根癌农杆菌介导的圆切捕虫堇高效遗传转化体系的建立

doi:10.16420/j.issn.0513-353x.2024-1040

摘要/Abstract

摘要：

以圆切捕虫堇（Pinguicula cyclosecta）的无菌苗叶片为外植体，通过探究卡那霉素（Kan）、头孢霉素（Cef）、菌液浓度与侵染时间、共培养时间和乙酰丁香酮（AS）等影响遗传转化的因素，成功建立了农杆菌介导的捕虫堇遗传转化体系。结果表明，Kan最优浓度为50 mg · L^-1，Cef最优浓度为200 mg · L^-1；菌液OD₆₀₀值为0.2，侵染时间15 min，共培养3 d，AS浓度为100 mg · L^-1时，圆切捕虫堇的阳性芽率最高，达到10.24%，经培养成功获得16株转基因植株。

关键词: 圆切捕虫堇, 遗传转化, 根癌农杆菌

Abstract:

In this study，sterile leaves of Pinguicula cyclosecta as explants were employed and successfully established an Agrobacterium-mediated transformation system for P. cyclosecta by examining the factors affecting genetic transformation，such as kanamycin（Kan），cephalosporin（Cef），bacterial solution concentration and infection time，co-culture time and acetyl syringone（AS）. The results indicated that the optimal concentration of Kan was 50 mg · L^-1，and the optimal concentration of Cef was 200 mg · L^-1. With a bacterial solution optical density at OD₆₀₀of 0.2，an infection duration of 15 minutes，a co-cultivation period of three days，and an AS concentration of 100 mg · L^-1，the highest positive bud rate for P. cyclosecta was achieved at 10.24%. A total of 16 individual transgenic plant lines were successfully obtained.

Key words: Pinguicula cyclosecta, genetic transformation, Agrobacterium tumefaciens

杨佳丽, 曾芝琳, 郑哲, 饶羽菲, 何燕红, 宁国贵. 根癌农杆菌介导的圆切捕虫堇高效遗传转化体系的建立[J]. 园艺学报, 2025, 52(12): 3363-3372.

YANG Jiali, ZENG Zhilin, ZHENG Zhe, RAO Yufei, HE Yanhong, NING Guogui. Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium tumefaciens in Pinguicula cyclosecta[J]. Acta Horticulturae Sinica, 2025, 52(12): 3363-3372.

图/表 6

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卡那霉素/ （mg · L^-1） Kan	存活率/% Survival rate	再生率/% Regeneration rate	生长状态 Growth state
0	100.00 ± 0.00 a	100.00 ± 0.00 a	无愈伤组织，叶片能直接再生不定芽 No callus tissue，leaves can directly regenerate adventitious buds
10	100.00 ± 0.00 a	85.33 ± 2.19 b	出现白色愈伤，部分再生不定芽 White callus tissue appears，with some regenerating adventitious buds
20	90.00 ± 6.67 b	55.98 ± 4.60 c	出现白色愈伤，部分再生不定芽 White callus tissue appears，with some regenerating adventitious buds
30	81.11 ± 6.94 c	25.56 ± 1.93 d	出现白色愈伤，不定芽再生受抑制 White callus tissue appears，and the regeneration of adventitious buds is inhibited
40	76.67 ± 6.67 cd	17.78 ± 5.09 e	无愈伤组织，不定芽再生受抑制 No callus tissue，and the regeneration of adventitious buds is inhibited
50	71.11 ± 1.92 d	2.22 ± 1.92 f	无愈伤组织，不定芽再生受抑制 No callus tissue，and the regeneration of adventitious buds is inhibited
100	38.89 ± 5.09 e	0 f	无愈伤组织，不能再生不定芽 No callus tissue，unable to regenerate adventitious buds
200	23.33 ± 3.34 f	0 f	无愈伤组织，不能再生不定芽 No callus tissue，unable to regenerate adventitious buds
300	16.52 ± 3.55 f	0 f	无愈伤组织，不能再生不定芽 No callus tissue，unable to regenerate adventitious buds

