The Scientific World Journal
Volume 2012, Article ID 578020, 6 pages
GerminationandPlantlet Regenerationof Encapsulated
Microshoots of AromaticRice(Oryzasativa L. Cv. MRQ 74)
Rosna Mat Taha,1AzaniSaleh,1,2NorainiMahmad,1
Nor AzlinaHasbullah,3and SadeghMohajer1
1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
3Department of Agricultural Sciences, Faculty of Technical and Vocational Education, Sultan Idris Education University,
35900 Tanjung Malim, Perak, Malaysia
Correspondence should be addressed to Azani Saleh, firstname.lastname@example.org
Received 7 June 2012; Accepted 27 June 2012
Academic Editors: A. Bekatorou and C. Drouza
Copyright © 2012 Rosna Mat Taha et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Plant tissues such as somatic embryos, apical shoot tips, axillary shoot buds, embryogenic calli, and protocom-like bodies are
potential micropropagules that have been considered for creating synthetic seeds. In the present study, 3–5mm microshoots of
Oryza sativa L. Cv. MRQ 74 were used as explant sources for obtaining synthetic seeds. Microshoots were induced from stem
explants on Murashige and Skoog (MS) medium supplemented with 1.5mg/L benzylaminopurine (BAP). They were encapsulated
regeneration of the encapsulated seeds were tested by culturing them on various germination media. The effect of storage period
(15–30 days) was also investigated. The maximum germination and plantlet regeneration (100.0%) were recorded on MS media
containing 3% sucrose and 0.8% agar with and without 0.1mg/L BAP. However, a low germination rate (6.67%) was obtained
using top soil as a sowing substrate. The germination rate of the encapsulated microshoots decreased from 93.33% to 3.33% after
30 days of storage at 4◦C in the dark. Therefore, further research is being done to improve the germination rate of the synthetic
Rice is the most important food crop for a large proportion
of the word’s human population especially in East, South,
Southeast Asia, the Middle East, Latin America, and the West
Indies. In 2008, international rice price rose greatly due to
general upward trend in grain prices caused by droughts,
increased use of grains animal feed, and so forth, has led to
worldwide food crisis. This caused the domestic rice price
in Malaysia increase almost double. The only way to protect
and stabilise local price is to increase local rice production.
Hence, synthetic seed technology could be an alternative and
potential tool for the propagation of rice.
Nowadays, the encapsulation technique for producing
synthetic seeds has become an important asset in micro-
propagation. Encapsulation of somatic embryos, apical and
axillary shoot buds, and regeneration of whole plants from
The use of unipolar axillary shoot buds and apical shoot tips
in creating the synthetic seeds have been reported in many
plants species such as Actinidia deliciosa, Brassica campestris,
Malus pumila Mill, Zingerber officinale Rose, and Syringa
vulgaris L. . Axillary shoot buds and apical shoot tips
are suitable for encapsulation studies of artificial seeds as
they possess great potential for plant development from pre-
existing meristematic tissue. In addition, the use of axillary
shoot buds and apical shoot tips would also ensure a genetic
uniformity and stability in the regenerants. However, infor-
mation about production of artificial seeds from apical shoot
tips or microshoots in rice is extremely limited. Therefore, in
the present study, different encapsulation matrix and efficacy
in the plantlet regeneration of encapsulated microshoots of
2 The Scientific World Journal
Oryza sativa L. Cv. MRQ 74 were investigated. Comparison
on morphological structure such as stomata density between
were also carried out.
74 were induced from stem explants. Prior to this, dehusked
mature seeds of rice were surface sterilized by soaking and
shaking in 70% (v/v) clorox with two drops of 1mL/L tween
20 followed by 50%, 30%, 20%, and 10% (v/v) clorox.
Shaking the material during sterilization would obviously
enhance the effectiveness of the process. Each treatment
lasted approximately one minute. The dehusked seeds were
three times in sterilised distilled water for complete removal
of clorox and ethanol in lamina flow.
The sterilised seeds were then cultured onto MS medium
 containing sucrose (30g/L) and agar (8g/L). Ten seeds
were placed per culture tube. The cultures were incubated in
the culture room at 25 ± 1◦C under 16 hours light and 8
hours dark with 1000lux of light intensity. The seeds started
to germinate after 3 to 4 days to form plantlets. The plantlets
were maintained in the culture room for 5 weeks before
they were used as the explants in obtaining microshoots for
synthetic seeds production.
