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Effect of Cytokinins on in Vitro Regeneration of
Cardamine Hirsuta from Nodal Explants
Weng Hing Thong and Kasturi Madhialagan INTI International University, Nilai, Negeri Sembilan, Malaysia
Email: [email protected]; [email protected]
Abstract—An efficient protocol has been established for the
regeneration of Cardamine hirsuta. The objective of this
study was to evaluate the effect of types and concentrations
of cytokinins on in vitro regeneration of C. hirsuta, an
important medicinal and edible plant. Nodal explants of C.
hirsuta were cultured in Murashige & Skoog (MS) media
containing kinetin or zeatin singly at concentrations of 1.0,
2.0, 3.0, 4.0, and 5.0 mg /L. MS medium devoid of plant
growth regulators served as control. The growth parameter
observed including number of shoots, leaves, and roots. 2.0
mg/L zeatin induced the highest number of shoots and
leaves. 1.0 mg/L kinetin-treated explants showed the highest
formation of roots. From the results obtained, it was learnt
that 2.0 mg/L zeatin was a more effective cytokinin
compared to kinetin for in vitro regeneration of C. hirsuta as
it induced the highest shoot and leaf growth while
minimizing root formation. Concentrations of zeatin above
optimal level not only caused stunted growth, but also
resulted in hyperhydration. In conclusion, in vitro
regeneration can serve as an alternative propagation
method for C. hirsuta.
Index Terms—Cardamine hirsuta, in vitro regeneration,
cytokinin, zeatin, kinetin
I. INTRODUCTION
Cardamine hirsuta L., commonly known as hairy
bittercress, is a herb that belongs to the genus Cardamine,
and family Brassicaceae (mustard) [1]. C. hirsuta is a
medicinal plant that has demonstrated antipyretic and
diuretic properties which are important in treating urinary
and bladder inflammations, dysentery, and white
discharges [2]. Besides, it can also be used to cure
insomnia and toothache [3]. It is a potherb that can be
eaten raw or cooked. The edible parts include flowers and
leaves, which are commonly added in salad for their
peppery taste [4]-[6].
In vitro regeneration (micropropagation) is a plant
tissue culture protocol to produce many identical copies
of a plant from its tissue segment via in vitro techniques
[7]-[9]. In general, a fragment of a germ-free plant tissue
or explant from the “mother plant” is cultured in an
axenic nutrient medium [8]. The medium composition
and physical conditions are manipulated to direct the
desired pattern of growth and reproduce the whole plant,
where the newly produced plants are clones of the
“mother plant” with identical genetic make-ups [8].
Manusript received September 13, 2014; revised December 3, 2014.
The development of a desired tissue is caused by the
action of plant growth regulators (PGRs) or plant
hormones [9]. Cytokinin is a type of PGR that functions
to induce cell division and protein synthesis, leading to
many plant processes such as delay of aging, chloroplast
formation, uptake of resources, wound healing, and
vascular growth [10] [11].
Cytokinin primarily stimulates development of shoots
and prevents the development of roots. When applied to a
shoot culture medium, apical dominance which is
controlled by auxin is inhibited, thus leading to lateral
bud shooting [7] [12]. Axillary bud formation is an
effective pathway of plant tissue culture initiated by
cytokinins [10].
In vitro regeneration technique is suggested to be
carried out to enable commercial exploitation and large
scale production of C. hirsuta for its medicinal
properties. The aim of this study was to study the effect
of different types and concentrations of cytokinins on in
vitro regeneration of C. hirsuta from nodal explants.
II. MATERIALS AND METHODS
A. Plant Material
Pre-cultured C. hirsuta was obtained from
Biotechnology lab of INTI International University as the
source of explants.
B. Effect of Types and Concentrations of Cytokinin on
the Growth of C. hirsuta
Nodal segments of approximately 1.0 cm long were
isolated from in vitro plantlets of C. hirsuta using sterile
scalpels in laminar air flow cabinet. Each excised stem
was placed vertically on culture medium supplemented
with either kinetin or zeatin at concentrations of 1.0, 2.0,
3.0, 4.0, or 5.0 mg/L. Randomization of culture vials was
done prior to culture initiation to ensure non-biased
culturing. The growth of plantlets in terms of number of
shoots, number of leaves, and number of roots was
observed weekly for 8 weeks continuously.
C. Statistical Analysis
Means among replicates was obtained using Microsoft
Excel 2010. Data collected were statistically analyzed by
one way analysis of variance (ANOVA) with 95%
confidence level.
