Establishment of In Vitro Regeneration System of Helenium aromaticum

doi:10.11983/CBB19211

Abstract

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

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

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB19211

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

Figures/Tables 10

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

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

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

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.

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.

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.

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	-	-	-

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

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.

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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