The stem explants were approximately cut into 5.0–
10.0mm segments and cultured onto MS medium supple-
mented with 3.0% (w/v) sucrose and 0.8% (w/v) technical
agar fortified with 1.5mg/L BAP for microshoots induction.
The pH of the medium employed in this experiment was
for 21 minutes. The cultures were kept in the culture room
at 25 ± 1◦C under 16 hours light and 8 hours dark with
1000lux of light intensity. Microshoots (approx., 5.0mm in
length) were excised from cultures after 2 weeks in culture.
The microshoots were carefully isolated and encapsulated.
2.2. Capsule Matrix and Encapsulation of Microshoots.
Microshoots of 3–5mm in length were used as explants
source in obtaining synthetic seeds. Different encapsulation
matrices were evaluated: (1) Ca-free MS (Duchefa) +
distilled water, (2) Ca-Free MS + 3% sucrose, and (3) Ca-
free MS + 3% Sucrose + 0.1mg/L BAP (Sigma) + 0.1mg/L
NAA (Sigma). Microshoots were mixed in the encapsulation
matrix consisted of 3% (w/v) sodium alginate (Sigma),
added with MS basal liquid medium with or without 3%
of calcium chloride solution (CaCl2·2H2O) was prepared in
autoclaved after adjusting the pH to 5.8. The microshoots
into CaCl2·2H2O solution using sterilized micro pipette.
The encapsulated microshoots were left for 30 minutes for
hardening. The beads containing one microshoot each were
washed in sterilized distilled water to avoid sticking together
and were retrieved using nylon mesh.
2.3. Germination Medium/Substrate. The beads were ger-
minated on various germination media and substrates: (1)
MS basal medium + 3% sucrose + 0.8% agar (MSO), (2)
MS + 3% sucrose + 0.8% agar + 0.1mg/L BAP, (3) MS +
0.8% agar, (4) tap water + 0.8% agar, (5) top soil, (6) top
soil + tap water, and (7) top soil + 1/2 strength MS + 3%
at 121kPa for 21 minutes prior to be used. In testing the
best encapsulation matrix, the nonencapsulated microshoots
were used as control. The encapsulated and nonencapsulated
microshoots were cultured onto MS basal medium. Mean-
while in determining the best culture medium and cultured
substrate, MS basal medium with 3% sucrose and 0.8%
agar was used as control using Ca-free MS + 3% sucrose
+ 3% sodium alginate + 0.1mg/L BAP + 0.1mg/L NAA as
encapsulation matrix. Thirty replicates were used for each
treatment. All the cultures were kept in the culture room at
25 ± 1◦C, 16 hours light and 8 hours dark. The germination
rate of the synthetic seeds and plantlets survival rate were
recorded after 10 days and 30 days of culture, respectively.
2.4. Storage Period. The beads were also cold-stored in the
incubator at 4◦C prior to germination process. Thirty seeds
were sown in MS basal medium for every 15 days interval.
2.5. Microscopic Studies (Scanning Electron Microscopy-SEM).
Scanning electron microscope (SEM) was used to observe
the differences between in vivo (intact) and in vitro leaf.
Observations and comparisons were made on the differences
of number of stomata and trichomes. Standard methods and
procedures for the preparation of samples for SEM process
as described by Islam et al.  were followed.
All the experiments were repeated trice and thirty replicates
were used. The effect of different treatments was quantified
as mean ± SE and the data were subjected to statistical
analysis using Duncan’s multiple range test (DMRT) at 5%
level significance .
4.Results and Discussion
4.1. Capsule Matrix and Encapsulation of Microshoots. In
the present study, the encapsulated and nonencapsulated
microshoots showed 100% germination rate and had a high
potential to be converted into plantlets on cultured medium.
The highest survival rate which was 100% found using Ca-
free MS + 3% sucrose, while nonencapsulated microshoots
(control) showed the lowest survival rate of plantlet (90.0%)
(Table 1). The survival rate of the plantlets significantly
reduced when the encapsulation matrix only contain 3%
sodium alginate with the addition of MS basal (free calcium)
and distilled water. It is interesting to note that the addition
The Scientific World Journal3
Table 1: Growth response of encapsulated microshoots of Oryza sativa L. Cv. MRQ 74 in different capsule matrix after being transplanted
onto MS media for 10 and 30 days.
