Sharma2013 Synseed Technology A Complete Synthesis

Biotechnology Advances 31 (2013) 186207
Contents lists available at SciVerse ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Synseed technologyA complete synthesis

Shiwali Sharma a, Anwar Shahzad a,, Jaime A. Teixeira da Silva b
a
b
Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh- 202 002, U.P., India
Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-Cho, Ikenobe, 2393, Kagawa-Ken, 761-0795, Japan
a r t i c l e
i n f o
Article history:
Received 29 December 2011
Received in revised form 23 September 2012
Accepted 26 September 2012
Available online 2 October 2012
Keywords:
Cryopreservation
Dehydration
Encapsulation
Hydrogel
Synthetic seed
a b s t r a c t
Progress in biotechnological research over the last two decades has provided greater scope for the improvement
of crops, forest trees and other important plant species. Plant propagation using synthetic seeds has opened new
vistas in the eld of agriculture. Synseed technology is a highly promising tool for the management of transgenic
and seedless plant species, polyploid plants with elite traits and plant lines that are difcult to propagate through
conventional propagation methods. Delivery of synseeds also alleviates issues like undertaking several passages
for scaling up in vitro cultures as well as acclimatization to ex vitro conditions. Optimization of synchronized
propagule development followed by automation of the whole process (sorting, harvesting, encapsulation and
conversion) can enhance the pace of synseed production. Cryopreservation of encapsulated germplasm has
now been increasingly used as an ex vitro conservation tool with the possible minimization of adverse effects
of cryoprotectants and post-preservation damages. Through synseed technology, germplasm exchange between
countries could be accelerated as a result of reduced plant quarantine requirements because of the aseptic
condition of the plant material.
2012 Elsevier Inc. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Types of synseed . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Encapsulated desiccated . . . . . . . . . . . . . . . . . . . . . .
2.2.
Encapsulated hydrated . . . . . . . . . . . . . . . . . . . . . . .
3.
Hydrogel encapsulation techniques . . . . . . . . . . . . . . . . . . . .
3.1.
Single layered synseed . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Double-layered synseed . . . . . . . . . . . . . . . . . . . . . .
3.3.
Hollow beads . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
Propagules used for synseed production . . . . . . . . . . . . . . . . . .
4.1.
Bipolar propagules . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Unipolar propagules . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.
Nodes with apical or axillary buds and microshoots . . . . .
4.2.2.
Microbulbs, microtubers, rhizomes and corms . . . . . . . .
4.2.3.
Meristemoids, cell aggregates and primordia . . . . . . . .
5.
Possible modes of synseed utilization . . . . . . . . . . . . . . . . . . .
5.1.
In vitro plant production . . . . . . . . . . . . . . . . . . . . . .
5.2.
Direct sowing . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Short-term germplasm conservation . . . . . . . . . . . . . . . .
5.4.
Cryopreservation: an effective approach for long-term germplasm storage .
6.
DNA marker technology in synseed experimentation . . . . . . . . . . . .
7.
Problems, limitations and future prospects . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +91 9837061683.

