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INT J CURR SCI 2016, 19(4): E 86-101
REVIEW ARTICLE ISSN 2250-1770
Development of Synthetic Seed Technology in Plants and its Applications: A Review
Potshangbam Nongdam
Department of Biotechnology, Manipur University, Imphal-795003, Manipur, India
Department of Biotechnology, Manipur University, Canchipur, Imphal-795003, Manipur, India
Corresponding author: [email protected]
Abstract
Artificial/ Synseed/ Synthetic seeds are produced by encapsulation of plant micropropagules (somatic embryos, shoot
buds/shoot tips, calli, nodal segments, embryogenic masses, protocorms and protocorm like bodies) with specific coating
materials. The outer coating matrix provides protection and nutrition to the encapsulated plant tissues. Calcium alginate gel
is the most preferred among other available protective coverings as it enhances capsule formation and provides sufficient
firmness to alginate beads to withstand mechanical injury to propagules. Nutrients, growth regulators, antibiotics and other
adjuvants are incorporated into coating matrix to facilitate normal development of plant propagules leading to synseed
germination and healthy plant formation. Somatic embryos are the commonly used explant propagule for synseed
development because of the availability of well-established in vitro culture protocols for somatic embryogenesis. Synthetic
seed technology has important application in large scale multiplication of commercially valuable plants which are difficult to
propagate through conventional vegetative methods. Another significant aspect of synthetic seeds is the possibility of
conserving germplasm of rare and endangered economically important plants through short term cold storage and
cryopreservation. Inspite of its bright prospect, synseed technology is besotted with several drawbacks due to limited
availability of appropriate micropropagules in large scale, immature and asynchronous somatic embryo development and
very low conversion rate of synseeds into normal plants. Refinement of technology is the need of hour to make its wide
applications at commercially level. The review aims to stress on various aspects of synseed technology development in
several commercially important plants using different explant propagules. The various applications and limitations of the
technology in plant science research have also been emphasized in the present article.
Keywords: synthetic seeds, desiccation, protocorms, somatic embryos, callus
Received: 13th September 2016; Revised: 28thSeptember; Accepted: 06th October; © IJCS New Liberty Group 2016
Introduction
Plants are generally propagated mostly through
seeds in nature. In some crops, propagation through seeds
has not achieved success because of seed heterozygosity,
minute size, and absence of endosperms and absolute
necessity of fungal infection for germination (Saiprasad,
2001). Some plants can be vegetatively propagated but
conventional methods are time consuming, expensive
and cannot produce plants at larger scale. Production of
artificial seeds/synseeds using synthetic seed technology
can play an important role as alternative to other
conventional methods for large scale propagation and
long term germplasm storage of useful crop varieties.
The typical plant seeds have embryos with one or two
cotyledons which are associated with endosperms
containing food reserves for developing embryos. The
whole structure is enclosed by rigid covering called testa
which protects the inner delicate structure from injury
and desiccation and also helps in maintaining embryo
viability till its germination. In synthetic seeds, artificial
coating materials like sodium alginate, agar, gelrite and
sodium pectate replace the function of seed coat by
encapsulating somatic embryos (SEs), shoot buds, nodal
segments or any other plant tissues (Gantait et al., 2015).
The naked somatic embryos or shoot buds when exposed
to natural environment will not survive as they are highly
sensitive to desiccation and other infectious pathogens.
The encapsulating agents give protection to enclosed
explant propagules apart from giving nutrition for their
proper development. The derived artificial seeds/
synseeds when sowed should possess the ability to
convert into plants under in vitro and ex vitro conditions
(Bapat and Mhatre, 2005). The plant micropropagules for
synthetic seed production are obtained through somatic
embryogenesis, organogenesis, axillary bud, protocorm
and protocorm like bodies (PLBs) proliferation systems
using in vitro culture methods. This encapsulation
technology has been applied to produce synthetic seeds
of a number of plants belonging to angiosperm and
gymnosperm. The present review aims to highlight the
past and present status of synthetic seed development,
use of different explant propagules for synseed
preparation along with limitations and applications of the
technology in the field of agriculture and forestry.
Synthetic seed production
The first synthetic seeds were produced by
encapsulated carrot somatic embryos with
polyoxyethylene followed by desiccation (Kitto and
Janick, 1982). The encapsulated somatic embryos and
calli of carrot were dried for several hours on telfon
surface under the lamina flow cabinet followed by
rehydration after inoculating them in freshly prepared
medium. The polyoxyethylene was favorable for use as
coating materials for synthetic seeds as it was non-toxic,
readily soluble in water, could be easily dried to form
thin film and did not support the growth of
microorganism. Redenbaugh et al. (1984) developed a
technique for encapsulation of single, hydrated somatic
embryo of alfalfa plant and since then encapsulation of
somatic embryos of several plant species in hydrogel
have been successfully performed by several workers.
The production of synthetic seeds was primarily based on
encapsulation of somatic embryos before it was extended
to other plant tissues generated under in vitro culture
condition. The somatic embryos were employed for
synthetic seed production mainly due to presence of
radicle and plumule which ultimately differentiated into
roots and shoots (Gray et al., 1993; Dodeman and et al.,
1997). They were developed from somatic cells in in
vitro condition unlike zygotic embryos which were
obtained from fusion of male and female gametes. The
normal embryos have protective seed coat and
endosperms which provide protection and nutrition to
developing embryos. The somatic embryos on other hand
lack both endosperms and protective covering which
make them inconvenient to store and handle normally.
