Establishment of Regeneration System of Chrysanthemum indicum in Pingtan with Various Ligulate Floret Form

doi:10.11983/CBB22135

Abstract

Abstract: Asteraceae plants have high ornamental value due to their rich petal variation in ray florets. Chrysanthemum indicum is a wild species and closely related to C. × morifolium. Ray florets mutants of C. indicum in natural populations, such as flat, spoon and tubular types, are excellent material for studying petal shapes. However, there is currently a lack of research on its regeneration system. In this experiment, considerable ray floret mutants of C. indicum were found on Pingtan Island, Fujian province, including the plants with flat, spoon and tubular types of ray florets coexisting in the same capitulum, in which leaves and transverse thin cell layers (tTCLs) were used as explants to establish a regeneration system. The results showed that the best medium for callus induction and adventitious bud differentiation of tTCLs was MS+1.0 mg∙L^-1 6-BA+0.5 mg∙L^-1 NAA, and the induction rate was 100% on the 14th day. The average differentiation time was 25 days, and the differentiation rate reached 82% on the 40th day. The best rooting medium was 1/2MS+0.5 mg∙L^-1 NAA, and the rooting time was 10 days. All of the rooted and regenerated seedlings were survived after transplanting and kept the mix type in capitulum. An efficient regeneration system for C. indicum in Pingtan with various ray floret forms was established in this experiment. The results not only laid the foundation for the establishment of a transformation system but also provided technology for future research on the morphological variation in ray florets.

Key words: Chrysanthemum indicum, plant growth regulator, regeneration system, stains with various ligulate floret form, transverse thin cell layer

Minling Liao, Ya Pu, Xiaoyun Wu, Chaofeng Ma, Wenkui Wang, Silan Dai. Establishment of Regeneration System of Chrysanthemum indicum in Pingtan with Various Ligulate Floret Form[J]. Chinese Bulletin of Botany, 2023, 58(3): 449-460.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22135

https://www.chinbullbotany.com/EN/Y2023/V58/I3/449

Figures/Tables 14

Table 1 The composition of the medium for callus induction and adventitious buds differentiation

No.	Basal medium	6-BA (mg?L^-1)	NAA (mg?L^-1)
Y1	MS	1.0	0.5
Y2	MS	1.0	1.0
Y3	MS	1.0	2.0
Y4	MS	2.0	0.5
Y5	MS	2.0	1.0
Y6	MS	2.0	2.0
Y7	MS	3.0	0.5
Y8	MS	3.0	1.0
Y9	MS	3.0	2.0
CK	MS	0	0

Table 2 The composition of the medium for callus induction with different concentrations of TDZ

No.	Basal medium	NAA (mg?L^-1)	6-BA (mg?L^-1)	TDZ (mg?L^-1)
T1	MS	2.0	1.0	0.1
T2	MS	2.0	1.0	0.2
T3	MS	2.0	1.0	0.4
T4	MS	2.0	2.0	0.1
T5	MS	2.0	2.0	0.2
T6	MS	2.0	2.0	0.4
T7	MS	2.0	3.0	0.1
T8	MS	2.0	3.0	0.2
T9	MS	2.0	3.0	0.4

Table 3 Formula of rooting medium

No.	Basal medium	NAA (mg?L^-1)
G1	1/2MS	0.2
G2	1/2MS	0.5
G3	1/2MS	1.0
1/2MS	1/2MS	0
MS	MS	0

Figure 1 Ray floret variations in Chrysanthemum indicum in Pingtan (A1) Flat type; (A2) Spoon type; (A3) Tubular type; (A4) Mix type; (B1), (B2) Chenille-like; (C1), (C2) Swallowtail-like; (D1) Evaginable; (D2) Incurvate; (E1) Difference in ray floret length; (E2), (E3) Difference in degree of curvature; (E4) Difference in cracking degree; (F1)-(F4) Difference in ray floret number; (G1)-(G4) Difference in proportion of each petal type in mix type; (H) Difference in diameter of capitulum. Bars=1 cm

Figure 2 Chrysanthemum indicum in Pingtan stains with various ligulate floret form (A) Plant (bar=3.5 cm); (B) Leaf shape (bar=1 cm); (C) Capitulum (bar=1 cm)

Table 4 Effects of different media on callus induction and adventitious buds differentiation for leaves and transverse thin cell layers (tTCLs) of Chrysanthemum indicum in Pingtan

