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Production of synthetic seeds from micro


shoots and somatic embryos of Gerbera
jamesonii bolus ex. Hook f.
ARTICLE in ACTA HORTICULTURAE JANUARY 2010

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3 AUTHORS:
Rosna Mat Taha

Nor Azlina Hasbullah

University of Malaya

Universiti Pendidikan Sultan Idris (UPSI)

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Asmah Awal
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Retrieved on: 26 September 2015

Production of Synthetic Seeds from Micro Shoots and Somatic Embryos


of Gerbera jamesonii Bolus ex. Hook f.
R.M. Taha1, N.A. Hasbullah1 and A. Awal2
1
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Malaysia
2
Faculty of Applied Science, Mara University of Technology, 40450 Shah Alam
Selangor, Malaysia
Keywords: sodium alginate, encapsulation matrix, Murashige and Skoog, growth
regulators, calcium chloride, germination
Abstract
Micro shoots of Gerbera jamesonii were obtained when petiole explants were
cultured on 3.0 mg/L BAP. Meanwhile, somatic embryos of Gerbera jamesonii were
induced from embryogenic callus obtained from leaf explants cultured on MS
medium fortified with 1.0 mg/L BAP and 0.1 mg/L NAA with the addition of 50 mM
L-Proline. Synthetic seeds were formed when these micro shoots and somatic
embryos were encapsulated using 3.0% sodium alginate solution and beads were
solidified using 100 mM calcium chloride (CaCl2.2H2O) solution. The effects of
different sodium alginate concentration, calcium chloride molarity, and different
composition of encapsulation matrix were studied. The addition of 3% sodium
alginate, Ca-free MS basal medium, 3% sucrose, 2.0 mg/L BAP, and 0.5 mg/L NAA
in the encapsulation matrix resulted in 74.52.6% and 54.21.3% of germination
rate respectively for micro shoots and somatic embryos. The viability of the seeds
after storage period at 4C was also determined. High germination rate (75-95%)
was achieved after one to three months storage, whereas low germination rate (850%) was obtained after four to six months storage. The synthetic seeds were also
successfully germinated onto three different substrates: MS basal, garden soil, and
vermiculite.
INTRODUCTION
Plant propagation through synthetic seeds or artificial seeds provides new
perspective in agriculture. The development of synthetic seeds offers great improvement
and novelty in producing transgenic plants, non-seed producing plants, and many others.
In vivo vegetative propagation techniques are time consuming and costly. Development
of synthetic seeds is now an efficient and effective mode for plant propagation in many
important horticultural crops. Successful reports of synthetic seed production and plantlet
regeneration have been reported for cereals, vegetables, fruits, ornamentals, aromatic
grass, and conifers (Redenbaugh et al., 1991; Redenbaugh, 1993; Ghosh and Sen, 1994;
Fowke et al., 1994; Castillo et al., 1998). Synthetic seeds could be established when
somatic embryos or micro shoots derived from tissues culture technique were
encapsulated in a protective coating. Gerbera jamesonii Bolus ex. Hook f., is commonly
known as Gerbera daisy or Barberton Daisy. This temperate perennial flowering plant
belongs to the Asteraceae family. They are planted in full sun and useful as cut flowers,
pot plants, and also bedding plants. Plant propagation through tissue culture of Gerbera is
mainly aimed at producing plants at a very high multiplication rate, since this plant is in
high demand worldwide. Through tissue culture techniques, Gerbera shoots were
regenerated, primarily from flower buds from greenhouse grown plants (Pierik et al.,
1973, 1975; Laliberte et al., 1985). Micro shoots were successfully obtained from petiole
explants through tissue culture techniques. As an alternative to in vitro propagation,
somatic embryogenesis was introduced. Somatic embryos were induced when leaf
explants were cultured on selected medium. The main objective of this study is to
establish the procedure for encapsulation of micro shoots and somatic embryos of
Gerbera jamesonii in appropriate sodium alginate encapsulation matrix for the production
Proc. VIth IS on In Vitro Cult. and Hort. Breed.
Eds.: R.J. Geijskes et al.
Acta Hort. 829, ISHS 2009

