	Bierner MW (1978). The taxonomy of Helenium sect. Cephalophora (Asteraceae). Syst Bot 3, 277-298.
[39]	Bloszyk E, Samek Z, Toman J, Holub M (1975). Linifolin a and helenalin from Helenium aromaticum. Phytochemistry 14, 1444-1445.
[40]	Bush SR, Earle ED, Langhans RW (1976). Plantlets from petal segments, petal epidermis, and shoot tips of the periclinal chimera, Chrysanthemum morifolium ‘Indianapolis’. Am J Bot 63, 729-737.
[41]	Chen J, Shen CZ, Guo YP, Rao GY (2018). Patterning the Asteraceae capitulum: duplications and differential expression of the flower symmetry CYC2-like genes. Front Plant Sci 9, 551.
[42]	Dewir YH, Nurmansyah, Naidoo Y, Teixeira da Silva JA (2018). Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep 37, 1451-1470.
[43]	Gómez-González S, Ojeda F, Torres-Morales P, Palma JE (2016). Seed pubescence and shape modulate adaptive responses to fire cues. PLoS One 11, e0159655.
[44]	Naing AH, Park KI, Mi YC, Lim KB, Kim CK (2016). Optimization of factors affecting efficient shoot regeneration in Chrysanthemum cv. ‘Shinma’. Braz J Bot 39, 975-984.
[45]	Renou JP, Brochard P, Jalouzot R (1993). Recovery of transgenic chrysanthemum (Dendranthema grandiflora Tzvelev) after hygromycin resistance selection. Plant Sci 89, 185-197.
[46]	Tanaka K, Kanno Y, Kudo S, Suzuki M (2000). Somatic embryogenesis and plant regeneration in chrysanthemum (Dendranthema grandiflorum ( Ramat.) Kitamura). Plant Cell Rep 19, 946-953. URL PMID