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
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.
. [J]. Frontiers of Agricultural Science and Engineering, 2020, 7(4): 513-522.
Zhaogui YAN, Shengyu LIU, Junlian ZHANG, Guan HUANG, Lijun DUAN, Yaomei YE. Optimizing hairy root production from explants of Phyllanthus hainanensis, a shrub used for traditional herbal medicine. Front. Agr. Sci. Eng. , 2020, 7(4): 513-522.
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
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
Fig.1
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
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
Fig.2
Fig.3
Fig.4
Fig.5
Fig.6
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