Establishment of Agrobacterium rhizogenes-Mediated Genetic Transformation System in Vegetable Pea

doi:10.16420/j.issn.0513-353x.2023-0944

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (10): 2439-2448.doi: 10.16420/j.issn.0513-353x.2023-0944

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Establishment of Agrobacterium rhizogenes-Mediated Genetic Transformation System in Vegetable Pea

FENG Zhijuan, LIU Na, BU Yuanpeng, ZHANG Guwen, WANG Bin, GONG Yaming^*()

Key Laboratory of Vegetable Legumes Germplasm Enhancement and Molecular Breeding in Southern China of Ministry of Agriculture and Rural Affairs，State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products，Institute of Vegetables，Zhejiang Academy of Agricultural Sciences，Hangzhou 310000，China

Received:2024-01-28 Revised:2024-07-29 Online:2024-12-13 Published:2024-10-21
Contact: GONG Yaming

Abstract

Abstract:

This study established a simple and efficient Agrobacterium rhizogenes-mediated genetic transformation system in vegetable pea. The vegetable pea seeds of‘Zhewan1’were sown into pots with wet sterile vermiculite. The hypocotyls of vegetable pea seedlings were cut at the junction of cotyledon node and hypocotyl and the residual seedlings were selected as explants. The hairy roots were induced with Agrobacterium rhizogenes AR1193 strain harboring the construct of pCAMBIA3301-GUS. The results indicated that 100% of the inoculated explants could produce hair roots. The effect of different seedling age of explant on hairy root induction was also investigated. It was found that the 7-day-old seedling was favorable for hair root induction and the number of hairy root per seedling was 14.60. Further，the hair roots were amplified by RT-PCR of GUS gene and stained with the GUS dye. It was proven that the exogenous GUS gene could be successfully expressed in the hair roots and 35.21% of the hair roots were transgenic positive roots.

Key words: vegetable pea, Agrobacterium rhizogenes, seeding age, hairy root, genetic transformation, transgenic root

FENG Zhijuan, LIU Na, BU Yuanpeng, ZHANG Guwen, WANG Bin, GONG Yaming. Establishment of Agrobacterium rhizogenes-Mediated Genetic Transformation System in Vegetable Pea[J]. Acta Horticulturae Sinica, 2024, 51(10): 2439-2448.

Figures/Tables 6

Fig. 1 Seedlings of vegetable pea cultivated in the vermiculite

Fig. 2 Agrobacterium rhizogenes-mediated genetic transformation process of vegetable pea A：Ideal stage（7-day-old seedling）of vegetable pea for transformation；B：Agrobacterium rhizogenes AR1193 strain containing the pCAMBIA3301-GUS vector cultured on the solid TY plate；C：Agrobacterium rhizogenes AR1193 strain containing pCAMBIA3301-GUS vector cultured in the liquid TY medium；D：The cotyledon and primary root were cut and the cut of residual seedling was scraped on the solid plate grown AR1193；E：Inoculated explants（coated the bacterial mass）were planted into sterile vermiculite and watered with liquid TY medium；F：Inoculated explants were covered with the plastic cover；G：Induced hairy roots；H：GUS staining of hairy roots.

Fig. 3 Effect of different seedling age（5，7 or 9 d）of explant inoculated with AR1193-pCAMBIA3301 strain for different treatment time（10，14 or 18 d）on hairy root induction

Table 1 Induction efficiency of hairy root from different seedling age of explant

苗龄 Seedling stage	培养时间 Cultivation time	侵染外植体数 Number of the explant inoculated	产生毛状根外植体数 Number of explant produced hairy root	外植体平均生根数 Number of hairy root per explant	GUS染色显蓝毛状根数 Number of GUS positive root per explant	毛状根诱导率/% Induction efficiency of hairy root	毛状根转化率/% Transformation frequency of hairy root
5 d	10 d	30	30	3.00 ± 1.61 d		100
	14 d	30	30	4.00 ± 1.70 cd		100
	18 d	30	30	6.50 ± 1.80 c		100
7 d	10 d	30	30	6.50 ± 2.20 c	5.14 ± 1.55	100	35.21
	14 d	30	30	14.60 ± 1.72 a		100
	18 d	30	30	14.60 ± 2.40 a		100
9 d	10 d	30	30	6.50 ± 1.62 c		100
	14 d	30	30	11.00 ± 2.60 b		100
	18 d	30	30	11.00 ± 2.10 b		100

Fig. 4 Validation of transgenic hairy roots of vegetable pea by GUS staining

Fig. 5 Validation of transgenic hairy roots of vegetable pea by RT-PCR M：DNA marker；1-5：Transgenic hairy roots inoculated by AR1193-pCAMBIA3301-GUS；6：pCAMBIA3301-GUS plasmid；7-9：Non-inoculated roots；10：AR1193 strain；11：H2O.

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Establishment of Agrobacterium rhizogenes-Mediated Genetic Transformation System in Vegetable Pea

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