E-mail addresses: shahzadanwar@rediffmail.com, ashahzad.bt@amu.ac.in (A. Shahzad).
0734-9750/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2012.09.007
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S. Sharma et al. / Biotechnology Advances 31 (2013) 186207
187
1. Introduction
2. Types of synseed
An active research front has emerged over the last decade with the
goal of developing non-zygotic embryogenesis into a commercially
useful method of plant propagation. From Haberlandt's postulate
(1902) of articial embryo cultivation to the concept proposed by
Murashige (1977), articial seeds have evolved from a futuristic
idea into a real eld of experimental research. The term articial
seed, which was rst coined by Murashige, is now also known by
other names including manufactured seed, synthetic seed or synseed.
The original denition of an articial seed, as given by Murashige
(1978), was an encapsulated single somatic embryo, i.e., a clonal
product that could be handled and used as real seed for transport,
storage and sowing and that, therefore, would eventually grow either
in vitro or ex vitro, into a plantlet (conversion). Gray and Purohit
(1991) also dened synseed as a somatic embryo that is engineered
for the practical use in commercial plant production. Thus, synseed
production was previously limited to those plants in which somatic
embryogenesis had been reported. However, many plant species remain recalcitrant to somatic embryogenesis. However, Bapat et al.
(1987) proposed that synseeds could be produced from in vitro derived
propagules other than somatic embryos, especially in non-embryogenic
species; in Morus indica, for example, they proposed the use of encapsulated axillary buds. Thus, a synseed is referred to as articially encapsulated somatic embryo, shoot bud or any other meristematic tissue that can
be used as functional mimic seed for sowing, possesses the ability to convert into a plant under in vitro or ex vitro conditions, and can be stored
(Ara et al., 2000; Capuano et al., 1998). This denition extends the concept of the synthetic seed from its bonds to somatic embryogenesis and
links the term to its use (storage, sowing) and product (plantlet). In response to this shortcoming, the possibility of using non-embryogenic
vegetative propagules such as shoot tips, nodal segments/axillary buds,
protocorm like bodies (PLBs), organogenic or embryogenic callus has
been explored as a suitable alternative to somatic embryos (Ahmad and
Anis, 2010; Ara et al., 2000; Danso and Ford-Llyod, 2003; Faisal and
Anis, 2007; Nhut et al., 2005; Ozudogru et al., 2011; Rai et al., 2008b;
Sharma et al., 2009a,b; West and Preece, 2009). To complete this denition, it should be emphasized that the propagule must be able to grow
into a plantlet after sowing (Piccioni, 1997).
Even though in vitro-derived propagules were used in most
synseed studies for encapsulation, it is also possible to encapsulate
propagules excised directly from in vivo cultivated mature plants.
For example, Pattnaik et al. (1995) successfully encapsulated the
dormant vegetative buds of an in vivo-grown three-year old mature
mulberry tree. More recently, Banerjee et al. (2012) produced
synseed containing young sprouted vegetative microshoots together
with a small basal rhizome portion excised from in vivo-grown rhizomes of Curcuma amada which were stored in lightly packed polythene packets.
Over the past two decades, extensive progress has been made in
synseed technology. Rai et al. (2009) presented a brief overview on
synseed technology development in fruit crops only while Ara et al.
(2000) and Saiprasad (2001) described the applications, prospects
and limitations of sysnseed technology, but both those reviews are
either incomplete, or outdated. The present review provides an upto-date, elaborate and refreshing perspective on synseed technology
covering as wide a range of plant species as possible.
Synseed technology is highly promising for the conservation and
mass clonal propagation (Singh et al., 2006) of valuable rare hybrids,
elite genotypes, sterile unstable genotypes and genetically engineered
plants for which seeds are either not available or that require a
mycorrhizal-fungal association for their germination as in the case of
orchids. Recently, encapsulation technology has attracted the interest
of researchers for germplasm delivery and for various analytical studies
(Ara et al., 2000). The possible applications of synseed are summarized
in Fig. 1.
Since the formulation of the concept of synseed by Murashige

