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Frontiers of Agricultural Science and Engineering

ISSN 2095-7505

ISSN 2095-977X(Online)

CN 10-1204/S

Postal Subscription Code 80-906

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Front. Agr. Sci. Eng.    2020, Vol. 7 Issue (4) : 513-522    https://doi.org/10.15302/J-FASE-2019272
RESEARCH ARTICLE
Optimizing hairy root production from explants of Phyllanthus hainanensis, a shrub used for traditional herbal medicine
Zhaogui YAN1,2, Shengyu LIU1,3, Junlian ZHANG4, Guan HUANG1, Lijun DUAN1, Yaomei YE1()
1. College of Horticulture and Forestry Sciences/Hubei Engineering Technology Research Center for Forestry Information, Huazhong Agricultural University, Wuhan 430070, China
2. School of Environmental & Rural Science, The University of New England, Armidale, NSW 2351, Australia
3. Wuhan Garden Constructional Engineering Company, Wuhan 430050, China
4. Hubei Wildlife and Plant Conservation Agency, Wuhan 430070, China
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Abstract

Phyllanthus hainanensis is a shrub that has been used in traditional herbal medicine. It has great pharmaceutical potential for treating diseases such as cancer and diabetes. As a prerequisite for propagation of this species on a large scale, hairy roots in P. hainanensis were induced using Rhizobium rhizogenes and various factors affecting hairy root induction and growth evaluated. Seven factors were tested: (1) type of explant, (2) type of culture medium, (3) duration of pre-culture, (4) R. rhizogenes inoculum cell density, (5) duration of infection, (6) acetosyringone concentration in the culture medium, and (7) duration of incubation. The optimal protocol for hairy root induction and growth was: young shoots, pre-cultured in Y1 for 2 d, inoculated with R. rhizogenes broth with an OD600 of 0.6 for 20 min, and incubated for 3 d. Putative transgenic hairy roots were initially identified by morphology and then confirmed by polymerase chain reaction. Successful and optimal production of hairy roots is a critical prerequisite for industrial scale clonal propagation of P. hainanensis. Being able to cultivate the plant on a large scale will provide rapid and ready supply of the plant materials that can be used in herbal medicine and in scientific and industrial exploitation.
Keywords hairy root induction      medicinal plant      MS growth medium      Phyllanthus hainanensis      Rhizobium rhizogenes     
Corresponding Author(s): Yaomei YE   
Just Accepted Date: 13 June 2019   Online First Date: 19 July 2019    Issue Date: 06 November 2020
 Cite this article:   
Zhaogui YAN,Shengyu LIU,Junlian ZHANG, et al. Optimizing hairy root production from explants of Phyllanthus hainanensis, a shrub used for traditional herbal medicine[J]. Front. Agr. Sci. Eng. , 2020, 7(4): 513-522.
 URL:  
https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2019272
https://academic.hep.com.cn/fase/EN/Y2020/V7/I4/513
Factor Level
Type of explant Leaves, young shoots and roots
Type of culture medium* MS and Y1
Duration of pre-culture 0, 1, 2, and 3 d
Inoculum cell density (OD600) 0.2, 0.4, 0.6, 0.8, and 1.0 in MS+ 100 mmol·L−1 AS broth
Infection time 5, 10, 15, 20, 25, and 30 min at OD600 of 0.5–0.6
Concentration of acetosyringone (AS) in the culture medium 50, 100, 150, and 200 mmol·L−1
Incubation time 0, 1, 2, 3, 4, and 5 d
Tab.1  Experimental design for testing factors affecting hairy root production of Phyllanthus hainanensis
Growth medium Explant type Hairy root induction rate/% Number of roots/Explant Average root length/mm
MS Mean 19.4±4.8 b 1.2±0.3 a 48±10 a
Leaf 2.8±2.8 c 0.3±0.3 b 9±9 b
Young shoot 33.3±4.8 ab 1.6±0.1 a 69±6 a
Root 22.2±2.8 b 1.8±0.4 a 66±5 a
Y1 Mean 29.6±6.7 a 1.5±0.2 a 59±9 a
Leaf 8.3±4.8 c 0.8±0.4 b 32±16 b
Young shoot 50.0±4.8 a 1.9±0.1 a 74±6 a
Root 30.6±7.3 b 1.9±0.2 a 70±7 a
Tab.2  Effect of explant type and pre-culture growth medium on hairy root development of Phyllanthus hainanensis
Fig.1  The effects of explants, growth medium, and Rhizobium rhizogenes incoulum cell density on hairy root development of Phyllanthus hainanensis. (a) Leaf explants, day 0; (b) young shoot explants, day 0; (c) hairy roots from young shoot explants inoculated with R. rhizogenes OD600 0.6 and incubated on MS medium, day 30; (d) hairy roots from young shoot explants are inoculated with R. rhizogenes OD600 0.6 and incubated on Y1 medium, day 30; (e) hairy roots from root explants are inoculated with R. rhizogenes OD600 0.6 and incubated on Y1 medium, day 30; (f) hairy roots from young shoot explants are inoculated with R. rhizogenes OD600 1.0 and incubated on Y1 medium, day 14, showing browning of explants and bacterial contamination; (g) P. hainanensis plants of 3 months old growing in pots. Scale bar= 10 mm.
Gene P1 (5′–3′) P2 (5′–3′)
RolA GCT CGT TGT CTC CGA CCT AT GGT CTG AAT ATT CCG GTC CA
RolC ATG GCG GAA TTT GAC CTA TG TTA GTT CCA TCT GCC CAT CC
Tab.3  Primers for PCR amplification
Pre-culture time/d Hairy root induction rate/% Mean number of root Average root length/mm
0 11.1±2.8 c 1.7±0.3 a 25±2 a
1 22.2±7.3 bc 1.8±0.1 a 27±2 a
2 38.9±5.5 a 2.1±0.1 a 32±3 a
3 30.5±2.8 ab 1.9±0.2 a 29±3 a
Tab.4  Effect of pre-culture time on hairy root induction in explants of Phyllanthus hainanensis
Fig.2  Effect of cell density of Rhizobium rhizogenes broth concentration on hairy root induction rate (a) and growth (b) of Phyllanthus hainanensis. Means (n = 3) with the same letter are not significantly different (P≥0.05).
Fig.3  Effect of infection time on (a) hairy root induction rate and contamination rate and (b) hairy root development of Phyllanthus hainanensis. Means (n = 3) with the same letter are not significantly different (P≥0.05).
Fig.4  Effect of acetosyringone (AS) concentration on (a) hairy root induction rate and (b) hairy root development of Phyllanthus hainanensis. Means (n = 3) with the same letter are not significantly different (P≥0.05).
Fig.5  Effect of incubation time on (a) hairy root induction rate and (b) hairy root development of Phyllanthus hainanensis. Means (n = 3) with the same letter are not significantly different (P≥0.05).
Fig.6  PCR gels for hairy roots of Phyllanthus hainanensis with Ri plasmid of Rhizobium rhizogenes strain K599. (a) RolA-specific primer; (b) RolC-specific primer. M, DL 2000 DNA marker; 1–2, hairy roots; 3, normal roots (negative control); 4, R. rhizogenes K599 (positive control).
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