The naked somatic embryos are generally enclosed
in protective coating materials to produce synthetic
seeds. The coating materials however should not produce
damaging effect to the embryos, be mild enough to allow
germination, be sufficiently durable for rough handling
during manufacture, storage, transportation and planting
and also give nutrition to embryos for germination
(Sharma et al., 2012). The synthetic seeds successfully
prepared should be transplantable using existing farm
machinery. Several gels like agar, alginate,
carboxymethyl cellulose, gelrite, guargum, sodium
pectate etc. have been tested for encapsulation of plant
propagules for synthetic seed development (Redenbaugh
et al., 1987). However, calcium alginate encapsulation is
the most favorable for providing protective covering as it
enhances capsule formation and provides sufficient
firmness to alginate beads to withstand mechanical injury
(Saiprasad, 2001). Also its moderate viscosity and
reduced toxicity to somatic embryos with low cost and
bio-compatibility characteristics make it the most sought
after hydrogel for synseed encapsulation (Khor and Loh,
2005; Kikowska and Thiem, 2011). The encapsulating
matrix is provided with nutrients and other growth
regulators which play the role of artificial endosperms
(Germana et al., 1998). Seed germination efficiency and
somatic embryo viability are enhanced with the
incorporation of nutrients and growth regulators into
coating materials. Other adjuvants like fungicides,
pesticides, antibiotics and microorganisms (eg. Rhizobia)
may be included into the encapsulation matrix to instill
different properties to synthetic seeds to suit to prevailing
conditions (Kikowska and Thiem, 2011). Addition of
antibiotics may prevent bacterial contamination of
encapsulated plant propagules (Bekheet, 2006). Khor and
Loh (2005) reported improvement of conversion
potential and vigor of synseeds when activated charcoal
was supplemented in coating gel. Charcoal can break up
the alginate and helps in increasing the respiration of
somatic embryos inside the coat. Charcoal is also
presumed to play a role in retaining nutrients within the
hydrogel capsule and also helps in releasing them slowly
to developing embryos. Absorption of harmful
polyphenolic exudates released by encapsulated explant
propagules in vitro is also facilitated by activated charcoal
(Ganapathi et al., 1992).
The primary goal of synthetic seed technology is to
produce somatic embryos that resemble more closely to
normal seed embryos in terms of storage and handling
capabilities by providing nutritive covering. The
synthetic seeds can later be used for large scale clonal
propagation and germplasm conservation of rare and
commercially useful crop varieties. Apart from using
popular somatic embryos, other unipolar structures like
apical shoot tips, axillary shoot bud, embryogenic calli,
protocorm and protocorm like structures (PLBs) are
widely utilized for development of synseeds.
Redenbaugh et al. (1991) first treated shoot tip and
axillary bud micropropagules for root induction before
they were encapsulated. Root induction treatment with
auxins was avoided for alginate encapsulation of shoot
tips of mulberry and banana to produce synseeds and
their successful conversation to plantlets (Bapat and Rao,
1990). The procedure for synthetic seed production
starting from different plant micropropagules are
diagrammatically represented in figure 1.
Different types of synthetic seeds
The plant propagules obtained from different
sources are encapsulated with coating materials which
act as artificial endosperms by proving nutrition to
embryos apart from their protective function. Synthetic
seeds are broadly classified into desiccated and hydrated
seeds depending on different approaches undertaken for
their development as per the requirement.
Desiccated synthetic seeds
Encapsulation of somatic embryos is performed
using polyoxyethylene followed by desiccation under
controlled conditions. Desiccation can be performed
slowly or rapidly depending upon the requirement. It
takes one or two week times to gradually desiccate the
encapsulated seeds in chamber of decreasing humidity
while instant desiccation involves the opening of sealed
petridish containing synseeds and leaving directly open
overnight for quick drying (Ara et al., 2000). The
desiccated synthetic seeds can be developed for those
plant species which have somatic embryos resistant
against desiccation. The carrot SEs can be encapsulated
with polyox by mixing equal volumes of embryonic
suspension and a 5% (w/v) solution of polyox to give a
final concentration of 2.5% polyox. The suspension was
dispensed as 0.2 ml drops from the pipette on to telfon
sheets and dried to wafers in a laminar flow hood. The
drying time was based on the ability of water to separate
from teflon plate and it usually took about 5 hours. The
embryo survival and conversion of seeds were
determined by redissolving the wafers in freshly prepared
embryogenic medium and culturing the rehydrated
embryos. High conversion frequency of somatic embryos
of celery could be obtained by incorporating abscisic acid
(ABA) and mannitol to maturation medium (Halal,
2011). The synthetic seeds with desiccation to 10-15%
can be stored at room temperature for about one year
without being lost in cell viability and germination
potential of seeds. More healthier seedlings could be
derived from dried somatic embryos when compared to
seedlings obtained from undried embryos (Senaratna
et al., 1990).
Hydrated synthetic seeds
Hydrated artificial seeds consist of somatic
embryos or suitable plant tissues enclosed by a hydrogel.
Many substances like potassium alginate, agar, gelrite,
sodium pectate have been examined but calcium alginate
was found to be most effective coating material for
hydrated synthetic seeds (Redenbaugh et al., 1987).
Alginate is a straight chain, hydrophilic, colloidal
polyuronic acid composed primarily of hydro-b-D
mannuronic acid residues with 1-4 linkages. For
production of hydrated seeds, the plant materials are
mixed with sodium alginate gel (0.5-5.0 % w/v) which is
followed by dropping into the calcium chloride solution
(30-100 mM) using pipette. Round and firm beads of
calcium alginate containing somatic embryos are formed
as the ion exchange occurs resulting in the replacement
of sodium ions with calcium ions (Reddy et al., 2012).