(1977), a number of studies have been undertaken in this area of
plant biotechnology. The basic hindrance to synseed technology was
the lack of a natural endosperm and protective coatings in somatic
embryos that made them inconvenient to store and handle
(Redenbaugh et al., 1993). Furthermore, the absence of a quiescent
resting phase and the inability of undergoing dehydration limited
the utility of somatic embryos as a source of synseed production.
Thus, the primary effort in synseed technology was to treat somatic
embryos in such a way that they mimicked zygotic embryos during
storage and other applications. This was the rst major step in the
success of synseed technology (Ara et al., 2000). Synseed technology
has been extended by several research groups for a variety of plant
species including cereals, fruits, vegetables, medicinal plants, forest
trees, orchids and other ornamentals (Ara et al., 1999; German et
al., 1998; Ipekci and Gozukirmizi, 2003; Janeiro et al., 1997; Rai et
al., 2008a,b; Utomo et al., 2008). Based on the literature available to
date, synseeds can be separated into two categories:
2.1. Encapsulated desiccated
Coated desiccated embryos represent an ideal form of synseed
(Pond and Cameron, 2003) for which somatic embryos are rst hardened to withstand desiccation before encapsulation. This induces quiescence in the embryos and provides more handling exibility in
large-scale production systems. Thus, the ability of somatic embryos
to withstand drying to low moisture content is an important factor
for storage and plays a critical role in the developmental transition
between maturation and conversion. Such types of synseeds can
only be produced in those plants whose somatic embryos are
desiccation-tolerant.
Desiccation can be achieved either slowly over a period of one or
two weeks sequentially using chambers of decreasing relative humidity, or rapidly by unsealing the Petri dishes and leaving them overnight to dry (Ara et al., 2000). The drying rate is one of the critical
factors for the efcient survival of somatic embryos. If the embryos
are immature, slow drying over one week is optimal, but if there is
large number of fully mature embryos, rapid drying in a laminar
ow bench is preferable (Senaratna et al., 1990).
Desiccation tolerance can also be induced with maturation medium
with high osmotic potential induced by either increased levels of permeating osmoticants (e.g., sucrose, mannitol), non-permeating osmoticants
(e.g., polyethylene glycol or PEG) or high gel strength media (to limit
water availability). While working with ginger synseeds, Sundararaj et
al. (2010) found that sucrose-dehydration was more effective than
air-dehydration in terms of re-growth ability by providing required nutrients; moreover, rapid moisture loss during air dehydration resulted
in poor conversion frequency. For sucrose-dehydration, Sundararaj et
al. (2010) transferred the synseeds to liquid nutrient medium containing
various concentrations of sucrose for 16 h and kept them in an
incubator-shaker for 16 h at 252 C. Synseeds dehydrated resulted
in 86% conversion whereas higher concentrations (0.50 M and 0.75 M)
resulted in no conversion.
Other sub-lethal stresses such as low temperature and nutrient deprivation also have a similar effect on desiccation tolerance (Pond and
Cameron, 2003). Properly pretreated embryos remain viable when
they are rapidly desiccated to less than 1015% moisture content.
Pretreatment with abscisic acid (ABA) also improves the conversion of
somatic embryos both in desiccated and hydrated systems (Nieves et
al., 2001; Pond and Cameron, 2003). Nieves et al. (2001) reported the
effect of ABA and jasmonic acid (JA) on partial desiccation of encapsulated sugarcane somatic embryos. Before encapsulation, embryogenic
callus with somatic embryos were placed on MS medium supplemented
with 3.8 M ABA and/or 4.7 M JA, as described by Tapia et al. (1999),
188
Synseeds
Propagation
Large-scale monoculture of
rare,endangered,
genetically engineered elite
genotypes.
Mixed genotype plantation
Uniform genetic
constitution of plantlets
Analytical tool
Conservation
Cost-effective approach for ex

situ germplasm conservation
Ensures economy of space,
medium, storage period
Protection from environmental
disasters
Extra protection to explants
against pests and diseases
Comparative study aid for

zygotic embryogeny
Determining the role of
endosperm in embryo
development and germination
Seed coat formation studies
Study of somaclonal
variation
Short to
medium-term storage
Long-term
storage
Slow-growth
conservation
Cryopreservation
Transport
Easy, cost-effective
disease-free germplasm
transportation
Direct greenhouse and
field delivery
Germplasm exchange
between countries without
obligations form quarantine
department
Two-step freezing
Simple desiccation
Encapsulation-dehydration
Vitrification
Encapsulation-vitrification
Maintenance under reduced

temperature and/or reduced light
intensity
Use of growth retardants such as ABA
Use of minimal growth medium
Use of osmoticum
Reduction in oxygen concentration
Combination of more than one
treatment
Fig. 1. Potential uses of synseeds.
and maintained over 5 days at 36 C. After encapsulation in calcium alginate enriched with articial endosperm medium and osmotically
dehydrated in 0.5 M sucrose over 24 h or non-dehydrated, somatic embryos were dehydrated over 96 h in chambers containing silica gel until
the beads reached either 60% or 30% of water content. Water content
was assessed according to wet weight and was evaluated until a thermodynamic balance was reached; thereafter the water activity (aw) of
somatic embryos was determined according to Timbert et al. (1996).
Survival of encapsulated-dehydrated embryos was achieved with ABA
rather than with JA. Further, they reported that ABA induced an increase
in protein, polyamine, free proline and starch levels in response to desiccation tolerance. Rai et al. (2008a) induced quiescence in somatic embryos of guava (Psidium guajava) using different concentrations of ABA
and sucrose. Maturation was possible by transferring earlier stages of
non-encapsulated somatic embryos (globular to cotyledonary stages)
to full-strength MS medium solidied with 0.8% agar and supplemented
with 1 mg l1 ABA and 10% sucrose for 4 weeks. As the concentration
of sucrose (39%) or ABA (0.011.0 mg l1) increased in MS medium,
the percentage conversion of encapsulated somatic embryos decreased
signicantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l1 ABA for different durations (060 days) converted to plantlets when transferred to MS
medium containing only 3% sucrose. About 20.8% and 37.5% of encapsulated somatic embryos converted after storage on MS medium
containing ABA (1 mg l1) or sucrose (9%) for 60 days.
After desiccation, embryos are coated with a protective and nutritive layer that inhibits mechanical damage during handling and provides nourishment during the early stages of conversion (Pond and
Cameron, 2003). The coating must be non-toxic, non-aqueous (to
prevent the rehydration of embryos), it should be able to melt at a

