菊花品种万代风光再生及遗传转化体系的建立

doi:10.11983/CBB24151

植物学报 ›› 2025, Vol. 60 ›› Issue (4): 597-610.DOI: 10.11983/CBB24151 cstr: 32102.14.CBB24151

菊花品种万代风光再生及遗传转化体系的建立

李晶晶^†, 李艳飞^†, 王安琪, 王佳颖, 邓成燕, 卢敏, 马剑英, 戴思兰^*()

北京林业大学园林学院, 城乡生态环境北京实验室, 花卉种质创新与分子育种北京市重点实验室, 国家花卉工程技术研究中心, 北京 100083

收稿日期:2024-10-10 接受日期:2025-01-20 出版日期:2025-07-10 发布日期:2025-01-21
通讯作者: 戴思兰
作者简介:第一联系人：
†共同第一作者
基金资助:
国家自然科学基金(32171849);国家自然科学基金(32371948);福建省科技计划(2022S0004)

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

Jingjing Li^†, Yanfei Li^†, Anqi Wang, Jiaying Wang, Chengyan Deng, Min Lu, Jianying Ma, Silan Dai^*()

National Engineering Research Center for Floriculture, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing Laboratory of Urban and Rural Ecological Environment, School of Landscape Architecture, Beijing Forestry University, Beijing 100083, China

Received:2024-10-10 Accepted:2025-01-20 Online:2025-07-10 Published:2025-01-21
Contact: Silan Dai
About author:First author contact:
†These authors contributed equally to this paper

摘要/Abstract

摘要： 菊花品种万代风光(Chrysanthemum × morifolium ‘Wandai Fengguang’)的色素背景适宜利用分子育种技术调控花瓣铁离子浓度进而培育蓝色花, 且其在夏秋两季均可开花, 是研究菊花开花期分子调控机理的重要材料, 但缺少高效的再生体系和遗传转化体系。以该品种为实验材料, 研究不同外植体类型和植物生长调节剂组合对其再生的影响, 并探讨农杆菌介导的遗传转化方法中相关因素对遗传转化效率的影响。结果表明, 适宜菊花万代风光再生的最适外植体为茎间薄层, 最适培养基为MS+1.5 mg∙L^-16-BA+0.6 mg∙L^-1NAA, 分化率为70.06%, 不定芽生成系数为3.37; 实验确定茎间薄层分化和不定芽生根的卡那霉素选择压分别为7.5 mg∙L^-1和5.0 mg∙L^-1。预培养1天、OD₆₀₀=0.8、处理5分钟及黑暗条件下共培养3天为最佳遗传转化体系。经过卡那霉素筛选共获得抗性苗15株, PCR鉴定发现2株阳性苗, 转化效率为13.33%。研究结果为利用这一独特品种资源解析菊花基因功能和进行定向改良的分子育种奠定了基础, 也为其它菊花品种的再生和转化体系建立提供参考。

关键词: 菊花, 外植体, 遗传转化体系, 再生体系, 茎间薄层

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

李晶晶, 李艳飞, 王安琪, 王佳颖, 邓成燕, 卢敏, 马剑英, 戴思兰. 菊花品种万代风光再生及遗传转化体系的建立. 植物学报, 2025, 60(4): 597-610.

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’. Chinese Bulletin of Botany, 2025, 60(4): 597-610.

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

图/表 17

表1 愈伤组织诱导及不定芽分化培养基配方

Table 1 Culture media for callus induction and adventitious bud differentiation

No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

图1 植物表达载体pBI121的结构 RB: T-DNA区段右边界; NOS-pro: 农杆菌胭脂碱合成酶基因启动子; KanR: 卡那霉素抗性筛选标记基因; NOS-ter: 农杆菌胭脂碱合成酶基因终止子; CaMV35S-pro: 花椰菜花叶病毒35S启动子; CcVIT: 矢车菊液泡铁离子转运蛋白基因; LB: T-DNA区段左边界

Figure 1 The structure of plant expression vector pBI121 RB: Right border of T-DNA region; NOS-pro: Agrobacterium tumefaciens nopaline synthase gene promoter; KanR: Kanamycin resistance screening marker gene; NOS-ter: A. tumefaciens nopaline synthase gene terminator; CaMV35S-pro: Cauliflower mosaic virus 35S promoter; CcVIT: Centaurea cyanus vacuolar iron transport protein gene; LB: Left border of T-DNA region