(after 10 days)
Plantlets survival rate
(after 30 days)
Ca-free MS + distilled water
Ca-free MS + 3% sucrose
Ca-free MS + 3% sucrose + 0.1mg/L BAP + 0.1mg/L NAA
Mean ± SE, n = 30. Mean with the same letter in the columns are not significantly different at P = 0.05.
Figure 1: Synthetic seed germination on, (a) MS medium, two weeks old, (b) MS medium after one month, (c) MS medium + 0.1mg/L
BAP, (d) tap water + agar, (e) capsule matrix containing 0.1mg/L BAP + 0.1mg/L NAA, (f) top soil + tap water.
of 0.1mg/L BAP and 0.1mg/L NAA did not influence the
germination rate of the synthetic seeds (Figure 1(e)).
In the previous experiment, the optimum regeneration
medium of rice (Oryza sativa L. Cv. MRQ 74) was MS
medium supplemented with 0.1mg/L BAP + 0.1mg/L
NAA (Data not shown). Therefore, the same types and
concentrations of hormones were added in capsule matrix
to stimulate the emergence of shoot and root of encapsulated
nificant effect on synthetic seeds germination. All treatments
with and without hormone showed 100% germination rate.
In fact, plantlets survival rate decreased to 96.67% compared
to capsule matrix containing MS medium without growth
hormone. For production of synthetic seeds from apical
shoot tips and axillary shoot buds, these organs are usually
first treated with auxins for root induction. However, Bapat
and Rao  reported that mulberry plantlets were obtained
from alginate encapsulated shoot buds without any specific
root induction treatment. The same finding was reported on
banana . Roy and Mandal  also stated that embryos
and pro-embryos of elite indica rice (Oryza sativa L. Var IR
72) developed into plantlets on MS basal medium without
any phytohormones. These results were in line with the
findings of the present study.
Synthetic seeds germination was affected by sucrose con-
centration in the capsule matrix.
Synthetic seeds with no sucrose in the capsule matrix
had significantly lower plantlets survival rate as compared
with synthetic seeds with sucrose in encapsulation matrix.
It has been previously reported that the inoculated somatic
embryos with various concentrations of sucrose (0, 30 and
60mg/L) gave synthetic seeds germination to 43, 57, and
46% respectively . Other studies have shown that low
germination and conversion capacity of synthetic seeds is
4 The Scientific World Journal
Table 2: Effect of different sowing media/substrates on germination rate of synthetic seeds of Oryza sativa L. Cv. MRQ 74.
Germination rate (after 10 days)
Plantlets survival rate (after 30 days)
100.00 ± 0.00f
100.00 ± 0.00f
MS basal medium + 3% sucrose + 0.8% agar (control)
MS + 3% sucrose + 0.8% agar + 0.1mg/L BAP
MS + 0.8% agar
Tap water + 0.8% agar
Top soil + tap water
Top soil + 1/2 strength MS + 3% sucrose
Mean ±SE, n = 30. Mean with the same letter in columns are not significantly different at P = 0.05.
Table 3: Effect of storage period (day) at 4±1◦C on germination of
rate (% ±SE)
Mean ± SE, n = 30. Mean with the same letter in the column is not
significantly different at P = 0.05.
due to absence of nutritive tissues like the endosperm of
the natural seed . These results indicated that coating
material and the concentration of the coating material are
important limiting factors for the synthetic seed technology.
4.2. Germination Medium/Substrate. The highest germina-
tion rate (100%) was recorded on MS basal medium (MSO),
MS medium supplemented with 0.1mg/L BAP, tap water
+ 0.8% agar, top soil + tap water, and top soil + 1/2
preferred germination substrate with 6.67% germination
to 0.0%. The maximum survival rate (100%) was observed
on MS media with (Figure 1(c)) and without 0.1mg/L BAP
(Figures 1(a) and 1(b)) followed by tap water + 0.8% agar
(93.33%, Figure 1(d)), MS + 0.8% agar (90.0%), top soil +
tap water (36.67%, Figure 1(f)), top soil + 1/2 strength MS +
3% sucrose (30.0%), and top soil (0.0%).
Sucrose is not only an important substance in capsule
matrix but also in culture media. The presence of sucrose
in germination medium showed significant effect on germi-
nation rate and plantlets survival rate of Oryza sativa L. Cv.