relatively low temperature (so the embryos do not suffer thermal
damage during coating) and must be able to adhere to embryos.
Moreover, the rigidity of the coating should be soft enough in order
to allow the emergence of shoot and root primordia.
The rst report on synseed production was published by Kitto and
Janick (1982). They produced desiccated synseeds of carrot (Daucus
carota) by encapsulating multiple somatic embryos in a water-soluble
resin, polyoxyethyelene glycol (Polyox) then desiccating these embryos. Of the various compounds tested for encapsulation of celery
(Apium graveolens) embryos, Kitto and Janick (1985) selected
polyoxyethylene which is readily soluble in water and dries to form a
thin lm, does not support the growth of microorganisms and is
non-toxic to the embryo. Later on, Janick et al. (1993) extended this
technology for encapsulating a PEG-based coating mixture for carrot
somatic embryos and embryogenic callus that was allowed to dry for
several hours on a Teon surface in a sterile hood. The dried mixture
was then placed onto a culture medium, allowed to rehydrate and
then scored for embryo survival.
Three years later, Timbert et al. (1996) suggested that the survival percentage of encapsulated carrot somatic embryos was dependent on the
type of encapsulating matrix and on the speed of dehydration. Some
hydrogels delayed dehydration and preserved the water content of
somatic embryos. They found alginate with gellan gum to be the most efcient hydrogel in maintaining high water activity (aw) around somatic
embryos. To exert different dehydration speeds, they used a slow dehydration protocol (9515% relative humidity (RH) into the chamber within
11.5 days). In the absence of any maturing pretreatment, 72.9% of
alginate-gellan gum-encapsulated carrot somatic embryos, dehydrated
to 15% RH and rehydrated in moisture air (90% RH), germinated. Desiccation in alginate-encapsulated and non-encapsulated somatic embryos from two cell lines (P28H9 and P29H17) of oak tree (Quercus
robur) was also induced by Prewein and Wilhelm (2003). They compared the effect of desiccation under two treatments: either after
1 week desiccation in a multi-well chamber (partial drying) at 27 C
in the dark at a relative humidity of more than 95% or after drying for
2 h in a laminar ow cabinet. No signicant effect of desiccation compared to alginate-encapsulation was observed, either on the germination rate or on the conversion rate. Both cell lines responded poorly
with only 8% and 7% conversion rates following rapid-drying. When
the two desiccation methods used with oak synseeds of cell line
P29H17 were compared, no differences were detected. The conversion
of encapsulated somatic embryos was signicantly higher with slow
desiccation than non-encapsulated controls. This can be attributed to
the effect of desiccation rather than the effect of encapsulation in
general.
2.2. Encapsulated hydrated
Redenbaugh et al. (1984) developed hydrogel encapsulation of individual somatic embryos of alfalfa (Medicago sativa) and patented
this technology in 1988. Since then it remains the most studied strategy
of synseed production (Ara et al., 2000; Rai et al., 2009). A number of
coating agents such as agar, sodium alginate, potassium alginate,
sodium pectate, carrageenan, sodium alginate with carboxymethyl
cellulose, gelatin, gelrite, guargum, tragacanth gum, etc. have been tested as hydrogels (Ara et al., 2000; Rai et al., 2009). Among all, sodium alginate has been frequently selected because of its moderate viscosity,
low spin ability of solution, no toxicity for propagules, quick gellation,
low cost and bio-compatibility (Saiprasad, 2001; Swamy et al., 2009).
Sodium alginate and calcium salt are reported to be the best combination for encapsulation since these ions are non-damaging, have a low
price, are easy to use and result in high embryo-to-plant conversion.
The capsule gel potentially serves as a reservoir of nutrients that helps
in the survival and speedy growth of embryos (Redenbaugh et al.,
1987).
3. Hydrogel encapsulation techniques
Since hydrogel encapsulation is the most successful and widely accepted approach to synseed production, the next section presents an
overview of hydrogel encapsulation and its modications.
3.1. Single layered synseed
This is the simplest way to achieve hydrogel encapsulation. To produce single layered synseeds, propagules are carefully isolated either
from an in vitro or an in vivo source and mixed with a suitable hydrogel
such as sodium alginate (0.55.0%, w/v). Alginate is a straight chain, hydrophilic, colloidal polyuronic acid primarily composed of hyrdo-D-mannuronic acid residues with 14 linkages (Cameron, 2008;
Martinsen et al., 1989). Alginate solution is prepared either in double
distilled water (DDW) or in liquid nutrient medium and dropped,
along with the propagule, as a bead into a complexion agent such as
calcium chloride (CaCl22H2O) or calcium nitrate [Ca(NO3)2] solution
(30150 mM). The pH of both the alginate matrix and complexing
agent is adjusted to 5.8 prior to sterilization. Generally, for sterilization,
both the gel matrix and complexing agent are autoclaved at
1.06 kg cm2 and 121 C for 20 min (Kavyashree et al., 2006; Sharma
et al., 2009a,b), although Rihan et al. (2011) sterilized the sodium
alginate solution by tyndallisation and calcium chloride by autoclaving.
The major principle involved in the alginate encapsulation process is
the formation of round and rm calcium alginate beads due to an ion
exchange process between sodium (Na+) and calcium (Ca++) ions.
The permeability, hardness or rigidity of the beads and the subsequent
189
success of the encapsulation method is affected by alginate and