The hardness and rigidity of capsules mainly depend
upon the number of sodium ions exchanged with calcium
ions. So, hardness of calcium alginate gel can be
modulated by changing the concentration of sodium
alginate and calcium chloride solution along with change
in duration of complexing. In most cases 2% of sodium
alginate gel when complexed with 100 mM Calcium
chloride solution produced desirable quality synthetic
seeds for many plant species (Redenbaugh et al., 1987;
Oceania et al., 2015). Ca-alginate capsules are difficult to
handle as they are very wet and tend to stick together
slightly. Moreover, the Ca-alginate loses water rapidly
and dries very fast to form hard pellet within few hours
when the beads are exposed to normal atmosphere.
However, these problems can be solved by coating the
capsules with Elvax 4260. Antibiotic mixture containing
rifampicin, cefatoxine and tetracycline-HCl can also be
added to the matrix to avoid bacterial contamination
(Bekheet, 2006). When there is encapsulation of plant
materials exuding high phenolic compounds, activated
charcoal (0.1-0.4%) may be incorporated to the matrix to
absorb the phenolic exudes (Ganapathi et al., 1992).
Propagules for synseed production
The somatic embryos were earlier employed as the
only explant for synthetic seed development in several
plants. But subsequent reports by different workers
showed the use of varieties of plant micropropagules like
unipolar apical shoot tips and buds, nodal segments,
embryogenic masses and calli along with hosts of other
explants like protocorms or protocorm like bodies, bulb,
bulblets, hairy roots and microtubers (Reddy et al., 2012;
Gantait and Sinniah, 2013)..
Somatic embryos
Embryos which are obtained asexually from
somatic cells without the union of gametes are called
somatic embryos. In vitro development of plant somatic
embryos was first reported by Reinert (1958) and
Steward et al. (1958) independently. The direct SEs
develop directly from explanted cells while indirect SEs
derive from explant tissues through intervening callus
phase (William and Maheswaran, 1986). The SEs
undergo globular, heart, torpedo and cotelydonous stages
like zygotic embryos to germinate and develop into
fertile plants. SEs however do not go through desiccation
and dormancy unlike normal embryos and enter the
germinating phase as soon as they are fully formed
(Zimmerman, 1993). They are the most favorable
propagules use for production of synthetic seeds due to
presence of radicle and plumule which develop in single
step into roots and shoots respectively without subjecting
to any specific treatment (Standardi and Piccioni, 1998).
With the advancement of plant tissue culture technology,
somatic embryos have been induced successfully in
number of plants making somatic embryos more
preferable for artificial seed production as they can be
more easily available. The SEs can be preserved in viable
state for longer duration if the moisture content can be
maintained at 10% like normal seeds by drying (Ara
et al., 2000).
Attempts have been made to desiccate somatic
embryos before encapsulation to exploit this potential.
Desiccation tolerance was induced in somatic embryos of
Alfalfa plant by exogenous application of abscisic acid
(Senaratna et al., 1989). When the somatic embryos after
encapsulation were dried to 10-15% and stored in dry
state for about 4 weeks, 65% of somatic embryos
survived and germinated like true seeds. Sometimes
emergence of shoot and root meristem during synthetic
seed germination was prevented by nutritive outer
covering. In order to avoid this scenario, gel capsules had
been prepared which were self-breaking under humid
conditions. This involved the rinsing of beads thoroughly
in running tap water which was followed by immersion
of beads in a 200 mM solution for 60 mins and finally
desalting them by rinsing them in running tap water for
40 minutes (Onishi et al., 1994). The synthetic seeds
showed 50% conversion in two weeks when sowed in
green house conditions. The use of somatic embryos as
explant propagules have been demonstrated in several
plants like Armoracia rusticana (Shigeta and Sato,
1994); Asparagus officinalis (Mamiya and Sakamoto,
2001); Oryza sativa (Suprasanna et al., 2002); Solanum
melongena (Huda and Bari, 2007); Zea mays
(Thobunluepop et al., 2009); Oryza nivara (Jaseela et al.,
2009); Vitis vinefera (Nirala et al., 2010); Tylophora
indica (Devendra et al., 2011); Rhinacanthus nasutus
(Cheravathur et al., 2013); Asethum graveolens (Dhir et
al., 2014); Apple rootstock MM.106 (Farahani et al.,
2015); Litchi (Das et al., 2016); Citrus (Micheli and
Standardi, 2016).
Protocorms and protocorm-like bodies
The miniature exalbuminous orchid seeds when
inoculated in culture medium in vitro started swelling in
one or two weeks indicating successful germination due
to imbibition of water and nutrients (Nongdam and
Chongtham, 2011). The embryos underwent several
division to develop into irregularly shaped
parenchymatus cell mass called spherules (Nongdam and
Tikendra, 2014). The hairy globular spherules developed
into protocorms which are oval, elongated, branched and
spindle shaped bodies considered to be an intermediate
structure between embryos and plants (Fig. 1a). The
protocorms directly differentiated into complete
seedlings after undergoing morphogenetic changes (Fig.
1b). Protocorm-like bodies are similar to protocorms in
their function and morphology but are developed from
plant parts other than seeds of orchids under in vitro
conditions. In different orchids like Cymbidium
giganteum, Dendrobium wardianum and Spathoglottis
plicata, synthetic seeds have been produced by
encapsulating protocorms or protocorm-like bodies with
calcium alginate gel (Sharma et al., 1992; Corrie and
Tandon, 1993; Nagananda et al., 2011). The encapsulated
protocorms of C. giganteum developed into healthy
plantlets when grown on nutrient medium in vitro or in
sterile soil and sand mixture under greenhouse
conditions. The frequency of synthetic seed conversion
was higher in vitro as compared to conversion of
germinated seeds in sand and soil mixture. Mohanty et al.
