THE EFFECT OF SUBCULTURE ON HAIRY ROOT

DEVELOPMENT OF JAVA GINSENG

(Talinum paniculatum Gaertn.)
A. Yachya* and Y.S.W. Manuhara$
*Department of Biology
Airlangga University, Surabaya, Indonesia
*e-mail: a_yachya@yahoo.com
$e-mail: wulanmanuhara@unair.ac.id

Abstract
Java ginseng (Talinum paniculatum Gaertn.) root was reported to have efficacy similar to the Korean ginseng (Panax
ginseng) root. Hairy root induction of Java ginseng on free hormone solid medium Murashige and Skoog (MS) using
genetic transformation mediated by Agrobacterium rhizogenes had succesfully performed. 2 - 3 cm in lenght of hairy
roots were initiated from cut edge of leaves after two weeks incubation and were used for subculture experiments on
solid and liquid MS medium without hormone. Subcultures were performed 4 times for two weeks period. Final
incubation of the fourth subculture showed differences of medium type (solid and liqiud) affected hairy root
development. Hairy roots on solid MS medium grew straight and had no brancing, whereas hairy roots on liquid
medium had branching and forming ball like structures (2-3 structures/cm). Hairy roots which were subcultured on
liquid medium looked thicker than on solid medium. The thickness of hairy root, branching and formation ball like
structure indicated suitability liquid medium as subculture medium than solid medium.
Keywords: hairy root, subculture, Talinum paniculatum Gaertn.

1.

Introduction

disease (Choi et al., 2008). Talinum paniculatum

Gaertn., as known "Java Ginseng" was reported its root
has medicinal property similar to the Korean Ginseng
(Panax ginseng) root (Wijayakusuma, 1994). Hairy
root cultures of T. paniculatum and its optimation on
biomass growth and secondary metabolite synthesis
have been conducted in our laboratory. Generally, the
next step after hairy root induction is subculture. The
decontaminated hairy roots can be subcultured for
periodic culture maintenance or batch reactor runs (Hu
and Du, 2006., Rijwani et al., 1998). It was reported
that subculture cycle of Catharanthus roseus hairy
root on liquid medium effected on growth rate and the
production of secondary metabolites (Rijwani et al
1998). The effect of subculture using different type of
medium (solid and liquid) on T. paniculatum hairy
root development has not been reported previously.
The objective of this study was to observe the effect of
subculture using solid and liquid hormone free medium
on development of T. paniculatum hairy root.

Hairy root is a plant disease caused by

Agrobacterium rhizogenes Conn., a Gram-negative soil
bacterium. When the bacterium infects the plant, the TDNA between the TR and TL regions of the Riplasmid in the bacterium is transferred and integrated
into the nuclear genome of the host plant. Hairy roots
grow rapidly, show plagiotropic growth, and are highly
branched on phytohormone-free medium (Hu and Du,
2006). A. rhizogenes transformed hairy roots
synthesize the same component as does the roots of the
intact plants and have a fast growth property in
hormone-free medium. Hairy roots provide an efficient
way of biomass production due to fast growth and
displays high biosynthetic capabilities that are
comparable to those of natural roots (Choi et al., 2008).
Hairy root culture can be an efective method of
produce useful secondary metabolites in medicinally
important plants like ginseng that grow slowly
(Washida et al., 1998; Court, 2006). Cultivation of
ginseng requires at least more than four years under
shade condition and also requires the careful control of

2.

Hairy root subculture

Hairy roots selected were transferred into 250
mL Erlenmeyer flask containing 100 mL liquid MS
medium without hormone and 100 mL bottle
containing 10 mL MS agar medium without hormone.
10 hairy root were inserted into a Erlenmeyer flask and
10 bottles (each bottle contained 1 hairy root).
Cultures were subcultured 4 times. Hairy roots on solid
medium were subcultured by transferring to fresh solid
medium every 2 week. Hairy root in liquid medium
were subcultured by pipetting aseptically and
substituted with fresh liquid medium. The liquid
culture was shaken at 110 rpm in the dark at 28C and
solid cultures had incubated similar with liquid culture
without agitation. Observation of hairy roots
morphology was done at the end of fourth subculture
day.

Materials and Methods

Hairy root induction

The isolate A. rhizogenes LB 510 was obtained
from collection of the Research Center of
Biotechnology, LIPI Bogor. A bacterial suspension
was prepared by inoculating a loop of the colony into
the Lauria-Bertani (LB) broth (10 g/L Bacto tryptone,
5 g/L Bacto yeast-extract, 5 g/L NaCl). Culture was
carried out in 250 mL Erlemeyer flask containing 50
mL Lauria Bertani medium, at 30C in rotary shaker
incubator 120 rpm for 2 days. 3 mL bacterial
suspention was diluted with 27 mL liquid Murashige
and Skoog (MS) free hormone medium to make
inoculation medium for explants and placed in 250 mL
Erlenmeyer flask. Leaves of T. paniculatum were cut
into small pieces using scalpel (approximately 1 cm2 in
size) and transferred into flask contain inoculation
medium. 100 uM acetosyringone was added into the
flask. This suspension was agitated slowly by hand for
5 minutes. After inoculation, the explants was removed
on sterile filter paper for blotting and then placed onto
MS agar without hormone. The cultures was incubated
at 28C at dark condition for 2 weeks. Hairy roots that
appeared on the cut edges of the plant were selected.
Hairy roots whose length 2-3 cm were detached and
used for subculture experiments.

3.