calcium chloride concentration used and curing time and it may vary
with different propagules as well as with the different plant species
(Table 1). Hence, the concentrations of these two solutions and
complexion time must be optimized for the formation of an ideal bead
(Saiprasad, 2001). In most of the reports, 3% (w/v) sodium alginate and
100 mM calcium chloride for 2030 min has proved to be the best
combination for the formation of an ideal synseed (Ahmad and Anis,
2010; Alatar and Faisal, 2012; Hung and Trueman, 2011, 2012a,b;
Ozudogru et al., 2011; Sarkar and Naik, 1998; Tabassum et al., 2010).
However, in several studies, 3% sodium alginate upon complexion
with 75 mM calcium chloride for 2030 min was the optimum
combination for proper hardening of beads viz., Dendrobium,
Oncidium and Cattleya orchids (Saiprasad and Polisetty, 2003) and
Fragaria ananassa (strawberry) and Rubus idaeus (raspberry)
(Lisek and Orlikowska, 2004). In contrast, for the encapsulation of
nodal segments of Pogostemon cablin (Swamy et al., 2009) and
Spilanthes acmella (Sharma et al., 2009b) and the microshoots of
Zingiber ofcinale (ginger) (Sundararaj et al., 2010), 4% sodium
alginate with 100 mM calcium chloride was optimum. This
variation in sodium alginate concentration for synseed formation in
different plant species might be due to the variation in commercial
source from which the chemicals were purchased, as reported
earlier by Ghosh and Sen (1994) and Mandal et al. (2000). In
Ocimum spp. (basil), the best results with respect to capsule
formation were obtained with Sigma's (Sigma, St. Louis, USA) 4%
sodium alginate (with 75 mM calcium chloride) than with the
product of CDH (Central Drug House, Mumbai, India) (Mandal et
al., 2000). In Spilanthes mauritiana, optimum bead formation was
possible with 3% sodium alginate of Loba Chemie (Mumbai, India)
and 100 mM calcium chloride but for S. acmella this was possible
with the CDH product (4% sodium alginate with 100 mM calcium
chloride) (Sharma et al., 2009a,b). At lower concentrations (12%),
sodium alginate became unsuitable for encapsulation because of a
reduction in its gelling ability following exposure to high temperature during autoclaving (Larkin et al., 1998). On the contrary, high
concentrations of sodium alginate (56%) beads were isodiametric
but too hard, causing considerable delay in sprouting of shoots
(Ahmad and Anis, 2010; Sharma et al., 2009a,b).
To make synseed beads, peristaltic pumps (Blandino et al., 2000)
and pipettes are usually employed (Ara et al., 2000; Hung and
Trueman, 2012a,b). The solution in which beads are formed is constantly stirred/shaken in order to avoid them sticking to each other
and to increase the formation of spherically-shaped beads during polymerization (Malln et al., 2007). The size of the beads is controlled
by varying the inner diameter of the pipette nozzle (Ara et al., 2000).
Nishitha et al. (2006) used a sterilized pipette, 10 mm in diameter, for
shoot tip encapsulation in Chonemorpha grandiora while Hung and
Trueman (2011, 2012a, 2012b) used a 7 mm diameter Pasteur pipette
for the encapsulation of shoot tips and nodal segments of Khaya
senegalensis (mahogany) and Corymbia torelliana C. citriodora (eucalypt). For the encapsulation of gametophyte buds of an endangered
moss, Splachnum ampullaceum, Malln et al. (2007) used a drip because: a) peristaltic pumps delivered the material too quickly to be
employed with fragile plant material and since sterile conditions required for in vitro culture are difcult to obtain; b) The use of pipettes
is tiring and the precision necessary to form uniform beads (i.e. with a
low standard deviation in diameter of 0.2 or 0.3 mm) is unlikely to be
reached. These rm beads are washed with sterile DDW to remove
traces of adhering chemicals (Fig. 2) and placed immediately either
on a nutrient medium or sown in different planting substrates such
as wet lter paper, cotton or Soilrite moistened with nutrient solution or DDW (Rai et al., 2009).
The composition of the gel matrix is an important factor that signicantly affects the conversion performance of encapsulated tissue.
For effective re-growth and conversion of the encapsulated plant
190
Table 1
Optimum conditions for ideal synseed or ca-alginate bead formation in different plant species (2000 onwards, in chronological order and alphabetical order within each year).
Proliferation medium of
the mother culture before
synseed production
Propagule Pretreatment or
Propagule
size
after treatment
used for
(as required)
encapsulation
Actinidia deliciosa
(Hayward kiwifruit)
Macro- and
micro-elements as in QL
medium, organic
compounds as in the
2nd formulation of
Walkej (1972)+
sucrose (25 g/l)+
1 mg/l GA3 +1 mg/l BA
MC (apical
and axillary
buds)
Ocimum
americanum
(hoary basil),
O. basilicum
(sweet basil), O.
gratissimum
(shrubby basil), O.
sanctum (sacred basil)
Explant taken from in vivo Axillary bud