卡那霉素/ （mg · L^-1） Kan	存活率/% Survival rate	再生率/% Regeneration rate	生长状态 Growth state
0	100.00 ± 0.00 a	100.00 ± 0.00 a	无愈伤组织，叶片能直接再生不定芽 No callus tissue，leaves can directly regenerate adventitious buds
10	100.00 ± 0.00 a	85.33 ± 2.19 b	出现白色愈伤，部分再生不定芽 White callus tissue appears，with some regenerating adventitious buds
20	90.00 ± 6.67 b	55.98 ± 4.60 c	出现白色愈伤，部分再生不定芽 White callus tissue appears，with some regenerating adventitious buds
30	81.11 ± 6.94 c	25.56 ± 1.93 d	出现白色愈伤，不定芽再生受抑制 White callus tissue appears，and the regeneration of adventitious buds is inhibited
40	76.67 ± 6.67 cd	17.78 ± 5.09 e	无愈伤组织，不定芽再生受抑制 No callus tissue，and the regeneration of adventitious buds is inhibited
50	71.11 ± 1.92 d	2.22 ± 1.92 f	无愈伤组织，不定芽再生受抑制 No callus tissue，and the regeneration of adventitious buds is inhibited
100	38.89 ± 5.09 e	0 f	无愈伤组织，不能再生不定芽 No callus tissue，unable to regenerate adventitious buds
200	23.33 ± 3.34 f	0 f	无愈伤组织，不能再生不定芽 No callus tissue，unable to regenerate adventitious buds
300	16.52 ± 3.55 f	0 f	无愈伤组织，不能再生不定芽 No callus tissue，unable to regenerate adventitious buds

头孢霉素/（mg · L^-1） Cef	再生率/% Regeneration rate	生长状态 Growth state	农杆菌生长情况 Growth of bacteria
0	100.00 ± 0.00 a	叶片再生正常，不定芽生长健壮 Normal leaf regeneration, and the adventitious buds grow vigorously	生长良好 Grow well
100	100.00 ± 0.00 a	叶片再生正常，不定芽细弱 Normal leaf regeneration，and the adventitious buds are thin and weak	生长良好 Grow well
200	96.67 ± 3.34 ab	叶片再生正常，不定芽细弱 Normal leaf regeneration，and the adventitious buds are thin and weak	生长受到抑制 Completely inhibited
300	93.44 ± 3.18 b	叶片再生正常，不定芽细弱 Normal leaf regeneration，and the adventitious buds are thin and weak	生长受到抑制 Completely inhibited
400	92.22 ± 3.84 c	叶片再生受到抑制 Leaf regeneration is inhibited	生长受到抑制 Completely inhibited

头孢霉素/（mg · L^-1） Cef	再生率/% Regeneration rate	生长状态 Growth state	农杆菌生长情况 Growth of bacteria
0	100.00 ± 0.00 a	叶片再生正常，不定芽生长健壮 Normal leaf regeneration, and the adventitious buds grow vigorously	生长良好 Grow well
100	100.00 ± 0.00 a	叶片再生正常，不定芽细弱 Normal leaf regeneration，and the adventitious buds are thin and weak	生长良好 Grow well
200	96.67 ± 3.34 ab	叶片再生正常，不定芽细弱 Normal leaf regeneration，and the adventitious buds are thin and weak	生长受到抑制 Completely inhibited
300	93.44 ± 3.18 b	叶片再生正常，不定芽细弱 Normal leaf regeneration，and the adventitious buds are thin and weak	生长受到抑制 Completely inhibited
400	92.22 ± 3.84 c	叶片再生受到抑制 Leaf regeneration is inhibited	生长受到抑制 Completely inhibited

菌液浓度OD₆₀₀ Bacterial solution concentration	侵染时间/min Infection time	存活率/% Survival rate	抗性芽率/% Resistant bud rate	阳性芽率/% Positive bud rate
0.2	5	78.89 ± 5.09 a	37.78 ± 6.94 abc	0 b
	10	75.56 ± 9.62 ab	41.11 ± 6.94 a	0 b
	15	72.22 ± 5.09 abc	40.00 ± 5.77 ab	7.78 ± 1.92 a
	20	63.33 ± 6.67 cd	31.11 ± 1.92 abcd	1.11 ± 1.92 b
0.6	5	64.44 ± 1.92 bcd	37.78 ± 8.39 abc	0 b
	10	62.22 ± 1.92 cd	35.56 ± 5.09 abc	0 b
	15	65.56 ± 8.39 bcd	36.67 ± 8.8 2abc	0 b
	20	56.67 ± 8.82 de	27.78 ± 6.94 bcd	0 b
1.0	5	55.56 ± 3.85 de	30.00 ± 3.33 abcd	0 b
	10	56.67 ± 3.33 de	25.56 ± 10.72 cde	0 b
	15	54.44 ± 8.39 de	21.00 ± 5.09 de	0 b
	20	47.78 ± 6.94 e	15.56 ± 1.92 e	0 b