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.

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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

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