表2 菊花万代风光遗传转化体系筛选正交试验表

Table 2 Orthogonal experimental table for genetic transformation screening of Chrysanthemum × morifolium ‘Wandai Fengguang’

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1

表3 不同植物生长调节剂组合对菊花万代风光不同外植体再生的影响

Table 3 Effects of different plant growth regulators combinations on the regeneration of different explants of Chrysanthemum × morifolium ‘Wandai Fengguang’

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

图2 菊花万代风光不同外植体的再生状况 (A)-(C) 叶片、叶柄和茎间薄层在A7培养基(表1)中培养0天的状态; (D)-(F) 叶片、叶柄和茎间薄层在A7培养基中培养35天的再生状况。Bars=1 cm

Figure 2 Regeneration of different explants of Chrysanthemum × morifolium ‘Wandai Fengguang’ (A)-(C) State of leaf, petioles, and transverse thin cell layers cultured in A7 medium (Table 1) for 0 day; (D)-(F) Regeneration of leaf, petioles, and transverse thin cell layers cultured in A7 medium for 35 days. Bars=1 cm

表4 卡那霉素对菊花万代风光茎间薄层分化的影响

Table 4 Effect of kanamycin on transverse thin cell layers of Chrysanthemum × morifolium ‘Wandai Fengguang’

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

图3 卡那霉素对菊花万代风光茎间薄层分化的影响 (A)-(F) 茎间薄层在含0、2.5、5、7.5、10和15 mg∙L-1卡那霉素的A3培养基(表1)中的生长状况。Bars=1 cm

Figure 3 Effect of kanamycin on transverse thin cell layers of Chrysanthemum × morifolium ‘Wandai Fengguang’ (A)-(F) Growth of transverse thin cell layers in A3 culture medium (Table 1) containing 0, 2.5, 5, 7.5, 10, and 15 mg∙L-1 kanamycin. Bars=1 cm

表5 卡那霉素对菊花万代风光不定芽生根的影响

Table 5 Effect of kanamycin on rooting of adventitious bud of Chrysanthemum × morifolium ‘Wandai Fengguang’

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

图4 卡那霉素对菊花万代风光不定芽生根的影响 (A)-(F) 不定芽在含0、3、5、7.5、10和12 mg∙L-1卡那霉素的MS培养基中的生根状况。Bars=1 cm

Figure 4 Effect of kanamycin on rooting of adventitious bud of Chrysanthemum × morifolium ‘Wandai Fengguang’ (A)-(F) Rooting of adventitious buds in MS culture media containing 0, 3, 5, 7.5, 10, and 12 mg∙L-1 kanamycin. Bars=1 cm

表6 不同转化条件对菊花万代风光抗性芽生长的影响

Table 6 Effect of different transformation conditions on the growth of resistant buds of Chrysanthemum × morifolium ‘Wandai Fengguang’

Treatments	Callus formation rate (%)	Differentiation rate (%)	Coefficient of adventitious bud production	Browning rate (%)
1	80.91±10.48 bc	3.33±5.77 c	0.33±0.58 b	0.00±0.00 c
2	94.84±4.51 a	0.00±0.00 c	0.00±0.00 b	0.00±0.00 c
3	86.96±5.54 b	1.85±3.21 c	0.33±0.58 b	0.00±0.00 c
4	71.11±7.70 cd	0.00±0.00 c	0.00±0.00 b	0.00±0.00 c
5	79.55±9.91 bc	8.84±0.44 bc	1.33±0.58 a	0.00±0.00 c
6	64.07±7.56 d	14.44±6.76 ab	1.83±1.04 a	28.52±5.70 a
7	81.85±3.61 bc	0.00±0.00 c	0.00±0.00 b	18.15±3.61 b
8	95.96±3.51 a	21.00±9.78 a	1.38±0.40 a	0.00±0.00 c
9	96.08±6.79 a	1.96±3.40 c	0.33±0.58 b	0.00±0.00 c

图5 菊花万代风光遗传转化体系筛选 (A)-(I) 茎间薄层在处理1-9 (同表2)中的生长状况。Bars=1 cm

Figure 5 Genetic transformation screening of Chrysanthemum × morifolium ‘Wandai Fengguang’ (A)-(I) Growth of transverse thin cell layers in treatments 1-9 (see Table 2). Bars=1 cm

表7 预培养时间对菊花万代风光抗性芽生长的影响

Table 7 Effect of pre-culture time on the growth of resistant buds in Chrysanthemum × morifolium ‘Wandai Fengguang’