(2013) produced synseeds in Dendrobium nobile using
PLBs and observed conversion of synseeds significantly
high at 80% as PLBs being highly potential for direct
plantlet generation. Mohanraj et al. (2009) encapsulated
PLBs of Coelogyne breviscapa with 3% sodium alginate
matrix and stored for 60 days before being germinated to
seedling on MS medium supplemented with different
growth regulators. The germination percentage of
synthetic seeds decreased gradually with increase in
storage time.
Embryogenic masses and Calli: Regenerative and stable
embryogenic masses can be used for production of clonal
plants and for studies of genetic transformation. But
maintaining them for longer duration in bioreactors and
culture vessels is difficult due to frequent subculturings
(Ara et al., 2000). The laborious and expensive
subculturing procedure can be overcome by
encapsulating the embryogenic masses with sodium
alginate and store them at 40oC after 6-benzyl amino
purine (BAP) treatment (Redenbaugh et al., 1991). The
synthetic seeds can be stored for around 2 months
without losing its viability and original proliferative
capacity. But more research needs to be done to
understand whether the storage period of synthetic seeds
can be extended and efficiency and proliferative nature of
embryogenic masses decrease with the increase in the
storage period. Regenerative embryogenic masses have
been utilized for synthetic seed production in few plants
like Anthurium andreanum (Nhut et al., 2004) and
Arnebi euchroma (Manjkhola et al., 2005). Plant callus is
unorganized mass of proliferative cells produced by
isolated cells, tissues or organs under the influence of in
vitro culture conditions (Fig. 1c). Callus formation is
associated with the development of progressively more
random planes of cell division, lower cell specializations
and loss of organized structures (Wagley et al., 1987).
The acceptance of calli as explant propagules for synseed
preparation is limited due to their undifferentiated nature
and low differentiation potential (Gantait et al., 2015).
The use of calli for synseed development was
successfully observed for the first time in Allium sativum
by Kim and Park (2002) showing high conversion and
regeneration rate of synseeds to plants. Zych et al. (2005)
and Reedy et al. (2005) also attempted successfully to
formulate synseeds from callus derived from in vitro
culture of Rhodiola kirilowii and Rauvolfia serpentina
respectively.
Apical shoot tips/ shoot buds and nodal segment
The shoot buds and apical shoot tips are unipolar
structure without root meristem. The conventional shoot
tip culture in vitro requires higher space and culture
media as compared to micropropagation of shoot tip/buds
encapsulated synseeds (Gantait et al., 2015). The
requirement of smaller space ensures easy transportation
of plant propagules from one center to another. The shoot
tip explants were induced to root before encapsulation by
exposing to indole-3-butyric acid (IBA) treatment for 3-6
days. Successful conversion to plantlets from synseeds
developed by encapsulating banana and mulberry buds
without auxin treatment was also reported (Bapat and
Rao, 1990; Ganapathi et al., 1992). Figure 1(d) represents
synseeds developed by enclosing in vitro derived shoot
tips with calcium alginate covering. The use of apical
shoot tips/ buds as explants for synseeds development
have been successfully reported in different plant such as
Catalpa ovate (Wysokinska et al., 2002); Stevia
rebaudiana (Andlib et al., 2011); Glycyrrhiza glabra
(Mehrotra et al., 2012); Terminalia aryria (Gupta et al.,
2014); Cucumis sativus (Adhikari et al., 2014) and
Solanum tuberosum (Ghanbarali et al., 2016). The nodal
explants derived either from natural or in vitro
regenerated plants can be encapsulated for synseed
production (Fig. 1e). The plantlet conversion frequency
of in vitro derived nodal explants would be significantly
higher as compared to that of mature nodal tissues
obtained from field grown plants due to higher
meristematic and organogenetic potential. Nodal explants
have been employed for the production of synseeds in
number of plants like Hibiscus moschentus (Prrece and
West, 2006); Pogostemon cablin (Swamy et al., 2009);
Eclipta alba (Singh et al., 2010); Vitex negundo (Ahmad
and Anis, 2010); Picrorhiza kurrooa (Mishra et al.,
2011); Stevia rebandiana (Khan et al., 2013);
Phullanthus fraternus (Upadhya et al., 2014); Blackberry
(Jadan et al., 2015); Physalis peruviana (Yucesan et al.,
2015) and Tomato (Oceania et al., 2015).
Applications of synthetic seeds
There are increasing numbers of medicinal plants
whose population are declining in an alarming rate and
are in the verse of extinction. Rampant deforestation,
rapid expansion of cities and industries in the expense of
natural forested areas, excessive exploitation of rare and
medicinally important plants for economic gains are the
main reasons for increasing number of plants becoming
rare and endangered in their natural habitats. Large scale
propagation for medicinal plants through conventional
methods has limitations as majority of plants are either
seedless varieties, or have reduced endosperms and lower
germination rate (Rai et al., 2009).
Fig.1. The procedure of synthetic seed production using different plant propagules
Fig. 2 (a-e). (a) In vitro protocorm formation in orchid (bar 4 mm), (b) Protocorms directly differentiated into seedlings (bar
6 mm), (c) In vitro callus formation in pea (bar 6 mm), (d) Synthetic seeds with shoot tip explant propagules (bar 5 mm) and
(e) Synseeds developed by encapsulating nodal segments (bar 6 mm).
(b) (a)
(c) (d) (e)
Many of them are also desiccation-sensitive or have
recalcitrant seeds because of which they cannot be stored
for longer period. The synthetic seed technology can be
employed for mass propagation and conservation of rare
and threatened medicinal plants by encapsulating somatic
embryos and meristematic vegetative propagules with
suitable coating materials. Artificial synseeds have been
successfully developed using different plant propagules
in several medicinally important plants like Allium
sativum, Cannabis sativa, Catalpa ovata, Rauwolfia
serpentine and Slevia rebaudiana (Wysokinska et al.,
2002; Belkeet, 2006; Ray and Battarcharya, 2008; Lata
et al., 2009; Andlib et al., 2011).