III. RESULTS
742014 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 2, No. 2, December 2014
doi: 10.12720/jolst.2.2.74-77
A. Effect of Types and Concentrations of Cytokinin on
the Growth of C. hirsuta
At the end of observation period (week 8), plantlets
treated with different types and concentrations of
cytokinins were compared with control plantlet which
was not treated with any plant growth regulators (Fig. 1).
Figure 1. (A) Effect of different concentrations of kinetin on the growth of C. hirsuta on week 8. (B) Effect of different concentrations of
zeatin on the growth of C. hirsute on week 8.
Fig. 2 shows that there was no significant difference in
shoot numbers within kinetin-treated plantlets, while
there was significant difference within zeatin-treated
plantlets. The highest number of shoots with a total of 31
shoots was formed by nodal explants treated with zeatin
at an optimal concentration of 2.0 mg/L. However,
kinetin induced a much lower number of shoot, 15 shoots
at its optimal concentration of 3.0 mg/L. the control
medium without cytokinins only produced 1 shoot per
explant.
On the other hand, there was no significant difference
in leaf numbers obtained within the tested concentration
range of each cytokinin. Plantlets treated with 2.0 mg/L
zeatin, with 52 leaves, recorded the highest number of
leaves among other treatments. Plantlets treated with 3.0
mg/L kinetin produced 32 leaves in average. MS medium
with cytokinins induced the lowest number of leaves, 17
per explant.
The maximum number of roots was achieved in
explants treated with 1.0 mg/L kinetin, with 15 roots.
Meanwhile, zeatin yielded the lowest number of roots for
all concentrations, compared to kinetin-treated plantlets
and control. 1.0 mg/L zeatin only induced 4 roots per
explant in average.
Figure 2. Different morphologies of C. hirsuta on the 8th week of
observation. (A) Normal morphology of C. hirsuta with multiple shoots in the presence of cytokinin. (B) Hyperhydricity of C. hirsuta at high
concentrations of cytokinin, causing plantlet to appear brittle and glassy. (C) Stunted growth of shoots and leaves of C. hirsuta at high
concentrations of cytokinin, causing plantlet to appear compact.
At the end of this study, abnormal morphologies of
plantlets were also observed, which include
hyperhydricity and stunted shoot growth (Fig. 2). These
phenomenons were seen in several plantlets treated with
zeatin at high concentrations (3.0 mg/L - 5.0 mg/L).
Hyperhydrated plantlets appeared glassy and brittle while
plantlets with stunted shoots appear compact, with
multiple shoots that were short, and small leaves.
IV. DISCUSSION
A. Effect of Types and Concentrations of Cytokinin on
the Growth of C. hirsuta
In this study, control medium that was not
supplemented with cytokinin responded to growth.
However, it resulted in formation of a single shoot. The
growth of shoot was due to the presence of endogenous
cytokinin that naturally existed in the plant. However,
newly excised explants usually require more time to
manufacture cytokinins, and the absence of exogenous
cytokinin made it more difficult to undergo shoot
formation. This confirmed that exogenous cytokinin is
required in culture medium to achieve the adequate shoot
formation [13]. Hence, without the inclusion of cytokinin
in culture, explants will still be able to form shoots but
with lower yield. This explained the formation of a single
shoot in the control medium. On top of that, high
cytokinin levels generally induce multiple shooting by
inhibiting shoot apical dominance which leads to
formation of lateral shoots [10].
It was observed that the development of shoots was
consistent with leaves. The highest levels of shoot and
leaf numbers were observed in plantlets treated with
zeatin, with an optimal concentration of 2.0 mg/L (31
shoots and 52 leaves). In accordance to the results
obtained, a study done by [14] also indicated that zeatin
induced higher shoot proliferation than kinetin. [15]
stated that zeatin provided higher shoot proliferation
compared to BAP and kinetin. A study of effects on PGRs
on Labisia pumila var. alata demonstrated a higher
formation of shoots in media containing zeatin compared
to media containing kinetin [16]. Natural cytokinin,
zeatin, promoted higher rate of growth and survival of
shoot cultures of Asparagus plumosus compared to
synthetic cytokinins, kinetin and BAP [12]. A similar
result was obtained in plants of the Ericaceae family, in
which zeatin induced higher shoot proliferation rate than
other synthetic cytokinins [12]. High number of multiple
shoot formation in zeatin-treated medium was due to its
ability to induce rapid cell division in the meristem of the
nodal segment of C. hirsuta compared to kinetin and
BAP [17]. [18] demonstrated that formation of leaves was
slow in Arabidopsis plants without cytokinin supplement,
and the number of leaf cells reduced tremendously. [19]
demonstrated that expression of cytokinin gene (IPT7) in
tomato leaves increased the number of leaflets. These
signified that cytokinin acts as a positive regulator in both
shoot and leaf development.
