Establishment of Regeneration and Genetic Transformation System for Chrysanthemum × morifolium ‘Wandai Fengguang’

doi:10.11983/CBB24151

Abstract

Abstract: INTRODUCTION Chrysanthemum × morifolium is one of the ten most famous traditional flowers in China, and it has a rich variety of cultivars with diverse floral colour and shapes. However, varieties with blue floral colour have not been found in the natural chrysanthemum, therefore, breeding blue chrysanthemums has always been a goal pursued by researchers. RATIONALE The total flavonoid extract of C. × morifolium ‘Wandai Fengguang’ could turn blue when adding appropriate concentration of Fe³⁺, and its living petal cells could also turn blue with the participation of Fe³⁺, which proved the feasibility of breeding blue chrysanthemums with Fe³⁺. Meanwhile, C. × morifolium ‘Wandai Fengguang’ can bloom both in summer and autumn, with early flowering and long flowering period, which is also an important material for the study of flowering period. Therefore, in order to cultivate blue chrysanthemums and achieve the targeted improvement of flowering period, it is particularly important to establish an efficient and stable regeneration and genetic transformation system for the C. × morifolium ‘Wandai Fengguang’. However, chrysanthemum has a long history of cultivation and complex genetic background, so the regeneration and genetic transformation system is not universal among different varieties.RESULTS In this study, C. × morifolium ‘Wandai Fengguang’ was used as the experimental material to study the effects of different explant types with different combinations of plant growth regulators on its regeneration, and to investigate the effects of relevant factors on the efficiency of genetic transformation with the Agrobacterium-mediated genetic transformation method. The experimental results showed that the most suitable explants for the regeneration of C. × morifolium ‘Wandai Fengguang’ was the transverse thin cell layers (tTCLs), and the optimal culture medium was MS+1.5 mg∙L^-1 6-BA+0.6 mg∙L^-1 NAA. The highest differentiation rate was 70.06% and an adventitious bud coefficient was 3.37. The kanamycin selection pressures for the differentiation of the tTCLs and the adventitious bud rooting were 7.5 mg∙L^-1 and 5.0 mg∙L^-1, respectively. The optimal procedure for genetic transformation was pre-culture for 1 day, OD₆₀₀=0.8, treatment for 5 minutes, and co-culture in the dark for 3 days. Fifteen resistant plantlets were screened on kanamycin medium, and two positive plantlets were confirmed by PCR amplification, with a transformation efficiency of 13.33%.CONCLUSION This study laid the foundation for the gene function analysis and targeted improvement molecular breeding of chrysanthemum by using this kind of unique variety resource, and provided reference for the establishment of regeneration and transformation system for other chrysanthemum varieties.

Key words: chrysanthemum, explant, genetic transformation system, regeneration system, transverse thin cell layers

Jingjing Li, Yanfei Li, Anqi Wang, Jiaying Wang, Chengyan Deng, Min Lu, Jianying Ma, Silan Dai. Establishment of Regeneration and Genetic Transformation System for Chrysanthemum × morifolium ‘Wandai Fengguang’[J]. Chinese Bulletin of Botany, 2025, 60(4): 597-610.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24151

