Optimization of Sweet Potato Genetic Transformation System Mediated by Agrobacterium rhizogenes

doi:10.13560/j.cnki.biotech.bull.1985.2023-0369

Abstract

Abstract:

Genetic transformation system is of great significance for the verification of plant gene function. In order to establish Agrobacterium rhizogenes mediated genetic transformation system in sweet potato（Ipomoea batatas L.）, a three-factor design of 3×3×5 was used to study the effects of infection time, explant type, and A. rhizogenes strains on the induction rate of hairy roots in sweet potato. The appropriate explant type and infection time were determined, and the 5×5 two-factor design was used to study the effects of five A. rhizomei strains and sweet potato varieties（lines）on the induction rates of sweet potato. The detection of induced hairy roots by PCR confirmed that the rolB gene of the A. rhizogenes Ri plasmid was integrated into the genome of sweet potato cells. The results showed that the infection time, explant type, A. rhizogenes strain, and sweet potato variety all affected the induction rate of hairy roots, and there was an interaction between the factors. Stem segment was the most suitable explants for hair roots, and the optimal infection time was 20 min. MSU440 was the suitable strain for ‘Xushu 22’. The suitable strain for ‘Taizhong 6’ ‘YS’ were K599. The suitable strain for‘1610’ was C58C1. The suitable strain for ‘Huishu’was C58C1. Using the A. rhizogenes strain MSU440, stem segments of ‘Xushu 22’were used as explants and Agrobacterium infection for 20 min, and the highest induction rate of hairy root was 79.63%. The transgenic efficiency of the hairy root of sweet potato was found to be 38.1% by PCR amplification of the target gene. A genetic transformation system of sweet potato mediated by A. rhizogenes was established, which lays the foundation for further transgenic breeding of sweet potato.

Key words: sweet potato, Agrobacterium rhizogenes, hairy root, induction rate, transgenic root

TAO Na, LI Mao-xing, GUO Hua-chun. Optimization of Sweet Potato Genetic Transformation System Mediated by Agrobacterium rhizogenes[J]. Biotechnology Bulletin, 2023, 39(10): 175-183.

Figures/Tables 9

References 31

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发根农杆菌菌株 Agrobacterium strain	冠瘿碱类型 Crown gallin type	抗性 Resistance
Ar.1193	农杆碱型 Agropine	链霉素 Streptomycin
K599	黄瓜碱型 Cucumopine	链霉素 Streptomycin
C58C1	农杆碱型 Agropine	链霉素 Streptomycin
ArQual	农杆碱型 Agropine	链霉素 Streptomycin
MSU440	农杆碱型 Agropine	链霉素 Streptomycin

发根农杆菌菌株 Agrobacterium strain	冠瘿碱类型 Crown gallin type	抗性 Resistance
Ar.1193	农杆碱型 Agropine	链霉素 Streptomycin
K599	黄瓜碱型 Cucumopine	链霉素 Streptomycin
C58C1	农杆碱型 Agropine	链霉素 Streptomycin
ArQual	农杆碱型 Agropine	链霉素 Streptomycin
MSU440	农杆碱型 Agropine	链霉素 Streptomycin

引物名称 Primer name	引物序列 Primer sequences（5'-3'）	产物长度 Product length/bp
rolB-F	AAGTGCTGAAGGAACAATC	585
rolB-R	CAAGTGAATGAACAAGGAAC	585
HBFD1-F	ATGGCTCCTGTTCTCCTTGG	1 224
HBFD1-R	CGCTCCTTGTGATGCTCG	1 224

引物名称 Primer name	引物序列 Primer sequences（5'-3'）	产物长度 Product length/bp
rolB-F	AAGTGCTGAAGGAACAATC	585
rolB-R	CAAGTGAATGAACAAGGAAC	585
HBFD1-F	ATGGCTCCTGTTCTCCTTGG	1 224
HBFD1-R	CGCTCCTTGTGATGCTCG	1 224

发根农杆菌菌株 Agrobacterium rhizogenes strain	甘薯品种（系）Sweet potato varieties（lines）
发根农杆菌菌株 Agrobacterium rhizogenes strain	泰中6号 Taizhong No. 6	1610 1610	徐薯22 Xushu22	YS YS	灰薯 Huishu	平均诱导率 Mean induction rate
Ar.1193	56.85±4.38B	25.93±2.26EF	43.34±0.68CD	10.39±0.46HI	12.81±2.0GH	29.86
K599	59.19±4.51B	48.73±3.09BC	75.24±5.24A	25.43±1.70EF	15.46±1.67FG	44.81
C58C1	58.89±4.84B	57.16±4.76B	11.80±1.28HI	19.31±1.60FG	20.0±0.00FG	33.43
ArQual	42.94±3.84CD	22.26±1.73FG	79.40±1.29A	19.0±2.58FG	14.02±2.04FG	35.52
MSU440	36.53±4.28DE	24.60±2.75FG	79.63±1.85A	5.69±0.29I	18.38±2.38FG	32.97
平均诱导率 Average induction rate	50.88	35.74	57.89	15.97	16.13