Construction of Transient Transformation System of Petals in Prunus mume

doi:10.16420/j.issn.0513-353x.2024-0448

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (4): 908-920.doi: 10.16420/j.issn.0513-353x.2024-0448

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Construction of Transient Transformation System of Petals in Prunus mume

TIAN Zhuang, FANG Chenxi, LIU Yating, WANG Yutong, XUE Linlei, SHI Jiaxin, REN Tianfang, ZHANG Junwei, BAO Manzhu, ZHANG Jie^*()

National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops，College of Horticulture and Forestry Sciences，Huazhong Agricultural University，Wuhan 430070，China

Received:2024-12-25 Revised:2025-01-24 Online:2025-05-08 Published:2025-04-25
Contact: ZHANG Jie

Abstract

Abstract:

This study takes the petals of Prunus mume as the receptor and GUS gene as the reporter gene. The influences of 7 main factors on the transient transformation efficiency of Prunus mume petals were conducted through an orthogonal design and single factor analysis method. The effects of infection mode，infection time，development stage and genotype on the transient transformation efficiency of Prunus mume petals were investigated by single factor experiment. The results showed that the whole flower vacuum method，infestation time of 15 min，development stage of initial blooming，and‘XueMei’ genotype had the highest transient expression rate of GUS gene，respectively. The orthogonal experiment was further designed to explore the effect of the combination of Agrobacterium strain type，OD₆₀₀ value，Coculture time on the transient transformation efficiency of Prunus mume petals. The results showed that the three factors affected GUS transient expression in the order of coculture time，Agrobacterium strains type，OD₆₀₀ value. Among them，the combination of Agrobacterium EHA105，OD₆₀₀values is 0.6 and coculture time is 5 d showed the highest GUS transient expression effect of Prunus mume petals（32.67%）. Then，the optimal combination screened was verified by applying an anthocyanin synthesis gene（PmANS）as the target gene.

Key words: Prunus mume, petal, Agrobacterium-mediated method, transient transformation, GUS

TIAN Zhuang, FANG Chenxi, LIU Yating, WANG Yutong, XUE Linlei, SHI Jiaxin, REN Tianfang, ZHANG Junwei, BAO Manzhu, ZHANG Jie. Construction of Transient Transformation System of Petals in Prunus mume[J]. Acta Horticulturae Sinica, 2025, 52(4): 908-920.

Figures/Tables 13

References 9

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引物名称Primer	序列（5′-3′）Sequence
ANS-T	F：ATGGTGAGCTCTGATTCAGTGG；R：TCACTTGTTGAGCAGAGCCTC
pBI-PmANS	F：GAGAACACGGGGGAC TCTAGAATGGTGAGCTCTGATTCAGTGGA；
pBI-PmANS	R：ATAAGGGACTGACCA CCCGGGTCACTTGTTGAGCAGAGCCTCTT
ANS（121）	F：ATGGTGAGCTCTGATTCAGTGG；R：ACTTTTCCCGGCAATAACATACGG
ANS-1-2	F：CCAGGCCTCTGGCAAAATTC；R：GGCCAAATGGACAAGTCACG

引物名称Primer	序列（5′-3′）Sequence
ANS-T	F：ATGGTGAGCTCTGATTCAGTGG；R：TCACTTGTTGAGCAGAGCCTC
pBI-PmANS	F：GAGAACACGGGGGAC TCTAGAATGGTGAGCTCTGATTCAGTGGA；
pBI-PmANS	R：ATAAGGGACTGACCA CCCGGGTCACTTGTTGAGCAGAGCCTCTT
ANS（121）	F：ATGGTGAGCTCTGATTCAGTGG；R：ACTTTTCCCGGCAATAACATACGG
ANS-1-2	F：CCAGGCCTCTGGCAAAATTC；R：GGCCAAATGGACAAGTCACG