Treat-ments	Callus formation rate on the 14th day (%)		Differentiation rate on the 40th day (%)		Treatments	Callus formation rate on the 14th day (%)
Treat-ments	Leaves	tTCLs	Leaves	tTCLs	Treatments	Callus formation rate on the 14th day (%)
Y1	100.00±0.00 a	100.00±0.00 a	0	82.00±3.46 a	T1	100.00±0.00 a
Y2	100.00±0.00 a	100.00±0.00 a	0	53.50±4.95 c	T2	100.00±0.00 a
Y3	100.00±0.00 a	90.50±4.95 b	3.71±0.19 b	33.50±6.36 d	T3	97.00±0.04 ab
Y4	100.00±0.00 a	100.00±0.00 a	0	63.50±4.95 bc	T4	83.50±0.06 ab
Y5	100.00±0.00 a	100.00±0.00 a	0	67.00±5.66 b	T5	97.50±0.03 ab
Y6	100.00±0.00 a	98.50±2.12 a	9.39±3.35 b	57.50±0.71 bc	T6	87.50±0.09 ab
Y7	100.00±0.00 a	100.00±0.00 a	0	61.00±1.41 bc	T7	81.50±0.05 b
Y8	100.00±0.00 a	93.50±0.71 b	0	16.50±3.54 e	T8	85.00±0.14 ab
Y9	75.48±4.30 b	98.50±2.12 a	19.09±1.29 a	41.00±4.24 d	T9	90.00±0.06 ab
CK	0.00±0.00 c	0.00±0.00 c	0	0	-	-

Figure 3 Regeneration of Chrysanthemum indicum in Pingtan leaves in different media (A) Leaves in different media; (B) Leaves in media with TDZ. Y1-Y9, and CK are the same as in Table 1; T1-T9 are the same as in Table 2. Bars=4.5 cm

Figure 4 Adventitious bud differentiation for leaves of Chrysanthemum indicum in Pingtan in different media (A) Y3; (B) Y6; (C) Y9. Y3, Y6 and Y9 are the same as in Table 1. From left to right: Adventitious bud differentiation of leaves in different media at 21, 28, 35, 42 and 49 d. Bar=1 cm

Table 5 The browning rate for leaves of Chrysanthemum indicum in Pingtan in different media

Treatments	Browning rate on the 21th day (%)	Treat- ments	Browning rate on the 24th day (%)
Y1	83.04±7.92 abcd	T1	52.78±3.93 f
Y2	96.43±5.05 a	T2	56.08±3.88 ef
Y3	80.36±6.84 bcde	T3	81.80±0.78 c
Y4	90.11±8.58 abc	T4	72.14±2.18 d
Y5	77.56±3.95 cde	T5	89.45±0.78 b
Y6	67.86±9.22 e	T6	61.51±0.56 e
Y7	74.22±2.49 de	T7	97.50±3.54 a
Y8	42.50±5.00 f	T8	100.00±0.00 a
Y9	34.17±1.18 f	T9	87.30±2.25 bc
CK	92.73±9.50 ab

Figure 5 Adventitious bud differentiation of transverse thin cell layers of Chrysanthemum indicum in Pingtan in different media (40 d) Y1-Y9 are the same as in Table 1. Bar=1 cm

Figure 6 Rooting and plant status of Chrysanthemum indicum in Pingtan adventitious buds (A1)-(E1) Plant status (0 d); (A2)-(E2) Rooting condition (10 d); (A3)-(E3) Plant status (26 d); (A4)-(E4) Rooting condition (26 d). From left to right: MS, 1/2MS, G1, G2, and G3 (the same as in Table 3). Bars=1 cm

Table 6 Rooting and plant status of Chrysanthemum indicum in Pingtan adventitious buds

Treat- ments	Time of rooting (d)	Rate of rooting (%)	Average root length (cm)	The situation of root and plant	Number of branches
MS	6	66.7	12.59±2.60 a	The root system is dense, slender, and long; the plant is weak with branches and small leaves	3±1 a
1/2MS	6	100	5.49±0.86 b	The root system is sparse and slender; the plant is weak with many branches and small leaves	6±2 a
G1	14	100	4.41±0.80 b	The root system is crude and short, with many root hairs; the plant is weak with branches	4±2 a
G2	10	100	3.21±2.62 b	The root system is crude and short, with many root hairs; the plant is robust and higher, with larger leaves	2±1 a
G3	24	100	3.80±2.22 b	The root system is crude and short, with many root hairs; the plant is robust and highest, with largest leaves	2±1 a

Figure 7 The capitulum of regenerated plants and the proportion of each petal type in Chrysanthemum indicum in Pingtan (A1), (A2) Plant status after transplanting for 60 days, from left to right, their rooting media were MS, 1/2MS, G1, G2, and G3, respectively (bars=3.5 cm); (B) The capitulum of regenerated plants (bar=1 cm); (C) The proportion of flat petals, spoon petals and tubular petals in each capitulum; (D) Average proportion of each petal type in all capitulums. MS, 1/2MS, G1, G2, and G3 are the same as in Table 3.

Figure 8 Regeneration system of transverse thin cell layers in Chrysanthemum indicum in Pingtan stains with various ligulate floret forms (A), (B) Callus induction and adventitious bud differentiation in Y1; (C) Adventitious buds grown to 0.8 cm; (D), (E) Rooting and plant status of adventitious buds in G2; (F), (G) Plant status after transplanting for 30 and 90 days; (H) Flower buds; (I) Capitulum of regenerated plant. Y1 is the same as in Table 1; G2 is the same as in Table 3. Bars=2 cm

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