https://www.chinbullbotany.com/EN/Y2025/V60/I4/597

Figures/Tables 17

References 53

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No.	Leaf		Petioles		Transverse thin cell layers
No.	Differentiation rate (%)	Coefficient of adventitious bud production	Differentiation rate (%)	Coefficient of adventitious bud production	Differentiation rate (%)	Coefficient of adventitious bud production
A1	0.00±0.00 c	0.00±0.00 c	57.32±6.87 a	1.85±0.29 a	36.51±5.50 c	3.37±0.28 a
A2	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	42.79±4.28 b	2.53±0.32 b
A3	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	70.06±2.84 a	3.37±0.22 a
A4	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	0.00±0.00 f	0.00±0.00 g
A5	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	9.79±2.29 e	1.11±0.19 ef
A6	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	0.00±0.00 f	0.00±0.00 g
A7	0.00±0.00 c	0.00±0.00 c	36.35±3.38 b	1.62±0.60 a	45.93±1.28 b	1.51±0.31 d
A8	8.59±0.44 b	1.00±0.00 b	0.00±0.00 e	0.00±0.00 c	41.95±1.88 b	1.62±0.21 d
A9	19.44±4.81 a	1.28±0.25 a	22.56±4.55 c	1.11±0.19 b	46.30±3.21 b	2.73±0.22 b
A10	8.33±0.00 b	1.00±0.00 b	0.00±0.00 e	0.00±0.00 c	31.31±3.50 c	1.44±0.27 de
A11	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	16.67±0.00 d	1.00±0.00 f
A12	0.00±0.00 c	0.00±0.00 c	8.33±0.00 d	1.00±0.00 b	34.98±3.90 c	2.07±0.12 c

No.	Leaf		Petioles		Transverse thin cell layers
No.	Differentiation rate (%)	Coefficient of adventitious bud production	Differentiation rate (%)	Coefficient of adventitious bud production	Differentiation rate (%)	Coefficient of adventitious bud production
A1	0.00±0.00 c	0.00±0.00 c	57.32±6.87 a	1.85±0.29 a	36.51±5.50 c	3.37±0.28 a
A2	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	42.79±4.28 b	2.53±0.32 b
A3	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	70.06±2.84 a	3.37±0.22 a
A4	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	0.00±0.00 f	0.00±0.00 g
A5	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	9.79±2.29 e	1.11±0.19 ef
A6	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	0.00±0.00 f	0.00±0.00 g
A7	0.00±0.00 c	0.00±0.00 c	36.35±3.38 b	1.62±0.60 a	45.93±1.28 b	1.51±0.31 d
A8	8.59±0.44 b	1.00±0.00 b	0.00±0.00 e	0.00±0.00 c	41.95±1.88 b	1.62±0.21 d
A9	19.44±4.81 a	1.28±0.25 a	22.56±4.55 c	1.11±0.19 b	46.30±3.21 b	2.73±0.22 b
A10	8.33±0.00 b	1.00±0.00 b	0.00±0.00 e	0.00±0.00 c	31.31±3.50 c	1.44±0.27 de
A11	0.00±0.00 c	0.00±0.00 c	0.00±0.00 e	0.00±0.00 c	16.67±0.00 d	1.00±0.00 f
A12	0.00±0.00 c	0.00±0.00 c	8.33±0.00 d	1.00±0.00 b	34.98±3.90 c	2.07±0.12 c

Concentration of kanamycin (mg∙L^-1)	Callus formation rate (%)	Differentiation rate (%)
0	97.22±4.81 ab	69.44±12.73 a
2.5	100.00±0.00 a	16.67±8.34 b
5	80.44±4.72 bc	5.56±4.81 bc
7.5	63.89±12.73 cd	0.00±0.00 c
10	50.00±8.33 de	0.00±0.00 c
15	33.33±16.67 e	0.00±0.00 c

Concentration of kanamycin (mg∙L^-1)	Callus formation rate (%)	Differentiation rate (%)
0	97.22±4.81 ab	69.44±12.73 a
2.5	100.00±0.00 a	16.67±8.34 b
5	80.44±4.72 bc	5.56±4.81 bc
7.5	63.89±12.73 cd	0.00±0.00 c
10	50.00±8.33 de	0.00±0.00 c
15	33.33±16.67 e	0.00±0.00 c

Concentration of kanamycin (mg∙L^-1)	Rooting rate (%)	Average number of roots
0	88.89±19.25 a	3.56±1.02 a
3	30.44±4.72 b	0.50±0.17 b
5	0.00±0.00 c	0.00±0.00 b
7.5	0.00±0.00 c	0.00±0.00 b
10	0.00±0.00 c	0.00±0.00 b
12	0.00±0.00 c	0.00±0.00 b