The germplasm preservation of difficult to
propagate ornamental plants and recalcitrant species like
mango, cocoa and coconut can be performed by
cryopreserving their synseeds in liquid nitrogen (-1960C).
The metabolic function of cells during cryopreservation
is arrested and supplementation of sucrose, salicyclic,
mannitol and other nutrients to encapsulating matrix is
essential which may improve cell viability and tolerance
to dehydration and other abiotic stresses (Janda et al.,
2007; Katouzi et al., 2011). There are several potential
uses of synthetic seeds for important crops such as citrus,
grapes, mango etc. which are vegetatively propagated
and have long juvenile periods. The planting efficiency
can be increased by use of synthetic seeds instead of
cuttings. The synthetic seeds are considered to be highly
advantageous for germplasm conservation in grapes and
other similar crops. Banana is considered as one of the
most important crops because of its large consumption
and considerable export potential. They are normally
propagated by suckers as they do not produce viable
seeds (Matsumoto et al., 1995). Conventional method of
propagation does not guarantee large scale production of
banana, so synthetic seed technology can be used as
another option by encapsulating the shoot-tips excised
from tissue culture raised plants and somatic embryos
developed from callus tissues obtained from in vitro
culture of male flower buds (Ganapathi et al., 2001;
Hassanein et al., 2005; Sandavol-Yugar et al., 2009).
Bapat and Rao (1980) used undifferentiated callus
produced from stem segments of sandalwood - one of the
most commercially valuable forest trees to produce
synseeds. High genetic variations among the progenies
were observed when seeds were used for propagation of
sandalwood. But germination of synthetic seeds
produced by encapsulating the somatic embryos with
calcium alginate produced plants with genetic
uniformity. Mulberry plants are important components of
silk industry as their leaves are chief source of food for
silkworms. The multiplication of mulberry cultivars is
difficult due to restriction in rooting response though
cutting and grafting are used for vegetative propagation.
Moreover 30-40% cutting can only survive the time
period between pruning, transportation and final
transplantation (Bapat and Rao, 1990). The synthetic
seeds produced by encapsulation of in vitro raised
axillary buds could be easily packed in bottles and
transported which reduced space requirement, increases
seed viability and survival rate.
The synthetic seed technology has provided space
and equipment saving option for storage of plant
materials of different rare and commercially important
plants at low temperature. It has the potential for short
term and long term storage of plant germplasm in the
form of synseeds without losing cell viability by
cryopreserving them in liquid nitrogen (Arora et al.,
2010). Other advantages of synthetic seeds are easy
handling because of smaller size beads, higher scale up
capacity, possibility of automation of the whole
production process and direct delivery to field. The
artificial seeds with vegetative propagules enclosed
within them can be used for exchange of axenic plant
materials between the laboratories (Reddy et al., 2012).
The transport of synthetic seeds between the countries
does not require quarantine department permission. This
technology is also independent of seasonal variations as
they can be produced in controlled laboratory
environments.
Limitations of synthetic seeds
The synthetic seed technology inspite of having
bright prospects in large scale propagation and
germplasm conservation of rare and important plants is
associated with many limitations. Production of good
quality microprogagules in high number is prerequisite to
production of synthetic seeds. However, there are
limitations in the generation of viable micropropagules
for use in synseed production. Somatic embryos which
are commonly used explant propagules for synthetic seed
production should attain proper maturation to germinate
out of the coating materials to form healthy plantlets.
Improper maturation of somatic embryos reduces the
success rate of conversion of synthetic into normal
plants. Mature embryos will control germination and
conversion rate which are highly significant for
successful synseed production. However, asynchronous
development of embryos limits the production of normal
plants post synseed formation even after induction of
successful somatic embryogenesis (Ara et al., 2000).
Somatic embryos exhibited immense variation in their
morphology in response to prevailing conditions in
culture system. Long term treatment of ABA either
induced anomalous cotyledon formation in somatic
embryos or activated normal embryo development in in
vitro condition (Crouch and Sussex, 1981).
The conversion of synthetic seeds into plants after
successful germination is the most important aspect of
synthetic seed technology. Due to various reasons the
conversion rate is always very low which limits the
commercial application of this technology. Most of the
studies reported somatic embryogenesis of plants and
their subsequent encapsulation to produce synseeds with
very low success in conversion rate. The choice of
appropriate coating materials is one of important limiting
factors for synseed generation. Low success rate in the
use of synthetic seed technology may be attributed to
failure in the choice of effective encapsulating materials
which should be non-damaging, provide sufficient
protection and nutrition to developing embryos
(Rendenbaugh et al., 1991). Proper storage of synthetic
seeds is affected by lack of dormancy and stress
tolerance in somatic embryos. Also storage of synthetic
seeds at low temperature reduces significantly the
viability of synthetic seeds and its potential for
conversion to normal plants (Makowwczynska and
Andrezejewska-Golec, 2006).
Conclusion
The artificial seed technology is considered as an
alternative to slow and expensive conventional plant
propagation methods by generating several important
plants rapidly in large scale. Artificial seeds also offer
tremendous potential in micropropagation and
germplasm conservation of economically significant rare
and endangered plants. However, lack of production of
high quality micropropagules for synthetic seed
production coupled with low conversion rate to normal
plants has restricted the widespread use of synthetic seed
technology in the field of agriculture. Further detailed
investigations are required to refine this useful
technology for its wide applications at commercial scale.