侵染方式 Infection mode	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material-necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion	GUS-3等级占比/% GUS-3 grade proportion
活体注射In vivo injection	49.33 ± 9.23 ab	18.67 ± 6.11 ab	37.33 ± 10.01 a	10.67 ± 4.61 ab	1.33 ± 1.01 a
带枝水培Hydroponics with branches	49.34 ± 10.58 a	12.00 ± 4 ab	38.67 ± 8.33 a	4.00 ± 3.93 b	6.67 ± 4.62 a
整花真空Whole flower vacuum	66.67 ± 18.33 a	21.33 ± 6.11 a	33.33 ± 6.11 a	24.00 ± 13.86 a	9.33 ± 8.33 a
花瓣真空Petal vacuum	10.67 ± 9.24 b	0 b	10.67 ± 9.24 b	0 b	0 a

侵染方式 Infection mode	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material-necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion	GUS-3等级占比/% GUS-3 grade proportion
活体注射In vivo injection	49.33 ± 9.23 ab	18.67 ± 6.11 ab	37.33 ± 10.01 a	10.67 ± 4.61 ab	1.33 ± 1.01 a
带枝水培Hydroponics with branches	49.34 ± 10.58 a	12.00 ± 4 ab	38.67 ± 8.33 a	4.00 ± 3.93 b	6.67 ± 4.62 a
整花真空Whole flower vacuum	66.67 ± 18.33 a	21.33 ± 6.11 a	33.33 ± 6.11 a	24.00 ± 13.86 a	9.33 ± 8.33 a
花瓣真空Petal vacuum	10.67 ± 9.24 b	0 b	10.67 ± 9.24 b	0 b	0 a

侵染时间/min Infection time	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion
10	34.67 ± 2.31 a	20.00 ± 6.11 a	34.67 ± 2.31 a	0 b
15	45.34 ± 6.66 a	13.33 ± 4.00 a	38.67 ± 8.33 a	6.67 ± 4.62 a
20	42.67 ± 9.24 a	29.33 ± 6.11 a	42.67 ± 9.24 a	0 b
30	45.33 ± 10.07 a	29.33 ± 6.11 a	45.33 ± 10.07 b	0 b

Construction of Transient Transformation System of Petals in Prunus mume

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Figures/Tables 13

References 9

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发育阶段 Development stage	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion	GUS-3等级占/% GUS-3 grade proportion
气球期Balloon stage	49.33 ± 8 b	17.33 ± 2.31 a	45.33 ± 2.31 a	4.00 ± 0 a	0 a
初开期Initial blooming stage	77.33 ± 10.07 a	20.00 ± 2.31 ab	57.33 ± 10.07 a	14.67 ± 12.22 a	5.33 ± 5.11 a
碗状期Bowl stage	66.67 ± 2.31 b	17.33 ± 2.31 c	57.33 ± 9.24 a	5.33 ± 5.11 a	4.00 ± 0 a
盛开期Blooming stage	49.33 ± 9.24 b	6.67 ± 4.62 bc	46.67 ± 6.11 a	2.67 ± 2.31 a	0 a

编号 No.	菌株类型 Agrobacterium strain	OD₆₀₀	共培养时间/d Co-cultivation time	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material necrosis rate
1	EHA105	0.4	1	0 c	0 d
2	EHA105	0.6	5	32.67 ± 5.03 a	28.00 ± 2.00 b
3	EHA105	0.8	3	28.33 ± 11.15 ab	25.00 ± 3.61 bc
4	GV3101	0.4	5	20.67 ± 2.31 b	26.67 ± 5.13 bc
5	GV3101	0.6	3	26.00 ± 3.00 ab	21.00 ± 2.00 c
6	GV3101	0.8	1	0 c	0 d
7	EHA105	0.4	3	27.00 ± 7.21 ab	21.33 ± 4.93 bc
8	EHA105	0.6	1	0 c	0 d
9	EHA105	0.8	5	26.00 ± 2.65 ab	37.33 ± 7.02 a
K1(G)	20.333	15.890	0
K2(G)	15.557	19.557	27.110
K3(G)	17.667	18.110	26.447
R(G)	4.776	3.667	27.110
K1(M)	17.667	16.000	0
K2(M)	15.890	16.333	22.443
K3(M)	19.553	20.777	30.667
R(M)	3.663	4.777	30.667

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