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of synthetic seeds, a new innovation in agrotechnology. Several factors were identified in


this study, such as the effect of different concentrations of sodium alginate and calcium
chloride on seed formation, growth response of micro shoots and somatic embryo of
Gerbera encapsulated in different capsule matrix, the effect of different sowing medium
on germination rate of synthetic seeds, and also the effect of storage time (days) at 4C on
germination of synthetic seeds.
MATERIALS AND METHODS
Induction of Micro Shoots and Somatic Embryos
Standard tissue culture method was used in this study. Explants were obtained
from eight-week-old aseptic seedlings. Gerbera seeds were soaked in distilled water for
30 minutes with addition of one to two drops of Tween-20 followed by 40% (v/v) sodium
hypochlorite solution and gently agitated for ten minutes. The seeds were then rinsed
three times in distilled water and then soaked in 70% (v/v) alcohol for one minute.
Finally, the seeds were rinsed three times in sterile distilled water. Sterilized seeds were
cultured on MS (Murashige and Skoog, 1962) basal medium. The medium pH was
adjusted to 5.8 prior to autoclaving at 121oC for 21 minutes. Petioles obtained from
aseptic young plantlets formed from the seedlings were used as source of explants. Micro
shoots of Gerbera jamesonii were successfully obtained when petiole explants were
cultured on MS medium (Murashige and Skoog, 1962) supplemented with 3.0 mg/L BAP.
Meanwhile, somatic embryos of Gerbera jamesonii were induced when embryogenic
callus obtained from leaf explants were cultured on MS medium fortified with 1.0 mg/L
BAP and 0.1 mg/L NAA with the addition of 50 mM L-Proline.
Formation of Synthetic Seeds
Micro shoots and somatic embryos of Gerbera jamesonii Bolus ex. Hook f. were
first mixed with sodium alginate solution. Encapsulation was accomplished when each
micro shoots and somatic embryos were drawn up with some of the encapsulation matrix
(sodium alginate solution) and then dropped into the CaCl2.2H2O solution. This process
was done repeatedly. Each drop of the encapsulation matrix has to contain only one micro
shoot/somatic embryo (explant). Round shaped beads containing micro shoots were
formed when the droplets were dropped in the CaCl2.2H2O solution. CaCl2.2H2O solution
needs to be agitated slowly to prevent the formed droplets from fusing with each other.
During this process, ion exchange reaction between sodium ion (Na+) and calcium ion
(Ca2+) occurred. This reaction is important in developing insoluble sodium alginate. The
resulting beads containing explants were left agitated in the CaCl2.2H2O solution for 30
minutes. The beads were sieved using a nylon mesh. All encapsulated beads were rinsed
three times in sterile liquid MS medium. Various concentrations of sodium alginate
solution, CaCl2.2H2O solution, concentration and duration of exposure to CaCl2.2H2O
solution, different types of sowing medium, and various storage periods of the synthetic
seeds produced were investigated. The presences of growth regulators in the
encapsulation matrix were also studied.
Germination of Synthetic Seeds
For germination of synthetic seeds, the beads were germinated on various
germination medium and substrates. All the germinating substrates were autoclaved prior
to use. The beads were also stored at 4C (one to six months) prior to germination
process. All samples were incubated at 251C, under 16-h light photoperiod of light
intensity (1000 lux). Germination rate were recorded after six weeks of germination.