MRQ 74. Similar findings were reported on other species
such as Psidium guajava L. With increasing concentration of
sucrose (3–9%) in medium, the percentage of germination
of encapsulated somatic embryos of Psidium guajava L.
decreased significantly . Taha et al.  found that MS
without hormones supplemented with 30g/L sucrose was
the best substrate for germination of the synthetic seeds of
Saintpaulia ionantha Wendl. The use of tap water in culture
rate too (100%). However, plantlets survival rates dropped
to 36.67%, 30.00%, and 0.0% in top soil + tap water, top
soil + 1/2 strength MS + 3% sucrose, and top soil only,
respectively, after 30 days. The most abundant minerals
dissolved in water are salts of calcium, magnesium, ferrous
iron, and manganese. Among the macronutrients required
for plant cell and tissue growth are calcium and magnesium.
In this study, plantlet survival rate is significantly higher
in top soil + tap water (36.67%) compared to top soil +
1/2 strength MS + 3% sucrose (30.00%). However, we did
not determine the mineral content of tap water used in this
study. The mineral content of tap water varies considerably
from place to place. For example, mineral levels of tap water
vary among North American cities and even among different
water sources within the same city . The mineral content
of water reflects the nature of the geologic formation with
which the water has been in contact.
4.3. Storage Period. The encapsulated microshoots with MS
medium supplemented with 0.1mg/L BAP + 0.1mg/L NAA,
storage (Table 3). The viability of seeds had fallen from
93.33% to 3.33% after one month storage at 4◦C. Storage
conditions such as temperature and period of storage are
important factors to determine the regeneration frequency
of the stored encapsulated propagules. Storage of synthetic
seeds using an alginate encapsulation protocol has been
attempted in a few species, with minimal success [1, 16, 17].
The synthetic seeds of sweet corn were germinated to 43
and 55% after 2 weeks of storage under 15 ± 2◦C and 25 ±
2◦C, respectively . In the present study, low germination
rate (16.67%) was recorded after 2 weeks of storage at 4◦C.
Therefore, further research for the development of a better
technique is required. For example, increasing the storage
temperature may increase regeneration levels. Elvax 4260
(ethylene vinyl acetate acrylic acid terpolymer, Du Pont,
USA) could be used for coating the capsules to avoid rapid
water loss when calcium alginate capsules are exposed to the
ambient atmosphere .
4.4. Microscopic Studies (Scanning Electron Microscopy-SEM).
The microscopic studies of the structure of in vivo (intact)
and in vitro leaves showed that the stomata apparatuses were
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Figure 2: Scanning electron micrograph showing adaxial (a) and abaxial (b) surfaces of in vitro leaf of plantlet from synthetic seed of Oryza
sativa L. Cv. MRQ 74, adaxial (c) and abaxial (d) surfaces of leaf from in vivo (intact) plant, adaxial (e), and abaxial (f) surfaces of in vitro
leaf of plantlet from culture medium containing 0.1mg/L BAP and 0.1mg/L NAA. S: stomata, T: trichomes.
generally triangular in shape. The number of stomata was
higher on abaxial surfaces of in vivo and in vitro (plantlet
from synthetic seed) leaves. However, a comparable number
of stomata were observed on abaxial and adaxial surfaces of
chomes were seen on adaxial surface of intact leaf compared
with in vitro leaf, grown on MS medium supplemented with
0.1mg/L BAP + 0.1mg/L NAA (Figure 2).
It can be concluded that this study was successful in deter-
mining the most suitable capsule matrix (Ca-free MS + 3%
sucrose) and the optimum culture medium (MS medium +
3% sucrose with and without 0.1mg/L BAP) for the max-
imum germination rate (100%) and plantlets survival rate
(100%) of Oryza sativa Cv. MRQ 74. The encapsulated and
6 The Scientific World Journal Download full-text
nonencapsulated microshoots developed into plantlets on
MS basal medium without any growth hormones. Based on
scanning electron microscopic (SEM) studies, leaves derived
from stem explants cultured on MS medium supplemented
with 0.1mg/L BAP + 0.1mg/L NAA showed higher stomata
number on both surfaces. However, there was no significant
differences between in vivo and in vitro in terms of stomata
number on both adaxial and abaxial leaf surfaces, indicating
that no morphological changes had occurred. In order to
confirm these findings, further histological studies on leaf
tissues grown in vivo and in vitro should be conducted.