Although cytokinin inhibits root formation [7] [20], in
rare cases, it had been shown that cytokinin-treated plants
752014 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 2, No. 2, December 2014
to have positive effects on root development [21]. A study
done by [22] showed positive effect of kinetin on rooting
of Matthiola incana. Root formation of tobacco stem
segments had been seen in medium provided with kinetin
[23]. [24] demonstrated formation of pseudonodules
initiated by exogenous kinetin on tobacco roots. In this
study, zeatin-treated plantlets had roots too, but the
number of roots was much lesser (0-4 roots) as compared
to kinetin-treated plantlets (5-15 roots). This indicated a
higher activity of cytokinin in zeatin-treated explants
which resulted in high shoot formation and minimal root
formation. Compared to kinetin, zeatin showed a more
significant activity of cytokinin, as the antagonistic
interaction between shooting and rooting was clearly
observed in zeatin-treated plantlets. Apart from that,
plants with low cytokinin activity generate more roots.
Plants with larger root systems are generally unfavorable
for growth of the plants as factors such as intake of
minerals and water would be higher, thereby limiting
growth. Therefore, zeatin is a more efficient and effective
cytokinin than kinetin in C. hirsuta.
The shoot proliferation in cytokinin-treated plantlets
increased as the concentration of PGR increased due to
rapid cell division triggered by the exogenous cytokinin.
Concentrations of zeatin and kinetin above optimal level
resulted in reduced number of shoots as the highly
concentrated cytokinin turned toxic to the plantlets.
Concentrations of zeatin above optimal level (3.0 mg/L –
5.0 mg/L) also resulted in changes of plantlet morphology,
such as plantlets with hyperhydricity, and stunted growth
of plantlets. Hyperhydricity, also known as vitrification
phenomenon is a physiological malformation associated
with lack of chlorophyll, poor lignification, and over
hydration of the plant tissues, leading to poor
regeneration or normal mature plants [25]. High
cytokinin level produced many small shoots that fail to
elongate [12]. In an experiment conducted by [16], high
levels of zeatin caused changes in morphology of the
newly formed plant. Hence, excessive cytokinin
concentration causes negative effects to growth and
morphology of plantlets.
V. CONCLUSION
The results obtained suggested that cytokinins were
significant growth regulator for C. hirsuta meristem, with
opposing shooting and rooting activity. Zeatin at
concentration 2.0 mg/L was the most effective cytokinin,
which induced the highest shoot and leaf proliferation
while minimizing root formation in nodal explants of C.
hirsuta. Explant treated with 2.0 mg/L zeatin developed
31 shoots, and 52 leaves. Kinetin induced the highest root
growth at 1.0 mg/L concentration (15 roots) therefore it
was less efficient compared to zeatin. Concentrations of
cytokinin above the optimal level caused toxicity to nodal
explants of C.hirsuta, reduced growth of shoots and
leaves, and in rare occasions, developed abnormal
morphology.
In conclusion, in vitro regeneration can serve as an
alternative propagation method for commercialization of
C. hirsuta from nodal explants.
ACKNOWLEDGMENT
We wish to thank INTI International University for
funding this research under INTI research grant: INT-
FHLS-05-02-2012.
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Weng Hing Thong originated from Penang, Malaysia. He attained his doctoral degree in
plant biotechnology from University Putra
Malaysia in 2010. Currently he works as Senior Lecturer at INTI
International Univeristy, Malaysia. He is active in conducting research in Biotechnology
and Education. He has published
“Encapsulation of nodal segments of Lobelia chinensis” in proceeding of Developing real-
life learning experiences: Learning innovation for ASEAN in Bangkok in 2013 and “Online forum in biotechnology education: a study from the
students’ perspective” in Acadamic Research International with has
colleague in 2012.
Kasturi Madhialagan
originated from
Selangor, Malaysia. She attained Bachelor of
Biotechnology from INTI International University Malaysia in August 2014.
Currently she works as Research Assistant at INTI International Univeristy.
Ms Kasturi received “Dean’s Honor Role”
from INTI International Univeristy and “Outstanding Interactor” (Interact Club) from
Rotary Club of Klang, Malaysia.
772014 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 2, No. 2, December 2014