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RESULTS AND DISCUSSION


Formation of Beads
Micro shoots of Gerbera jamesonii bolus ex. Hook f. in vitro were successfully
produced when petiole explants were cultured on MS medium supplemented with 3.0
mg/L BAP (Fig. 1b). Meanwhile, somatic embryos were developed when embryogenic
calli derived from leaf explants of Gerbera were cultured on MS medium added with 1.0
mg/L BAP and 0.1 mg/L NAA fortified with 50 mM L-Proline (Fig. 1a). These micro
shoots and somatic embryos of Gerbera jamesonii were used as explant sources for the
production of synthetic seeds. Synthetic seeds were formed using encapsulation
technique. This technique involves the usage of sodium alginate (NaC6H7O6) and calcium
chloride dehydrate solution (CaCl2.2H2O). This technique was introduced by Lynch
(2002). Both micro shoots and somatic embryo explants were successfully encapsulated
in the encapsulation matrix containing 3% sodium alginate solution. However, early
preliminary experiment was done to study the effect of different concentrations of sodium
alginate and calcium chloride solution on seed formation. It was found that the most
suitable sodium alginate concentration for encapsulation matrix was at 3% and 4%, and
the most suitable calcium chloride (CaCl2.2H2O) solution concentration was between
100-125 mM. Calcium chloride (CaCl2.2H2O) acts as a complexing solution that allows
the hardening of encapsulated matrix. Synthetic seeds produced were left to harden for 30
minutes in the calcium chloride solution. In the present experiment, the production of
synthetic seed of Gerbera jamesonii gave optimum result when micro shoots and somatic
embryos explants were encapsulated with 3% sodium alginate and the seeds produced
were then left to harden in 100 mM calcium chloride solution for 30 minutes (Fig. 1c).
The seeds produced were in uniform round shape, with firm coat that is easy to handle
and also allowed the seeds to convert into plantlets (Table 1). Lower concentration of
sodium alginate and calcium chloride solution resulted in very fragile, soft, and produce
beads with no definite shapes. Meanwhile, concentration of sodium alginate and calcium
chloride solution that was too high caused the beads to be too rigid and stiff and caused
difficulties for germination of the seeds. Capsule formation was also influenced by the
concentration of the encapsulation matrix and complexing agent. Pattnaik and Chand
(2000) reported that axillary buds encapsulated with 4% sodium alginate solution upon
complexation with 75 mM CaCl2.2H2O solution produced firm, clear, uniform capsules
within an ion exchange duration of 30 minutes in Morus alba L., Morus australis Poir.,
Morus bombycis Koidz., Morus cathyana Hemsl., Morus latifolia Poir., and Morus nigra
L. Somatic embryos of Paulownia elongata were encapsulated with 3% sodium alginate
solution prepared in MS solution and submerged for 30 minutes in 50 mM calcium
chloride solution for hardening of the seeds (Ipekci and Gozukirmizi, 2003). Previous
research showed that different types of encapsulation matrix could be used as coating
agents but sodium alginate was the most popular for encapsulating matrix. Redenbaugh et
al. (1986) identified that the production of sodium alginate from different commercial
sources may be due to differential purity of alginic acid or the variation in the mannuronic
acid:guluronic acid ratio. Alginate was chosen as the encapsulation matrix because of its
moderate viscosity, low toxicity, quick gelation, and low cost (Onishi et al., 1992).
Germination of Beads
Table 2 shows the growth response of micro shoots and somatic embryo of
Gerbera jamesonii encapsulated in different encapsulation matrices after being
transplanted into MS medium for 10 and 30 days. Non-encapsulated (control) micro
shoots and somatic embryos cultured under aseptic conditions exhibited higher
germination rate (78.40.5) and (57.22.0) respectively. Higher survival rates were also
reported for non-encapsulated micro shoots (88.40.3) and somatic embryos (73.01.6) as
compared to the encapsulated explants. All of the treatments tested allowed the
germination and survival of the seeds. The highest germination rate of encapsulated micro
shoots (74.52.6) (Fig. 1d) and somatic embryos (54.21.3) were observed when the
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synthetic seeds were encapsulated with 3% sodium alginate with the addition of MS basal
(free calcium), 3% sucrose and 2.0 mg/L BAP, and 0.5 mg/L NAA (explant germinating
medium). The germination rate of the synthetic seeds significantly reduced for both micro
shoots (56.01.1) and somatic embryos (41.41.0) when the encapsulation matrix only
contained 3% sodium alginate with the addition of MS basal (free calcium) and distilled
water. The survival rates also reduced significantly. Synthetic seeds of Begonia x
Hiemalis Fotch. produced 90.48% germination rate (Asmah et al., 2007) when 3%
sodium alginate was used as the encapsulation matrix. The highest germination frequency
of encapsulated micro shoots and somatic embryos were then transferred to vials
containing sowing medium. Most of the beads managed to survive after eight weeks in
culture on MS basal medium, 100% (encapsulated micro shoots) and 90% (encapsulated
somatic embryo), sterile garden soil, 90% (encapsulated micro shoots), 83%
(encapsulated somatic embryo) (Table 3). The survival rate of seedlings reached 75%
after they were transferred to the green house. The success of retrieving complete
plantlets in vitro from encapsulated axillary buds or shoot tips on various planting
mediums has been achieved in few herbaceous plants (Sharma et al., 1994) and woody
species (Bapat et al., 1994).
Storage of Synthetic Seeds
The result showed that 75-95% germination rate could be obtained from one to
two months storage at 4C, whereas three months storage also showed precocious
germination and the seeds germinated at 77% (encapsulated micro shoots) and 60%
(encapsulated somatic embryos). The four- to six-month storage seeds showed lower
germination rate (8-50%). In contrast, the germination frequency of encapsulated micro
shoots and somatic embryos after 120 days storage was lower than after 30 days storage
(Table 4). It is thought that the decline in the germination frequency observed among
encapsulated micro shoots and somatic embryos of Gerbera jamesonii sotred at low
temperature may be due to inhibited respiration of plant tissue by alginate (Redenbaugh et
al., 1987). Storage of somatic embryos or shoot buds using alginate encapsulation
protocol has been attempted in a few species with various degree of success (Bapat and
Rao, 1988; Ghosh and Sen, 1994). Fewer than 5% of Santalum album L. (Bapat and Rao,
1988) encapsulated embryos germinated, followed by storage at 4oC for 45 days and only
8.3% of encapsulated Asparagus cooperi baker. somatic embryos germinated. Most of the
artificial seeds developed into normal seedlings. No phenotypic changes were observed
from the plantlets and over 80% of the plantlets developed into healthy plants. Plantlets
germinated from the germination of synthetic seeds of Gerbera jamesonii (Fig. 1e) were
morphologically identical to the mother plant and developed normally and produced
flowers after six months being transplanted to the green house (Fig. 1f). Results showed
that synthetic seed produced via tissue culture system could serve as a mean for a new
plant line produced through biotechnological improvements to be transplanted to the
field. Synthetic seeds produced could also be handled as true seeds. Furthermore, it has
the potential for long term storage without losing its viability.
CONCLUSION
Synthetic seed technology could be a valuable aid to economical large-scale clonal
propagation and germplasm conservation of Gerbera jamesonii Bolus ex. Hook f. The
possibilities of using both embryogenic and non-embryogenic, in vitro derived vegetative
propagules for encapsulation have been successfully reported in this study. Both explants
were successfully encapsulated with 3% sodium alginate with the addition of MS basal
(free calcium), 3% sucrose and 2.0 mg/L BAP, and 0.5 mg/L NAA. The highest
germination rate was 74.52.6 of encapsulated micro shoots and 54.21.3 of somatic
embryos.