source
Morus alba, M. australis, M.

bombycis, M. cathyana,
M. latifolia and M. nigra
(mulberry)
Modied MS
medium + 100 mg/l
myo-inositol + 30 g/l
sucrose + 8 g/l
agar + 4.4 M BA
MS medium + 4.5 M
2,4-D, 1 mg/l D-biotin,
100 mg/l
L-glutamine + 100 mg/l
malt extract (suspension
culture)
B5 medium + 4.65 M
Kn + 396.48 M PG
MS medium
supplemented with
5 106 M
NAA + 1 10-5 M Kn
Musa spp. AAB group

(banana cv. Rasthali)
Adhatoda vasica (vasaka)
Allium sativum (garlic)
4 mm
Encapsulating
matrix
composition
Concentration Polymerization Composition of

of CaCl22H2O time (min)
regrowth
medium for
or CaCl2
synseed
References
% Conversion
(complete
plantlet recovery
in
vitro)
2.5% SA + macro- and

microelements of
QL medium + 25 g/l
sucrose + 1 mg/l
GA3 + 1 mg/l BA
100 mM
30
-strength
proliferation
medium without
PGR
Adriani et al.
56.236.1%
(2000)
(after cold
treatment) 31.4
25.7% (after root
induction
treatment) 50
57% (after
adding sucrose
in sowing and
encapsulation
matrix)
4% SA + MS
medium + 100 mg/l
myo-inostiol +
30 g/l + 1.1 M BA
(O. americanum)
+2.2 M BA
(O. gratissimum)+4.4
M BA (O. basilicum and
O. sanctum)
4% SA + MS
medium + 30 g/l
sucrose + 4.4 M BA
75 mM
NA
MS medium + 30 g/l
sucrose + 1.1 M BA
(O. americanum)
+ 2.2 M BA
(O. gratissimum)
+ 4.4 M BA (O.
basilicum and O.
sanctum)
9599%
Mandal et al.
(2000)
75 mM
30
MS medium + 4.4 M
BA
78-98% (for
shoot
development)
Pattnaik and
Chand (2000)
MS medium
66%
Ganapathi et
al. (2001)
20
B5 medium
66.28%
Anand and
Bansal (2002)
30
-MS
95%
medium + 4.4 10-2 M
sucrose + 5 106 M
NAA + 1 10-5 M Kn
100%
NA
i. Cold treatment was given