References
Adhikari S, Bandyopadhyay TK, Ghosha P (2014).
Assessment of genetic stability of Cucumis
sativus L. regenerated from encapsulated shoot
tips. Sci Hortic 170: 115-122.
Ahmad N, Anis M (2010). Direct plant regeneration from
encapsulated nodal segments of Vitex negundo.
Biol Plt 748-752.
Andlib A, Verma RN, Batra A (2011). Synthetic seeds
an alternative source for quick regeneration of a
zero calorie herb-Stevia rebaudiana Bertoni. J
Parm Res 4(7): 2007-2009.
Ara H, Jaiswal U, Jaiswal VS (2000). Synthetic seed:
prospect and limitation. Curr Sci 78(12): 1438-
44.
Arora R, Mathur A, Mathur AK (2010). Emerging
trends in medicinal plant biotechnolgy. In: Arora
R, editor. Medicinal Plant Biotechnology.
Willingford; pp. 1-12.
Bapat VA, Mhatre M (2005). Bioencapsulation of
somatic embryos in woody plants. In: Jain SM,
Gupta PK, editors. Protocol for somatic
embryogenesis in woody plants. Springer; pp.
539-552.
Bapat VA, Rao PS (1988). Sandalwood plantlets from
synthetic seeds. Plt Cell Rep 7(6):434-436.
Bapat VA, Rao PS (1990). In vivo growth of
encapsulated axillary buds of mulberry (Morus
indica L.). Plt Cell Tiss Org Cult 20: 69-70.
Bekheet SA (2006). A synthetic seed method through
encapsulation of in vitro proliferated bulblets of
garlic (Allium sativum L.). Arab J Biotech 9(3).
Cheravathur MK, Kumar GK, Thomas TD (2013).
Somatic embryogenesis and synthetic seed
production in Rhinacanthus nasutus (L.) Kurz.
Plt Cell Tiss Org Cult 113: 63-71.
Corrie S, Tandon P (1993). Propagation of Cymbidium
giganteum Wall. through high frequency
conversion of encapsulated protocorms under in
vivo and in vitro conditions. Ind J Exp Biol 31:
61-64.
Crouch ML, Sussex IM (1981). Development and
storage-protein synthesis in Brassica napus L.
embryos in vivo and in vitro. Planta 153: 64-74.
Das DK, Rahman A, Kumara D, Kumari N (2016).
Synthetic seed preparation, germination and
plantlet regeneration of Litchi (Litchi chinensis
Sonn.). Amer J Plt Sci 7: 1395-1406.
Devendra BN, Srinivas N, Nail GR (2011). Direct
somatic embryogenesis and synthetic seed
production from Tylophora indica (Bum f.)
Merill, an endangered medicinally important
plant. Int J Bot 7: 216-222.
Dhir R, Shekhawat GS, Alam A (2014). Improved
protocol for somatic embryogenesis and calcium
alginate encapsulation in Anethum graveolens L.:
a medicinal herb. Appl Biochem Biotech 173:
2267-2278.
Dodeman VL, Ducreux G, Kreis M (1997). Zygotic
embryogenesis versus somatic embryogenesis. J
Expt Bot 48(313): 1493-1509.
Farahani F, Noormohamadi Z, Satari TN (2015).
Optimization of synthetic seed production in
MM.106 apple rootstocks from somatic embryos
and axillary buds. Bull Envn Phar Life Sci
4(6):68-78.
Ganapathi TR, Sriniva L, Suprasanna P, Bapat VA
(2001). Regeneration of plants from alginate-
encapsulated somatic embryos of banana cv.
Rasthali (Musa sp. AAB group). In Vitro Cell
Dev Biol Plt 37: 178-181.
Ganapathi TR, Suprasana P, Bapat VA, Rao PS (1992).
Propagation of banana through encapsulated
shoot tips. Plt Cell Rep 11:571-575.
Gantait S, Kundu S, Ali N, Sahu NH (2015). Synthetic
seed production of medicinal plants: a review on
influence of explants, encapsulation agent and
matrix. Acta Physio Plt 37: 98-110.
Gantait S, Sinniah UR (2013). Storability, post-storage
conversion and genetic stability assessment of
alginate-encapsulated shoot tips of monopodial
orchid hybrid Aranda Wan Chark Kuan Blue X
Vanda corrulea Grifft. Ex. Lindl. Plt Biotech Rep
7: 257-266.
Germana MA, Amanuele P, Alvaro S(1998). Effect of
encapsulation on citrus reticulate Balanco
somatic embryo conversion. Plt Cell Tiss Org
Cult 55:235-238.
Ghanbarali S, Abdollahi MR, Zolnorian H et al. (2016).
Optimization of the conditions for production of
synthetic seeds by encapsulation of axillary buds
derived from minituber sprouts in potato
(Solanum tuberosum). Plt Cell Tiss Org Cult
126(3): 449-458.
Gray DJ, Mc Colley DW, Compton ME (1993). High-
frequency Somatic Embryogenesis from
Quiescent Seed Cotyledons of Cucumis melo
Cultivars. J Amer Soc Hort Sci 118(3): 425-432.
Gupta AK, Harish M, Raj MP, Agarwal T, Shekhawat
NS (2014). In vitro propagation, encapsulation,
and genetic fidelity analysis of Terminalia
arjuna: a cardioprotective medicinal tree. Appl
Biochem Biotech 173: 1481-1494.
Halal NAS (2011). The green revolution via synthetic
(artificial) seeds: A review. Res J Agric Biol Sci
7: 464-477.
Hassanein AM, Ibrahiem IA, Galal AA, Salem JMM
(2005). Micropropagation factors essential for
mass propagation of banana. J Plt Biotech 7:175-
181.
