Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots

doi:10.11983/CBB24092

Abstract

Abstract: INTRODUCTION:An efficient Agrobacterium rhizogenes-mediated transformation system for Pueraria lobata was established. RATIONALE: In this study, tissue-cultured plantlets of P. lobata were used as explants to investigate the effects of different genotypes, A. rhizogenes strains, explants, precultivation times, infection times, culture days, subculture times, and culture methods on the efficiency of hairy root genetic transformation in P. lobata. RESULTS: The results indicated that the induction rate of hairy root formation was the highest when the immature leaves of YG-19 were used as the explant material, reaching 10.2%. A. rhizogenes K599 was identified as the most suitable strain. The optimal explant material was immature leaves that had just unfolded from the first to second nodes of the 5^th to 13^th generation tissue culture plantlets subcultured for 8 days. After 3 days of pre-culture and 15 minutes of bacterial infection, the highest induction rate of hairy roots reached 22.4%. The optimal type of culture medium for the proliferation of hairy roots in P. lobatawas solid medium culture, and the fresh weight of hairy roots grown on solid medium was 75 times greater than that of hairy roots grown in liquid medium. PCR detection and fluorescence microscopy assays revealed that the expression of GFP and rolB genes in the hairy roots of P. lobata was stable, and the rate of cotransformation was 80%.CONCLUSION: Genotype, A. rhizogenes strain, and culture duration were the most critical factors for the efficient genetic transformation of hairy roots in P. lobata.

Key words: Pueraria lobata, Agrobacterium rhizogenes, hairy roots, genetic transformation

Zeng Wendan, Yan Huabing, Wu Zhengdan, Shang Xiaohong, Cao Sheng, Lu Liuying, Xiao Liang, Shi Pingli, Cheng Dong, Long Ziyuan, Li Jieyu. Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots[J]. Chinese Bulletin of Botany, 2025, 60(3): 425-434.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24092

https://www.chinbullbotany.com/EN/Y2025/V60/I3/425

Figures/Tables 12

References 33

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Primer name	Primer sequence (5′−3′)
GFP	F: CAGTGCTTCAGCCGCTAC R: TTCTCGTTGGGGTCTTTG
rolB	F: AAGTGCTGAGGAACAATC R: CAAGTGAATGAACAAGGAAC
virG	F: CCTTGGGCGTCGTCATAC R: TCGTCCTCGGTCGTTTCC

Primer name	Primer sequence (5′−3′)
GFP	F: CAGTGCTTCAGCCGCTAC R: TTCTCGTTGGGGTCTTTG
rolB	F: AAGTGCTGAGGAACAATC R: CAAGTGAATGAACAAGGAAC
virG	F: CCTTGGGCGTCGTCATAC R: TCGTCCTCGGTCGTTTCC

Genotype	Hairy root frequency of different explants (%)
Genotype	Unfolding im- mature leaves	Mature leaves	Stems	Petioles	Shoot me- ristems
YG-18	0	0	0	0	0
YG-19	10.2±0.9	6.1±0.1	0	5.2±1.2	0
YG-50	0	0	0	0	0
YG-51	1.9±0.6	0	0	3.5±0.4	0
YG-81	2.1±0.3	1.1±0.4	0	1.9±0.1	0

Genotype	Hairy root frequency of different explants (%)
Genotype	Unfolding im- mature leaves	Mature leaves	Stems	Petioles	Shoot me- ristems
YG-18	0	0	0	0	0
YG-19	10.2±0.9	6.1±0.1	0	5.2±1.2	0
YG-50	0	0	0	0	0
YG-51	1.9±0.6	0	0	3.5±0.4	0
YG-81	2.1±0.3	1.1±0.4	0	1.9±0.1	0

Agrobacterium rhizogenes	Rooting time (d)	Hairy root frequency (%)	Hairy root density
CK	-	0 c	0 c
K599	14	12.1±0.6 a	2.2±0.1 a
R1601	16	8.3±0.5 b	1.1±0.1 b
ATCC15834	-	0 c	0 c
Ar4	-	0 c	0 c