to 1 month old proliferation
culture by placing in cold
chamber for 1 month of
darkness at 4 C. ii. MCs
were pretreated with 15 g/l
sucrose and 1 mg/l or
3 mg/l IBA. The cultures
were kept in darkness
inside the growth chamber
on a 100 rpm rotatory
shaker for 24 or 48 h. After
root induction MCs
submitted to root initiation
on sterile lter paper
moistened with nutrients.
iii. 30 g/l sucrose increase
in the sowing medium as
well as in articial
endosperm.
-
NS or axillary
bud
5 mm
SE
-MS medium + 10 M
Zea + 30 g/l sucrose
(maturation medium)
5% SA + MS medium
Shoot bud
14 mm
Calli
NA
1.1%
4% SA + B5
Medium + 4.65 M
Kn + 50 mg/l PG
1.5% SA + half-strength *50 mM
MS medium
+ 5 106 M
NAA + 1 10-5 M Kn
MS
2-5 mm
30
Kim and Park

(2002)
Plant species
(common name)
synseed production
Propagule used for

encapsulation
Ananas comosus
(pineapple)
MS basal medium + MS
vitamins + 0.56 mM
myo-inositol + 0.06 M
sucrose + 9.67 M
NAA + 9.84 M
IBA + 9.29 M Kn for the
induction of multiple
shoots
Paulownia elongata
(Royal Empress tree)
MS medium + 500 mg/l

casein
hydrolysate + 10 mg/l
TDZ + 3% sucrose
-MS medium + 6.97
M Kn
Ipsea malabarica
(Malabar daffodil
orchid)
Dendrobium, Oncidium
and Cattleya (orchids)
Pretreatment or
after treatment
(as required)
Encapsulating
matrix
composition

regrowth
or CaCl2
medium for
synseed
Pretreatment of shoots in
liquid medium of W basal
medium + W
vitamins + 0.56 mM
sucrose + 10.8 M
NAA + 39.4 M IBA
and agitated on a
gyratory shaker at
100 rpm for 12 h
MS basal medium
(maturation)
3% SA + MS basal
medium + 0.56 mM
sucrose
1.36 g/
150 ml
3% SA + MS medium
*50 mM
30
Peat + perlite (1:1)
53.3%
Ipekci and
Gozukirmizi
(2003)
Shoots developed
on proliferation
medium transferred
on -MS + 6.97
M + 6 or 8%
sucrose
P24 medium + 0.9 M
BA + 3% sucrose
(maturation medium)
3% SA + -MS
medium + 6.97 M Kn
*0.7%
30
-MS medium
with or without
6.97 M Kn
100%
Martin
(2003)
4% SA + P24
medium + 3% sucrose
50 mM
20
26%
Prewein and
Wilhelm
(2003)
NA
75 mM
30
100% (for all

three species)
Saiprasad
and Polisetty
(2003)
Nodal segments were

placed on -MS
supplemented with
1.0 mg/l IBA for 10 days
Pre-conditioning with
10 mg/l mannitol added to
proliferation media
3% SA + -MS
medium + 0.44 M BA
with 0.54 M NAA (for
Dendrobium), 2.69 M
NAA (for Oncidium)
and 5.38 M (for
Cattleya)
3% SA + half-strength
MS medium
*75 mM
20
P24 medium
+ 0.1 M IBA
+ 0.9 M BA
+ 3% sucrose
MS + 0.44 M
BA + 0.54 M NAA
(for Dendrobium)
MS + 2.69 M NAA
(for Onicidium)
MS + 5.38 M NAA
(for Cattleya)
-MS medium
85%
Chand and
Singh (2004)
3% SA + 4%
glucose + water
(strawberry) or 3%
SA + MS medium
(raspberry)
75 mM
30
Proliferation
medium
3.7 shoots/bead
(after 9 month
of storage and
2nd subculture)
5.6 shoots/bead
(after 9 month
of storage and
2nd subculture)
Lisek and
Orlikowska
(2004)
10
Whatman lter
paper irrigated
with -MS medium
+ vitamins
+ 2% sucrose
47%
Arun Kumar
et al. (2005)
30
-MS
medium
100%
Chithra et al.
(2005)
SE
1.5
2.0 mm
Bulbs
NA
Embryogenic culture lines SE

