ISSN: 2224-0616

Int. J. Agril. Res. Innov. & Tech. 1 (1&2): 64-68, December, 2011 Available at http://www.ijarit.webs.com

DEVELOPMENT OF EFFICIENT CALLUS INITIATION OF MALTA (Citrus

sinensis) THROUGH TISSUE CULTURE
Fazle Azim, M.M. Rahman, Shamsul H. Prodhan, Saif U. Sikdar,
Nayem Zobayer and M. Ashrafuzzaman*

Received 1 November 2011, Revised 24 December 2011, Accepted 25 December 2011, Published online 31 December 2011

Abstract
The effects of different hormonal concentration on shoot formation and callus induction
were studied on BARI Malta-1 (Citrus sinensis). Seeds containing seed coat and without
seed coat was treated by Murashige and Skoog (MS) media supplemented with 6-benzyl
adenine (BA) and Kinetin (KIN). Removal of seed coat showed an early response for shoot
formation. The highest (70%) shoot formation was obtained from seeds without seed coat
treated with MS basal media + BA 1.0 mg/l while KIN showed no response for shoot
formation in any supplemented concentration. However, in case of callus induction
internodes and apical shoot tips were used as explants and 2, 4-dichlorophenoxy acetic acid
(2, 4-D) was used as callus inducing hormone. MS basal media supplemented with 2, 4-D,
2.0 mg/l showed highest (68%) callus induction.
Key words: Malta, Citrus sinensis, In vitro, Seed shoot formation, Callus induction

Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology,
Bangladesh
*Corresponding author’s email: [email protected]
Reviewed by Dr. Iftekhar Ahmad, Shahjalal University of Science & Technology, Bangladesh
variations, somatic cell hybridization
Introduction (Kobayashi, et al., 1992; Deng et al., 2000),
Citrus sinensis is a member of Rutaceae family transformation of high yielding cultivars
(Citrus family) and has the common name like (Koltunow et al., 2002) disease free plants. But
sweet orange or naval orange (Christman, 2003) all these highly sophisticated technique requires
or Malta in Bangladesh. C. sinensis is one of the the presence of highly responsive regeneration
major commercial fruit crops that is widely protocol.
consumed both as fresh fruit or juice attributed to Through micropropagation method, there is a
its high vitamin C content and its antioxidant chance to establish a cell line of virus free Malta
potential (Kiong et al., 2008). As other fruits, or somaclonal varieties. The term somaclonal
citrus is attacked by several pre- and/or post variation was coined by Larkin and Scowcroft
harvest pathogens that affect fruit quality (Bekele, (1981) to define genetic variation present in
2007). regenerated plants that is either uncovered or
Advances in biotechnology have generated new induced by a tissue culture process. Somaclonal
opportunities for citrus genetic improvement. In variation has been reported in a wide range of
vitro propagation has therefore been a great traits including plant height, overall growth
potential tool to overcome problems related with habit, flower, fruit and leaf morphology,
the field culture for such species (Hidaka & juvenility, maturity date, disease resistance, yield
Omara, 1989). The genetic improvements of this and biochemical characteristics. However, most
perennial woody plant often take many years reports generally deal with either solanaceous or
using traditional plant-breeding methods (Kayim cereal crops and little information has been
and Koe, 2006). Hence, plant tissue culture reported in woody perennial fruit crops.
techniques can be applied as a helpful tool to Sweet orange is well-suited for studies of
reduce the time for improvement of Malta somaclonal variation because of its biology and
through somaclonal variation (Chandler et al., efficient performance in tissue culture. Sweet
1996). Techniques like in vitro culture made it orange plants have been regenerated via somatic
easy to improve citrus against different a biotic embryogenesis from protoplasts isolated from
stresses, low yield and conserve important citrus embryogenic callus or suspension cultures,
genotypes though exploiting somaclonal reported by Vardi et al. (1975). Citrus is also very