Huda AKMN, Bari MA (2007). Production of
synthetic seeds by encapsulating asexual
embryo in Eggplant (Solanum melongena L.).
Int J Biotech 2:832-837.
Jadan M, Ruiz R, Soria N, Mihai RA (2015). Synthetic
seeds production and the induction of
organogenesis in blackberry (Rubua glaucus
Benth). Rom Biotech Lett 20(1): 10143-10142.
Janda T, Horvath E, Szalai G, Paldi E (2007). Role of
salicyclic acid induction of abiotic stress
tolerance. In: Harat S, Ahmad A, editors.
Salicyclic acid-A plant hormone. Springer; pp.
91-150.
Jaseela F, Sumitha VR, Nair GM (2009). Somatic
embryogenesis and plantlet regeneration in an
agronomically important Wild rice species
Oryiza nivira. Asian J Biotech 1: 74-78.
Katouzi SSS, Majid A, Fallahian F, Bernard F (2011).
Encapsulation of shoot tips in alginate beads
containing salicylic acid for cold storage and
plant generation in sunflower (Helianthus annuus
L.) AJCS 11: 1469-1474.
Khan MK, Sharma T, Mishra P, Shukla PK, Singh Y,
Ramteke PW (2013). Production of plantlets on
different substrates from encapsulated in vitro
nodal explants of Stevia rebaudiana. Int J Recent
Sci Res 4: 211-215.
Khor E, Loh CS (2005). Artificial seeds. In: Nedovic V,
Willaert K editors. Application of Cell
Immobilization, Biotechnology, Springer; pp.
527-537.
Kikowska M, Thiem B (2011). Alginate-encapsulated
shoot tips and nodal segments in
micropropagation of medicinal plants: A review.
Kerba Polnca 57(4): 46-57.
Kim MA, Park JK (2002). High frequency plant
generation of garlic (Allium sativum L.) calli
immobilized in calcium aliginate gel. Biotech
Bioproc Eng 7: 206-211.
Kitto SK, Janick J (1982). Polyox as an artificial seed
coat for asexual embryos. Hort Sci 17: 488-490.
Lata H, Chandra S, Khan IA, Elsohly MA (2009).
Propagation through alginate encapsulation of
axillary buds of Cannabis sativus L- an important
medicinal plant. Physiol Mol Plt 15(1): 80-86.
Makowwczynska J, Andrezejewska-Golec E (2006).
Somatic seeds of Plantago asiatica L. Acta Soc
Bot Pol 75(1): 17-21.
Mamiya K, Sakamoto Y (2001). A method to produce
encapsulatable units for synthetic seeds in
Asparagus officinalis. Plt Cell Tiss Org Cult 64:
27-32.
Manjkhola S, Uppeandra D, Meena J (2005).
Organogenesis, embryogenesis and synthetis
seed production in Arnebia euchroma a critically
endangered medicinal plant of the Himalaya. In
Vitro Cell Dev Plt 41: 244-248.
Matsumoto K, Hirao C, Teixeira C (1995). In vitro
growth of encapsulated shoot tips in banana
(Musa sp.). Acta Hortic 370: 13-20.
Mehrotra S, Khaja O, Kukreja AK, Rahman L (2012).
ISSR and RAPD based evaluation of gentic
stability of encapsulated micro shoots of
Glycyrrhiza glabra following 6 months of
storage. Mol Biotech 52(3): 262-268.
Micheli M, Standardi A (2016). From somatic embryo to
synthetic seed in Citrus sp. through the
encapsulation technology. Methods Mol Biol
1359: 515-522.
Mishra J. Singh M, Palni LMS, Nandi SK (2011).
Assessment of genetic fidelity of encapsulated
microshoots of Picrorhiza kurrooa. Plt Cell Tiss
Org Cult 104: 181-186.
Mohanraj R, Ananthan R, Bai VN (2009). Production
and storage of synthetic seeds in Coelongyne
breviscapa Lindl. Asian J Biotech 11: 37-43.
Mohanty P, Nongkling P, Das MC, Kumaria S,Tandon P
(2013). Short-term storage of alginate-
ebcapsulated protocorm-like bodoes of
Dendrobium nobile Lindl: an endangered
medicinal orchid from northeast India. 3 Biotech
3: 235-239.
Nagananda GS, Satishchandra N, Rajath S (2011).
Regeneration of encapsulated protocorm like
bodies of medicinally important vulnerable
orchid Flickingeria (Dalz.) Seidenf. Int J Bot 7:
310-313.
Nhut DTN, Duy, Ha-vy HN, Khue CD, Khiem DV,
Hang NTT, Vinh DN (2004). Artificial seeds for
propagation of Anthurium TROPICAL. J Agric
Sci Technol 4: 73-78.
Nirala NK, Das DK, Reddy MK, Srivastava PS, Sopory
SK, Upadhyaya KC (2010). Encapsulated
somatic embryos of grape (Vitis vinifera L.) an
efficient way for storage, transport and
multiplication of pathogen free plant material.
Asia Pac J Mol Biol Biotech 18: 159-162.
Nongdam P, Chongtham N (2011). In vitro rapid
propagation of Cymbidium aloifolium (L) Sw.: A
medicinally important orchid via seed culture. J
Biol Sci 11: 254-260.
Nongdam P, Tikendra L (2014). Establishment of
efficient in vitro regeneration protocol for rapid
and mass propagation of Dendrobium
chrysotoxum Lindl. using seed culture. The
Scientific Wld J.
Oceania C, Doni T, Tikendra L, Nongdam P (2015).
Establishment of efficient in vitro culture and
plantlet generation of Tomato (Lycopersicon
esculentum Mill.) and development of synthetic
seeds. J Plt Sci 10: 14-24.