Results and discussion

The effect of subculture using solid and liquid

hormone free medium on development of T.
paniculatum hairy root was performed 4 times for 2
weeks period. Each hairy root morphology in flask and
ten bottles were observed at the end incubation of
fourth subculture day. Figure 1 shows hairy root
morphology that was subcultured on solid medium and
figure 2 on liquid medium.

5 mm
M 40X

rh

el
M 100X

mr

mt

Figure 1. Hairy root morphology was subcultured on solid MS medium without hormone. Subculture was
performed 4 times for 2 weeks period; A. whole hairy root at magnify 40x; B. zoom of part of
hairy root with red line mark at magnify 100x; el.elongation zone; mr. meristematic zone;
mt. maturation zone; and rh. root hair.

rh

bl

rb

M 40X
5 mm

B1

B2
mt

rb
rb

el

mr

mr
M 100X

M 100X

B4

B3
bl

bl

M 100X

M 100X

Figure 2. Hairy root morphology was subcultured on liquid MS medium without hormone. Subculture was
performed 4 times for 2 weeks period; A. whole hairy root at magnify 40x; B. zoom of part of hairy root with
red (1), blue (2), orange (3) and green (4) line mark at magnify 100x; el.elongation zone; mr. meristematic
zone; mt. maturation zone; bl. ball like structure; rb. root branching; and rh. root hair.

Hairy roots were subcultured on solid

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medium grew on agar medium and some hairy root
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tips troughted into agar. According Fiedler review

in liquid medium to grow faster .

Suitability between agitation rate and
nutrition level in liquid medium during subculture
also caused morpholgy change of T. paniculatum
hairy roots. Previous research reported an increase
in rocking rate of wafe bioreactor in accordance
with the medium feeding was characterised by
changes in morphology of Panax ginseng hairy
root clones, that was formation of ball-like
structures which show poor growth and changes in
branching, colour and root hair development (Eibl
and Eibl. 2008). The formation ball like structures
also appeared on T. paniculatum hairy roots that
were subculture in liquid medium. Base on
observation showed hairy root that subcultured in
liquid culture looked thicker than hairy root on
solid medium. It indicated suitability condition in
liquid culture could increase hairy root biomass.
Hairy root growth depended on the uptake of
sugar, water, and nutrients from medium. Sucrose
moves approximately 4-times faster in stationary
water than agar gel. Faster uptake of sucrose,
ammonium, nitrat, calcium and potassium by hairy
roots in liquid culture related to greater fresh
weight and results in higher dry weights (Adelberg,
2008). Formation ball like structure and the
thickness of hairy root in liquid culture indicated
easy uptake sucrose and other nutrition to hairy
root tissues. Its also could influence to synthesis
and accumulation of secondary metabolite.
Formation ball like structures coul be
representative of storage root. The thickness of
hairy root, branching and forming ball-like
structure indicated suitability liquid medium as
subculture medium than solid
medium as
intermediate culture for establishing bioreactor
techniques (Takayama and Akita, 2008).

(1938/1939), culture conditions on agar medium

got optimal oxygen supply, because the cells,
tissues and organs on the medium surface (Preil,
2005). Otherwise, limitation on liquid culture is
oxygen mass transfer and mixing (Curtis, 2005).
Liquid culture of root was success achieved by
White (1934), tomato root tips continuous growth
in liquid medium when excising and subculturing
newly-formed root meristems (Preil, 2005). Hairy
roots were subcultured on liquid medium grew
under submerged condition and have different
morphology with hairy root grew on solid medium.
Figure 1 shows, Hairy root on solid medium grew
straight and had no brancing. Figure 2 shows, hairy
root on liquid medium has branching and forming
ball like structure. Takayama and Akita, 2008
reported that the growth response of the plants in
liquid medium varied between species or genera.
Branching and formation ball like structure
of hairy roots that subcultured on liquid medium
indicated higher multiplication rates of hairy root
cells which were grew in liquid medium. Higher
multiplication rates in liquid medium compared to
cultures on agar gelled medium was reported for
many species (Preil, 2005). Douglas, 1984 reported
that some Rhododendron cultivars, shoot
production in liquid medium was 10-fold higher
than on agar-gelled medium. Lower multiplication
rates of hairy roots on solid medium caused by
limitation to access nutrition from medium
although the culture had optimal oxygen. It caused
by agar as gelling agent. Agar or other organic
gelling agents, are frequently used despite problems
of limited availability of solutes to the tissue (Smith
and Spomer. 1995; Williams. 1995; Leifert et al.,
1995). Solute movement through gelled media and
transfer to the plant is primarily by diffusion
(William, 1993) and the limitation by agar limited
multiplication of hairy root cells. On the contrary,
hairy roots that were subcultured in liquid medium
had well access of nutrition but limeted by oxygen
mass transfer and mixing. Apply agitation on
culture and 2 week subculture periodic could cover
oxygen demand of hairy root in medium.
Furthermore, agitation in shake-flask culture make
the entire plant surface is available for nutrient
exchange (Edelberg, 2008). Fulfilled oxygen demand and easy access nutrition made suitable
condition for hairy root cells that were subcultured

4.

Conclusion

Growth pattern of T. paniculatum hairy root

at subculture stage on solid and liquid medium
was observed in this research. Base on morphology
References
change was known, different type of medium had
effect to hairy root development. The result
showed liquid medium gave suitable condition for
haiy root development and qualified as medium
type of subculture stage than solid medium for
M 100X
culture maintenance or establishing bioreactor
techniques.

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