Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium tumefaciens in Pinguicula cyclosecta

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

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (12): 3363-3372.doi: 10.16420/j.issn.0513-353x.2024-1040

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Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium tumefaciens in Pinguicula cyclosecta

YANG Jiali¹^,², ZENG Zhilin¹^,², ZHENG Zhe¹^,², RAO Yufei¹^,², HE Yanhong¹^,²^,^**(), NING Guogui¹^,²^,^**()

¹ National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops，College of Horticulture and Forestry Sciences，Huazhong Agricultural University，Wuhan 430070，China
² The Institute of Flowers，Huazhong Agricultural University，Wuhan 430070，China

Received:2025-02-20 Revised:2025-05-21 Online:2025-12-25 Published:2025-12-20
Contact: HE Yanhong, NING Guogui

Abstract

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

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.

Figures/Tables 6

References 24

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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

Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium tumefaciens in Pinguicula cyclosecta

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