The authors are grateful to the Malaysian Agricultural
Research and Development Institute (MARDI) Seberang
Prai, Penang, Malaysia for providing rice mature seeds and
the University of Malaya for facilities provided and the
Institute of Research Management and Monitoring (IPPP),
University of Malaya for the research grant PV025/2011B.
 K. Redenbaugh, B. D. Paasch, J. W. Nichol, M. E. Kossler, P.
R. Viss, and K. A. Walkee, “Somatic seed: encapsulation of
asexual plant embryos,” Biotechnology, vol. 4, pp. 797–801,
 J. Mathur, P. S. Ahuja, N. Lal, and A. K. Mathur, “Propagation
of Valeriana wallichii DC. using encapsulated apical and axial
shoot buds,” Plant Science, vol. 60, no. 1, pp. 111–116, 1989.
 T. R. Ganapathi, P. Suprasanna, V. A. Bapat, and P. S. Rao,
“Propagation of banana through encapsulated shoot tips,”
Plant Cell Reports, vol. 11, no. 11, pp. 571–575, 1992.
 M. M. Lulsdorf, T. E. Tautorus, S. I. Kikcio, T. D. Bethune,
and D. I. Dunstan, “Germination of encapsulated embryos of
interior spruce (Picea glauca engelmannii complex) and black
spruce (Picea mariana Mill.),” Plant Cell Reports, vol. 12, no.
7-8, pp. 385–389, 1993.
 H. Ara, U. Jaiswal, and V. S. Jaiswal, “Synthetic seed: prospects
and limitations,” Current Science, vol. 78, no. 12, pp. 1438–
 T. Murashige and F. Skoog, “A revised medium for rapid
growth and bioassay with tobacco tissue culture,” Plant
Physiology, vol. 15, pp. 473–497, 1962.
 M. T. Islam, Y. Hashidoko, A. Deora, J. Ito, and S. Tahara,
“Suppression of damping-off disease in host plants by the
rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked
to plant colonization and antibiosis against soilborne per-
onosporomycetes,” Applied and Environmental Microbiology,
vol. 71, no. 7, pp. 3786–3796, 2005.
 K. A. Gomez and A. A. Gomez, Statistical Procedures for
Research Institute, Los Banos, Philippines, 1976.
 V. A. Bapat and P. S. Rao, “In vivo growth of encapsulated
axillary buds of mulberry (Morus indica L.),” Plant Cell, Tissue
and Organ Culture, vol. 20, no. 1, pp. 69–70, 1990.
 B. Roy and A. B. Mandal, “Development of synthetic seeds
involving androgenic and pro-embryos in elite indica rice,”
Indian Journal of Biotechnology, vol. 7, no. 4, pp. 515–519,
 P. Thobunluepop, E. Pawelzik, and S. Vearasilp, “Possibility
of sweet corn synthetic seed production,” Pakistan Journal of
Biological Sciences, vol. 12, no. 15, pp. 1085–1089, 2009.
 M. Sanada, Y. Sakamoto, M. Hayashi, T. Mashiko, A.
Okamoto, and N. Onish, “Celery and lettuce,” in Synseeds, K.
Redenbaugh, Ed., pp. 305–327, CRC Press, Boca Raton, Fla,
 M. K. Rai, V. S. Jaiswal, and U. Jaiswal, “Effect of ABA and
sucrose on germination of encapsulated somatic embryos of
guava (Psidium guajava L.),” Scientia Horticulturae, vol. 117,
no. 3, pp. 302–305, 2008.
 R. M. Taha, N. Daud, N. A. Hasbullah, and A. Awal,
“Somatic embryogenesis and production of artificial seeds
in Saintpaulia ionantha Wendl,” in Proceedings of the 6th
International Symposium on In Vitro Culture and Horticultural
Breeding, R. J. Geijskes, P. Lakshmanan, and A. Taji, Eds., vol.
829 of Acta Horticulturae, pp. 331–336, Brisbane, Australia,
 A. Arik, G. Philippe, and J. E. Mark, “Comparison of the
mineral content of tap water and bottled waters,” Journal of
General Internal Medicine, vol. 16, no. 3, pp. 168–175, 2001.
 K. Redenbaugh, D. Slade, P. Viss, and J. A. Fujii, “Encapsula-
tion of somatic embryos in synthetic seed coats,” Hortscience,
vol. 22, pp. 803–809, 1987.
 J. A. Fujii, D. T. Slade, K. Redenbaugh, and K. A. Walker, “Arti-
5, no. 12, pp. 335–339, 1987.