Pre-cultivation time (d)	Callus formation rate (%)	Differentiation rate (%)	Coefficient of adventitious bud production	Brow- ning rate (%)
0	87.57 a	1.73 a	0.22 b	0.00 a
1	71.58 b	7.76 a	1.06 a	9.51 a
2	91.29 a	7.66 a	0.57 ab	6.05 a

表8 农杆菌菌液浓度对菊花万代风光抗性芽生长的影响

Table 8 Effect of Agrobacterium concentration on the growth of resistant buds in Chrysanthemum × morifolium ‘Wandai Fengguang’

Concentration of Agrobacterium (OD₆₀₀)	Callus formation rate (%)	Differentiation rate (%)	Coefficient of adventitious bud production	Browning rate (%)
0.6	77.96 b	1.11 b	0.11 b	6.05 a
0.8	90.11 a	9.95 a	0.90 a	0.00 a
1.0	82.37 ab	6.09 ab	0.83 ab	9.51 a

表9 侵染时间对菊花万代风光抗性芽生长的影响

Table 9 Effect of infection time on the growth of resistant buds in Chrysanthemum × morifolium ‘Wandai Fengguang’

Infection time (min)	Callus formation rate (%)	Differentiation rate (%)	Coefficient of adventitious bud production	Browning rate (%)
5	80.31 a	12.93 a	1.18 a	9.51 a
10	87.34 a	0.65 b	0.11 b	0.00 a
15	82.78 a	3.56 b	0.56 ab	6.05 a

表10 共培养时间对菊花万代风光抗性芽生长的影响

Table 10 Effect of co-culture time on the growth of resistant buds in Chrysanthemum × morifolium ‘Wandai Fengguang’

Co-culture time (d)	Callus formation rate (%)	Differentiation rate (%)	Coefficient of adventitious bud production	Browning rate (%)
1	85.51 a	4.71 a	0.67 a	0.00 b
2	80.25 a	4.81 a	0.61 a	15.56 a
3	84.68 a	7.62 a	0.57 a	0.00 b

表11 不同转化条件下菊花万代风光抗性芽分化率的极差分析

Table 11 Range analysis of resistance bud differentiation rate of Chrysanthemum × morifolium ‘Wandai Fengguang’ under different transformation conditions

Factors	Pre-cultivation time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infectation time (min)	Co-culture time (d)
K1	5.18	3.33	38.77	14.13
K2	23.28	29.84	1.96	14.44
K3	22.96	18.25	10.69	22.85
k1	1.73	1.11	12.92	4.71
k2	7.76	9.95	0.65	4.81
k3	7.65	6.08	3.56	7.62
R	5.93	8.84	12.27	2.80

图6 菊花万代风光抗性苗PCR检测

Figure 6 PCR amplification on the resistant plantlets of Chrysanthemum × morifolium ‘Wandai Fengguang’ M: DL2000 plus DNA marker; 1-15: 抗性苗; CK1: ddH2O; CK2: 野生型; CK3: 质粒 M: DL2000 plus DNA marker; 1-15: Resistant plantlets; CK1: ddH2O; CK2: Wild type; CK3: Plasmid

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菊花品种万代风光再生及遗传转化体系的建立

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

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No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1

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No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1

No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

No.	6-BA (mg∙L^-1)	KT (mg∙L^-1)	NAA (mg∙L^-1)	2,4-D (mg∙L^-1)
A1	1.5	0.0	0.2	0.0
A2	1.5	0.0	0.4	0.0
A3	1.5	0.0	0.6	0.0
A4	1.5	0.0	0.0	0.2
A5	1.5	0.0	0.0	0.4
A6	1.5	0.0	0.0	0.6
A7	0.0	1.5	0.2	0.0
A8	0.0	1.5	0.4	0.0
A9	0.0	1.5	0.6	0.0
A10	0.0	1.5	0.0	0.2
A11	0.0	1.5	0.0	0.4
A12	0.0	1.5	0.0	0.6

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1

Treatments	Pre-cul- ture time (d)	Concentration of Agrobacterium tumefaciens (OD₆₀₀)	Infection time (min)	Co-culture time (d)
1	0	0.6	5	1
2	0	0.8	10	2
3	0	1.0	15	3
4	1	0.6	10	3
5	1	0.8	15	1
6	1	1.0	5	2
7	2	0.6	15	2
8	2	0.8	5	3
9	2	1.0	10	1