effect of cytokinins on in vitro regeneration of cardamine ... · pdf fileeffect of cytokinins...

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

mailto:[email protected]

A. Effect of Types and Concentrations of Cytokinin on


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


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



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.



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