Rapid Identification of Gene StHKT1’s Function via Potato Hairy Root System

doi:10.13560/j.cnki.biotech.bull.1985.2025-0658

Abstract

Abstract:

Objective To establish a simple, high-efficiency and tissue culture-free transformation method and validatethefunction of StHKT1 gene, which will provide a theoretical basis for large-scale gene function studies and germplasm improvement in potato. Method Terminal buds of potato were scraped on the plate containing Agrobacterium rhizogenes and then planted on MS solid medium to induce hairy roots. The induction and transformation efficiency of hairy roots were evaluated. The expressions of the StHKT1 gene in hairy roots were detected by qRT-PCR. Both control plants and composite plants overexpressing StHKT1 gene were treated with NaCl to analyze the tolerance to salt. Result The composite plants with abundant hairy roots were successfully obtained after infecting terminal buds for 30 d. The induction and transformation efficiency of the hairy roots were 100% and 87.4% respectively, and the relative expression of the StHKT1 gene in the hairy roots of the composite plants significantly increased. Under 100 mmol/L NaCl stress, the composite plants showed better growth performance in root length and fresh weight compared to control plants. The StHKT1 gene was inducted by different concentration salt stress, and the weight of the transgenic hairy roots was significantly higher than that of controls. Finally, Under 200 mmol/L NaCl stress, the growth status of the composite potato plants remained superior to controls. Meanwhile, the MDA content decreased significantly in the composite plants, while the chlorophyll content and SOD activity increased significantly. Conclusion Composite plants can be rapidly obtained via Agrobacterium rhizogenes-mediated hairy root transformation in potato, and the hairy-root composite potato plants overexpressing StHKT1 show the enhanced tolerance to salt.

Key words: hairy root transformation, Agrobacterium rhizogenes, potato, composite plants, StHKT1

SONG Qian-na, WU Shi-yun, CAO Shi-jin, DUAN Yong-hong, FENG Rui-yun. Rapid Identification of Gene StHKT1’s Function via Potato Hairy Root System[J]. Biotechnology Bulletin, 2025, 41(10): 233-241.

Figures/Tables 8

Fig. 1 Phylogenetic trees constructing and domain analysis of StHKT1 proteinA: Phylogenetic trees. B: Domain of StHKT1 protein. C: Sequence alignment of p-loop (PA, PB, PC and PD) of StHKT1 protein

Fig. 2 Genetic transformation of potato hairy roots and obtaining of composite plantsA: Ideal potato seedings for transformation. B: Explants on terminal bud. C: Infected by MSU440 Agrobacterium rhizogenes. D: Explants co-cultured for 2 d. E: Hairy roots of induction 10 d after inoculation. F: Selection of GFP-positive hairy roots. G: Potato plants with composite. H: The composite plants cultured continuously

Table 1 Evaluation on the induction efficiency and transformation efficiency of hairy roots

转化

Transformation

毛状根诱导效率

Induction efficiency of hairy roots

毛状根群体

Hairy root clones

转化效率

Transformation efficiency

Fig. 3 Detection of relative expressions of StHKT1 in the composite potato plantsA: PCR detection of StHKT1 geneintegration. P: Plasmid; W: wild type; K: ddH2O. B: Detecting relative expression of StHKT1 by RT-qPCR. The different lowercase letters indicate P<0.05. The same below

Fig. 4 Analysis of tolerance to salt and StHKT1 expression pattern of hairy roots in the composite potato plantsA: Increase ratio of hairy roots under different NaCl concentrations. B: Relative expression of StHKT1 in hairy roots under different NaCl concentrations. VC and OE contain mixed samples of hairy roots from three independent composite plants

Table 2 Growth indicators of the composite potato plants under salt stress

样本 Sample	NaCl浓度 Concentrations of NaCl （mmol/L）	根长 Length of root （cm）	根数 No. of roots	根鲜重 Fresh weight of root （g）	茎长 Length of shoot （cm）	植株鲜重 Fresh weight of plant （g）
VC	0	9.833±0.763a	3.000±1.000b	0.021±0.008a	4.567±0.551a	0.059±0.001a
VC	100	5.300±0.650bc	3.000±1.000b	0.006±0.001b	1.967±0.642b	0.039±0.002bc
OE	0	9.600±2.970a	5.000±0.001a	0.025±0.021a	5.100±0.529a	0.080±0.012a
OE	100	6.900±0.360b	4.000±1.000b	0.007±0.002b	2.967±0.586b	0.059±0.008b

Fig. 5 Phenotypic analysis of StHKT1-overexpressed composite potato plants under salt stressA-B: Growth status of composite plants treated with salt for two weeks. C: Growth status of hairy roots of composite treated with salt for two weeks

Fig. 6 Physiological characteristics of overexpressing StHKT1 composite potato plants under salt stressA: Relative expression of StHKT1 in the hairy roots under salt stress. B: Chlorophyll content in leaf. C, E: SOD activity in leaf and hairy roots. D, F: MDA content of leaf and hairy roots. OE-1, OE-2 and OE-3 refer to three independent hairy roots from composite plants; VC and OE contain mixed samples of leaves and hairy roots from three independent composite plants

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