Student ID: BI3-074

PLANT ENGINEERING
Hairy root culture
Agrobacterium-mediated transient expression

I.

Introduction

Hairy root culture is a common tissue culture method in plant genetic engineering.
Agrobacterium rhizogenes, a type of soil bacterium that contain root inducing (Ri) plasmids, are
often used to infect explants and promote the growth of hairy roots. These roots can be excised,
cleared of excess bacteria by antibiotics and grow indefinitely in vitro by subculture of root tip in
liquid medium. Various plant species have been successfully transformed to hairy roots, which
lead to a wide range of application in studying metabolic processes and producing valuable
secondary metabolites or recombinant proteins.
Transient gene expression is also another method used in plant engineering. Agrobacterium
tumefaciens carries T-DNA and when it infects a plant, the T-DNA will be transferred to a
random site in the plant genome. In further research, T-DNA is cut out of the plasmid and
replaced with the desired foreign gene to be expressed in the plant within a short period of time.
This method is reliable and often applied in tobacco. In addition, it does not require high cost or
concern ecological problems, and it is quite fast to observe gene expression.
In this experiment, we performed two techniques to make hair roots from leaf samples and see
the expression of influenza hemagglutinin in Nicotiana tabacum

II.
Hairy root culture
1. Materials
- Agrobacterium rhizogenes: wild type strain
- In vitro plant
- Mediums:
+ MS for plant tissue culture
+ YMB for bacteria culture
2. Procedure

a. Prepare bacteria cell suspension

- Bacteria stored at -80oC were activated by inoculation on solid YMB and cultured at
28oC for 2 days
- Transfer one single colony into YMB liquid media and culture on a shaking system at
150rpm and 28oC
- Remove YMB medium and harvest bacteria culture
- Dissolve bacteria with MS medium to make cell suspension
b.
-

Hairy root culture

Prepare explants based on leaf discs method: cut leaves into small pieces
Inoculate explants with Agrobacterium cell suspension for 10 minutes
After infection, explants were transferred to MS solid medium and co-cultured in 2
days
After 2 days, explants were cultured on hormone-free MS solid medium contain
antibiotics
Hairy roots should be formed after 3-4 weeks. Then, these roots will be screened,
selected and cultured on hormone-free medium

3. Result and discussion

Figure 1. Explants in antibiotic-containing MS medium

t
Figure 2. Samples after 19 days

The result was collected after 19-day culture in hormone-free MS medium. No contamination
was found. On the other hand, it can be seen that no hairy roots were observed. This could be due
to several reasons. Firstly, the time of culture in the medium might not be enough for hairy roots
formation. Secondly, transfection could also be not good. For example, the inoculation of
explants with bacteria occurs in a short period of time, therefore transfection cannot be effective.
However, with a long-time inoculation, the samples can be affected in a negative way. In short,
the duration of inoculation need to be appropriate. Apart from that, some of the explants
remained green color while the others turned yellow. This could be attributed to strong damage
happened while carrying out the experiment, which caused samples to die soon after they were
cultured.
III.
Agrobacterium-mediated transient expression
1. Materials
- Nicotiana tabacum
- Agrobacterium tumefaciens carrying binary vector expression for influenza
hemagglutinins (A/H7N9)
- Agrobacterium tumefaciens containing binary vector for expression of HcPro
suppressor of gene silencing were coinfiltrated
- Infiltration buffer: 10mM MES, 10mM MgSO4, pH 5.6
- Medium: LB (5g/L yeast extract, 10g/L tryptone, 10g/L NaCl, 9g/L agar,
autoclaving)
- Antibiotic: Kanamycin 50mg/mL, Rifamycin 50mg/mL, Carbemycin 50mg/mL
2. Procedure
- Agrobacteria carrying shuttle vectors for expression of HA and plant vector for
expression of HcPro were pre-cultivated separately in 5mL of YEB medium with

50g/mL Kanamycin, 50g/mL Carbemycin, 50g/mL Rifamycin, grown overnight

at 28oC and 140rpm
- Transter 1mL pre-cultured into 200mL medium culture containing appropriate
antibiotics, grow overnight at 28oC and 140rpm
- After 24h of cultivation, bacteria were harvested by centrifugation at 5000rpm,
10min, 4oC and resuspended in infiltration buffer
- Agrobacterium suspensions were diluted in infiltration buffer to a final OD600 of 0.6
- Nicotiana tabacum plants were infiltrated by completely submerging each plant in the
Agrobacterium-containing cup standed inside a desiccator
- Vacuum was applied during 2 minutes and then quickly released.
- The plants were then placed in the greenhouse at 21oC, 16h light per day
- 5 days after infiltration, leaf samples were harvested and stored at -80oC
- Protein were extracted from leaf samples. Electrophoresis (SDS-PAGE) and Western
blot were performed
3. Result and discusstion
M

(-)control (+)control

HA1

HA2

HA: 95 kDa

OD600 of bacterial solution was 0.59 and can be considered to present good concentration. After
extraction, proteins went under gel electrophoresis and Western blot with anti c-myc antibodies
as primary antibody and enzyme-conjugated secondary antibodies which oxidized an organic
compound called DAB to make brown color. It was calculated that the amount of protein per
well was 30g. There was no mark in the negative control (which is MES buffer) and the marker
was also good. It means that the kit worked well and there was no problem. HA was detected at
molecular weight of 95 kDa.
IV.

Conclusion

Hairy roots culture and transient gene expression are two common methods in research field of
plant engineering. What we have done was not completely successful but we have some
perspectives of how the experiments are carried out, how to interpret the results and gain some
experiences. To summarize, it is crucial to have good laboratory skills and concentration while
performing any experiments to avoid contamination and earn good results.