Onishi N, Sakamoto Y, Hirosawa T (1994). Synthetic
seeds as an application of mass production of
somatic embryos. Plt Cell Tiss Org Cult 39: 137.
Prrece JE, West TP (2006). Greenhouse growth and
acclimatization of encapsulated Hibiscus
moschatus nodal segments. Plt Cell Tiss Org Cult
87: 127-138.
Rai MK, Asthana P, Singh SK, Jaiswal VS, Jaiswal U
(2009). The encapsulation technology in fruit
plants-A review. Biotech Adv 27: 671-679.
Ray A, Bhattarcharya S (2008). Storage and plant
regeneration from encapsulated shoot tips of
Rauvolfia serpentine-an effective way of
conservation and mass propagation. South Afr J
Bot 74: 776-779.
Reddy GS, Kumar DD, Babu RS, Madhav M (2005).
Callus culture and synthetic seed production in
Rauvolfia serpentina. Ind J Hort 62:102-103.
Reddy MC, Murthy KSR, Pullaiah T (2012). Synthetic
seeds: A review in agriculture and forestry.
African J Biotech 11(78): 14254-14275.
Redenbaugh K, Nichol J, Kossler ME, Paasch BD
(1984). Encapsulation of somatic embryos for
artificial seed production. In Vitro Cell Dev Biol
Plt 20: 256-257.
Redenbaugh K, Fujii J, Slade D, Viss P, Kossler M
(1991). Artificial seeds-encapsulated embryos.
Biotech Agri For 17:395-416.
Redenbaugh K, Slade D, Viss PR, Fujii J (1987).
Encapsulation of somatic embryos in synthetic
seed coats. Hort Sci 22: 803-809.
Reinert J (1958). Untersuchungen u¨ber die
morphogenese a Gewebekulturen. Ber Dtsch Bot
Ges 71:65.
Saiprasad GVS (2001). Artificial seeds and their
applications. Resonanace 6: 39-46.
Sandavol-Yugar EW, Vesco LDD, Steinmacher DA,
Stolf EC, Guerra MP (2009). Microshoots
encapsulation and plant conversion of Musa sp.
Cv. Grand naine. Ciencia Rural 39: 998-1004.
Senaratna T, Mckersie BD, Bowley SR (1990). Artificial
seeds of alfalfa (Medicago sativa L.) induction of
desiccation tolerance in somatic embryos. In
Vitro Cell Dev Biol Plt 26:85-90.
Senaratna T, Mckersie BD, Bowley SR (1989).
Desiccation tolerance of alfalfa (Medicago sativa
L.) somatic embryos, Influence of abscisic acid,
stress pretreatments and drying rates. Plt Sci
65:253-259.
Sharma S, Shahzad A, Silva JAT (2012). Synseed
technology-A complete synthesis. Biotech Adv
31:186-207.
Sharma A, Tandon P, Kumar A (1992).Regeneration
of Dendrobium wardianum Warner
(Orchidaceae) from synthetic seeds. Ind J Exp
Biol 30: 747-748.
Shigeta J, Sato S (1994). Plant regeneration and
encapsulation of somatic embryos of horseradish.
Plt Sci 102: 109-115.
Singh SK, Rai MK, Asthana P, Sahoo L (2010).
Alginate encapsulation of nodal segments for
propagation, short term storage and germplasm
exchange and distribution of Eclipta alba (L.).
Acta Physiol Plt 32: 607-10.
Standardi A, Piccioni E (1998). Recent perspective on
synthetic seed technology using nonembryogenic
in vitro-derived explants. Int J Plt Sci 159: 968-
978.
Steward EC, Mapes MO, Mears K (1958). Growth and
organized development of cultured cells. II:
organization in cultures grown from freely
suspended cells. Am J Bot 45: 705-708.
Suprasanna P, Bharati G, Ganapathi TR, Bapat TA
(2002). In vitro development of encapsulated
somatic embryos in rice. Trop Agric Res Ext
5:76-78.
Swamy MK, Balasubramanya S, Anuradha M (2009).
Germplasm conservation of Patchouli
(Pogostemon cablin Benth.) by encapsulation of
in vitro nodal segments. Int J Biodvers Conserv
1: 224-230.
Thobunluepop P, Pawelzik E, Vearasilp S (2009).
Possibility of sweet corn synthetic seed
production. Pak J Biol Sci 12: 1085-1089.
Upadhya R, Kashyap SP, Singh CS, Tiwari KN, Singh
K, Singh M (2014). Ex situ conservation of
Phyllanthus fraternus Webster and evaluation of
genetic fidelity in regenerates using DNA- based
molecular markers. Appl Biochem Biotech.
Wagley LM, Gladfelter HJ, Phillips GC (1987). De novo
shoot organogenesis of Pinus eldarica Medw. In
vitro. II. Macro-and micro-photographic evidence
of de novo regeneration. Plt Cell Rep 6: 167-171.
William EG, Maheswaran G (1986). Somatic
embryogenesis: factors influencing coordinated
behaviour of cells as an embryogenic group. Ann
Bot 57: 443-462.
Wysokinska H, Lisowska K, Floryanowicz-Czekalska K
(2002). Plantlets from encapsulated shoot buds of
Catalpa ovata G. Don. Acta Soc Bot Pol 71: 181.
Yucesan BB, Mohammed A, Arslan M, Gurel E (2015).
Clonal propagation and synthetic seed production
from nodal segments of Cape gooseberry
(Physalis peruviana L.) a tropical plant. Turk J
Agric For 39:797-806.
Zimmerman JL (1993). Somatic embryogenesis: A
model for early development in higher plants. Plt
Cell 5(10): 1411-1423.