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ACKNOWLEDGEMENTS
This work was partially funded by Vote F grant number PS077/2007B. The
authors would like to thank the University of Malaya, Malaysia for the above financial
support. The authors would also like to thank the Ministry of Science Technology and
Innovation, Malaysia for the Science Fund Grant SF 12-02-03-2037.
Literature Cited
Awal, A., Taha, R.M. and Hasbullah, N.A. 2007. In vitro Formation of synthetic seed of
Begonia x hiemalis Fotch. CATRINA- International Journal of Environmental
Sciences. 2(2):189-192.
Bapat, V.A., Mhatre, M. and Rao P.S. 1987. Propagation of Morus indica L. (mulberry)
by encapsulated shoot buds. Plant Cell Rep. 6:393-395.
Castillo, B., Smith, M.A.L. and Yadav, U.L. 1998. Plant regeneration from encapsulated
somatic embryos of Carica papaya L. Plant Cell Rep. 17:172-176.
Fowke, L.C., Atree, S.M. and Pomeroy, M.K. 1994. Production of vigorous desiccation
tolerant white spruce (Picea glauca [Moench] Voss.) synthetic seeds in a bioreactor.
Plant Cell Rep. 13:601-606.
Gosh, B. and Sen, S. 1994. Plant regeneration from alginate encapsulated somatic
embryos of Asparagus cooperi baker. Plant Cell Rep. 13:381-385.
Ipekci, Z. and Gozukirmizi, N. 2003. Direct somatic embryogenesis and synthetic seed
production from Paulownia elongate. Plant Cell Rep. 22:16-24.
Laliberte, S., Chretien, L. and Vieth, J. 1985. In vitro plantlet production from young
capitulum explants of Gerbera jamesonii. Hort. Science (20):137-139.
Lynch, T.P. 2002. Plant Tissue Culture Workshop. 3rd-5th September. University of
Malaya, Kuala Lumpur, Malaysia.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays
with tabacco tissue cultures. Physiol. Plant. 51:473-497.
Onishi, N., Mashika, T. and Okamoto, A. 1992. Cultural system producing encapsulatable
units of synthetic seeds in celery. Acta Hort. 319:113-118.
Pattnaik, S. and Chand, P.K. 2000. Morphogenic response of the alginate-encapsulated
axillary buds from in vitro shoot cultures of six mulberries. Plant Cell Tissue and
Organ Culture 60:177-185.
Pierik, R.L.M., Jansen, J.L.M., Maasdam, A. and Binnendijk., C.K. 1975. Optimalization
of Gerbera plantlet production from excised capitulum explants. Scientia Hortic.
3:351-357.
Pierik, R.L.M. 1987. In vitro culture of higher plants. Martinus Nijhoff Publishers,
Dordrecht, the Netherlands, p. 219-222.
Redenbaugh, K., Slade D., Viss, P.R. and Fujii, J.A. 1987. Encapsulation of somatic
embryos in synthetic seed coats. HortScience 22:803-809.
Redenbaugh, K., Fuji, J.A. and Slade, D. 1991. Synthetic seed technology. p. 35-74. In:
I.K.Vasil (ed.), Scale-up and automation in Plant Propagation. San Diego Academic
Press.
Redenbaugh, K. 1993. Synseeds: application to synthetic seeds to crop improvement.
CRC Press, Boca Raton.
Sharma, T.R., Singh, B.M. and Chauhan, R.S. 1994. Production of disease free
encapsulated buds of Zingiber officinale Rosc. Plant Cell Rep. 13:300-302.