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responsive organogenically in-vitro, and Shoot formation media: Seed regeneration
adventitious buds can be induced in many citrus media were prepared by mixing all the
species from non-meristematic juvenile seedling components as the callus induction media except
explants (Grinblat, 1972; Barlass and Skene, hormones. In case of hormone BA varying in
1982). Recently, tissue culture techniques have concentration from 1.0 mg/l to 2.0 mg/l was
been adopted for consistent commercial used. The other steps i.e., agar melting,
production of economically important plants autoclaving etc were similar to callus induction
(Honda et al., 2001). Plant tissue culture media preparation. Explants were inoculated
technology has been successfully used for the and data were recorded.
commercial production of microbe free plants
Callus induction media: For callus induction
(Parmessur et al., 2002).
popular callus inducing hormone 2, 4-D was
The present study was done to develop an used. MS media (Murashige and Skoog, 1962)
efficient callus initiation system of Malta (Citrus supplemented with 2, 4-D varying in
sinensis) through tissue culture which might be concentration from 1.0 mg/l to 3.0 mg/l were
used in genetic transformation system and/or prepared separately in conical flasks. The 3%
efficient and suitable regeneration protocol of sucrose was added to each conical flask. The PH
malta in future. was adjusted to 5.6-5.8. Then 0.7% agar was
added. The agar was melted by boiling at 110ºC
Materials and Methods for 2-3 minute. After melting the agar, the media
This experiment was conducted at the Plant were poured into the test-tubes and autoclaved
Genetic Engineering Lab. of the Department of at 121ºC and 15 psi for 15 minute. After
Genetic Engineering and Biotechnology, Shahjalal autoclaving the media were allowed to cool down
University of Science & Technology (SUST), and coagulate in the Laminar Air Flow Cabinet.
Sylhet, Bangladesh. Inoculation of explant: Shoot tips and
internodes were inoculated in callus induction
Collection of explants media. For shoot formation of seeds, some seeds
were inoculated with seed coat and some were
not. The cultures were incubated at 25±2°C with
Sterilization of explants
16 h of photoperiod with the light intensity of
2000 lux under cool-white fluorescent lamps. All
the treatments were conducted with 5 replicates.
Preparation of culture media
Results and Discussion
Effect of different concentration of BA
Inoculation of explants and Kinetin on shoot formation: In vitro
shoot formation of seed (with seed coat and
without seed coat) on MS basal media
Observation supplemented with various concentrations of BA
and KIN were studied in this experiment. The
results of the treatments are summarized in the
The flow chart of method followed in this experiment table 1. Among all the treatments seeds treated
with BA 1.0 mg/l showed the highest (with seed
The detail of methods employed during this study
coat 36 ± 2.16 % and without seed coat 70 ± 3.16
is given below:
%) shoot formation (Fig. 1). Other
Collection of explant: The young malta (citrus concentrations of BA also showed various degree
sinensis) plants were collected from BRAC, of response on shoot formation. But KIN showed
Gazipur, Dhaka, Bangladesh before starting this no response for any kind of concentration.
experiment and grown in the pot. The malta seeds
Effect of seed coat on shoot formation of
were collected from local nursery of Sylhet. The
C. sinensis: C. sinensis contain two layers of
types of explants were seeds, internodes and seed coat. Some seed were inoculated with seed
apical shoot tips. The internodes and apical shoot
coat and some were without seed coat. The shoot
tips were aseptically excised and cultured on the
formation percentage was higher in seeds
callus induction medium.
without seed coat (Fig. 1). This is due to the fact
Explant sterilization: Seeds, internodes and that seed coat is barrier for nutrient passing to
shoot tips of C. sinensis were washed by using the seed. Therefore embryos of the seeds do not
detergents for 2 minutes. Then explants were get enough nutrients from the surrounding
immersed for 15 minutes with 2 or 3 drops of media and thus shoot formation process is
Tween-20. In order to remove all traces of delayed. Though seed coat protects embryo from
detergents and Tween-20 from the surface, unfavorable environments and microorganisms
explants were washed by sterile-distilled water for and helps to survive in nature, it is not necessary
3-4 times. for in vitro condition. Rather seed coat is the