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Tables

Table 1. Effect of different concentrations of sodium alginate and calcium chloride on


bead formation.
CaCl2.2H2O
(mM)
25
50
75
100
125

Sodium alginate concentration (%)


2.0
+
+
++
++
++

3.0
+++
+++
+++
++++
++++

4.0
+++
+++
+++
++++
++++

5.0
+++
+++
***
***
***

6.0
+++
+++
***
***
***

+Very fragile bead with no definite shape


+ +Fragile beads with no definite shape
+ + + Soft, solid and uniform shape
+ + + + Optimal, firm, uniform and round shape
* * * Rigid, stiff and unbreakable beads

Table 2. Growth response of micro shoots and somatic embryo of Gerbera encapsulated
in different encapsulation matrix. Unencapsulated embryos were used as control.
Encapsulation
matrix
Control
Ca-free MS +
distilled water
Ca-free MS +
3% sucrose
Ca-free MS +
3% sucrose +
2.0 mg/L BAP +
0.5 mg/L NAA

Germination rate (%) (10 days)


Encapsulated
Encapsulated
micro shoot
somatic embryo
78.40.5c
57.22.0c

Survival rate (%) (30 days)


Encapsulated
Encapsulated
micro shoot
somatic embryo
88.40.3c
73.01.6c

56.01.1a

41.41.0a

64.80.7a

46.10.5a

72.10.8a,b

50.61.5a,b

68.91.2a,b

54.51.1a,b

74.52.6c

54.21.3 c

75.00.5c

63.21.0c

Mean SE, n=30. Mean with different letters differ significantly at p=0.05

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Table 3. Effect of different sowing medium on germination rate of synthetic seeds of


Gerbera. 30 replicates were used in each experiment.
Sowing medium

Ca-free MS +
3% sucrose
Garden soil
Vermiculite

No. of germinated seeds


Encapsulated Encapsulated
micro shoot
somatic
embryo
30
27
27
21

25
19

Germination rate (%)


Encapsulated Encapsulated
micro shoot
somatic
embryo
100
90
90
70

83
63

Table 4. Effect of storage period (days) at 4C on germination of synthetic seeds of


Gerbera on germination MS basal medium. Thirty replicates were used in each
experiment.
Period of storage
(days)
0
30
60
90
120
150
180

No. of germinated seeds


Germination rate (%)
Encapsulated Encapsulated Encapsulated Encapsulated
micro shoot
somatic
micro shoot
somatic
embryo
embryo
28
25
93
83
27
23
90
77
27
21
90
70
23
18
77
60
16
11
53
37
7
3
23
10
4
2
13
7

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Figures

4.0 mm

5.0 mm

10 mm

10 mm

2.0 mm

5.0 cm

Fig. 1. a) Mature cotyledonary stage somatic embryo of Gerbera jamesonii. b) Micro


shoots of Gerbera jamesonii cultured on MS medium supplemented with
3.0 mg/L BAP. c) Encapsulated micro shoots and somatic embryos of Gerbera
jamesonii. d) Germinated synthetic seed. e) Plantlet obtained from germination of
synthetic seed after eight weeks in culture medium. f) Gerbera jamesonii
produced from synthetic seed six months after being transferred to soil.

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