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constraint of success for in vitro culture. Neidz embryo can get enough nutrients for shoot
(2008) also reported in vitro shoot formation formation with a relatively short time and thus
without seed coat. Since aseptic condition is percent seed shoot formation without seed coat
strictly maintained and all microorganisms are was increased within a short time. The frequency
eliminated from the culture media seed coat of shoot proliferation from the germinated seeds
should be removed during inoculation so that without seed coat was higher.
Table 1. Effect of BA and KIN on seed (without seed coat) germination of C. sinensis in MS medium

Hormone Concentration Number of explant % of shoot formation (A.M ± S.E.)

(mg/l) inoculated
0.5 20 64 ± 2.45
BA 1.0 20 70 ± 3.16
1.5 20 60 ± 3.16
2.0 20 40 ± 4.47
0.5 20 No shoot formation
KIN 1.0 20 No shoot formation
1.5 20 No shoot formation
2.0 20 No shoot formation
Here, A.M. = Arithmetic Mean and S.E.= Standard Error

90
80
70
60
% of germination

50
40 With seed coat
30
Without seed coat
20
10
0
BA 0.5 BA 1.0 BA 1.5 BA 2.0
Concentration of BA (mg/ l)

Fig. 1. Effect of seed coat on the shoot formation of C. sinensis. MS media supplemented with BA 1
mg/l showed best response for shoot formation where removal of seed coat showed 70± 3.16 % and
seeds containing seed coat showed 36 ± 2.16 % shoot formation

a b c

d e f

Fig. 2. Shoot formation of C. sinensis where a, b, c representing the poor shoot formation of seed
containing seed coat and d, e, f representing the rapid shoot formation of seed without seed coat. In
each case seeds were treated with MS basal media supplemented with BA 1.0 mg/l.

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Effect of different concentration of 2, 4-D During study of somatic embryogenesis Kiong et
on callus induction: Response of 2, 4-D on al. (2008) reported that 2, 4-D, 4.0 mg/l and 2,
callus induction by internodes and shoot tips as 4-D 3mg/l showed highest percent callus
explants was studied. Effects of 2, 4-D on callus induction. But they also found 2, 4-D, 2.0 mg/l
induction is showed in the table 2. The highest showed a good response. However, we also found
percent (68 ± 2.00 %) callus was obtained from 2, that 2, 4-D, 1.0 mg/l and 2, 4-D, 3.0 mg/l also
4-D, 2mg/l. This result is contradictory in the showed a moderate response. The callus
concentration of 2, 4-D with Kiong et al. (2008). induction from explants is showed in figure 3.

Table 2. Effect of 2, 4-D on callus induction from nodal segment of C. sinensis. 2, 4-D, 2.0 mg/l showed the best
response for callus induction
Concentration Number of Number of Survival Number of Percent of callus
of 2,4-D explant explant survived rate explants that give induction (A.M. ±
(mg/l) inoculated rise to callus S.E.)

1.0 25 20 80% 12 60± 4.47

2.0 25 25 100% 17 68± 2.00
3.0 25 20 80% 13 65± 2.44

Here, A.M.= Arithmatic mean and S.E.= Standard error

Fig. 3. Callus induction from nodal segment of C. sinensis. MS media supplemented with 2, 4-D 2.0 mg/l showed
best (68 ± 2.00 %) response for callus induction

Acknowledgement Deng, X.X., Yu, G.H. and Guo, W.W. 2000.

Somatic hybridization between diploids and
The authors are grateful to the authority of
allotetraploid somatic hybrids in Citrus. 9th
National Museum of Science and Technology,
ISC Congress Sun City Resort, South Africa,
Agargaon, Dhaka, Bangladesh for their financial
54: 115–21
support to carry out this study.
Grinblat U., 1972. Differentiation of citrus stem
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