were maintained
on P24 medium + 0.9 M
BA + 3% sucrose
MS medium + 4.44 M BA PLB
48 mm
Dalbergia sissoo (sissoo)
Explants taken from in

vivo source
NS
10 mm
Fragaria ananassa
(strawberry) and Rubus
idaeus (raspberry)
Boxus medium + 2.2 M

BA + 2.46 M IBA (for
strawberry) and MS medium with NH4NO3 and
KNO3 reduced by
50% + 3.55 M
BA + 0.49 M IBA, both
supplemented with WPM
vitamins
Embryogenic calli from
dehusked seed induced
on MS medium + 30 g/l
sucrose + 2 mg/l 2,4-D.
Calli were then transferred to MS
medium + 30 g/l
sucrose + 2 mg/l
BA + 0.5 mg/l
NAA + 0.5 mg/l
MS medium
+ 0.45 M 2,4-D
(induction medium);
liquid -MS
medium + 0.23 M 2,4-D
ST
3 mm
SE
NA
Self breaking treatment was

given to the prepared
synseed, by dipping them in
200 mM KNO3 for 60 min
and rising in sterile tap
water for 40 min till the
bead became swollen
SE
Semi-solid -MS medium
Oryza sativa (hybrid rice)
Rotula aquatic (takad)
4% SA + MS medium
*1.5%
(except CaCl2) + 30 g/l
sucrose + plant growth
regulators (0.5 mg/l
NAA, 0.5 mg/l IAA,
2 mg l
BA) + antibiotics
(1 mg/l bavistin,
1 mg/l streptomycin)
+ 1.25% AC
*50 mM
3% SA + 1/2-MS
medium (except
CaCl2) + 3% sucrose
% Conversion
References
(complete
plantlet recovery
in
vitro)
Soneji et al.
(2002)
MS basal medium, MS
vitamins + 0.56 mM
myo-inositol
+ 0.06 M
sucrose + 9.67 M
NAA + 9.84 M
IBA and 9.29 M Kn
191
(continued on next page)
Quercus robur
(pedunculate oak)
Propagule
size
192
Table 1 (continued)
regrowth
medium for
or CaCl2
synseed
References
% Conversion
(complete
plantlet recovery
in
vitro)
2.5% SA + MS medium
NA
30
86.13% (after
15 days of
storage at 8 C)
Gangopadhyay
et al. (2005)
2.5% SA + DCR basal

DCR medium + 175 mM
salts
maltose + 80 M
ABA + 9 g/l Gellan gum was
used for maturation. All
cultures were placed in the
dark at 25 2 C for
814 weeks
3% SA
100 mM
(calcium nitrate)
3% SA + MS + 1.0 mg/l
IAA
*100 mM
MS
medium + 0.022 mM
BA + 0.003 mM IAA.
For root induction,
microshoots were
placed in liquid MS
medium with
Luffa-sponge)
+ 0.01 mM IBA
-DCR basal medium
89%
Malabadi and
van Staden
(2005)
25
25
MS medium
60.6%
*75 mM
30
Perlite moistened with

MS medium
50% (without
IAA treatment)
Manjkhola et
al. (2005)
Pinker and
Abdel-Rahman
(2005)
35 mm
4% SA
1.036 g/
150 ml
3040
MS medium + 1 mg/l
BA + 0.5 mg/l IBA
Axillary bud
0.5 cm
40
Naik et al., 1999, 2000
NS
36 mm
*5 104 M
4% SA + LSBM
medium + 8.88 M
BA + 2.0 M TIBA
*100 mM
3% SA + MS
medium + 3% sucrose
and 100 mg/l
BA + 0.54 M NAA
Chonemorpha grandiora
(Bengal creeper)
MS medium + 13.3 M
BA + 2.46 M IBA
ST
NA
LSBM
medium + 8.88 M
BA + 2 M TIBA
MS medium + 100 mg/l
myo-inositol + 3%
sucrose + 4.44 M
BA + 0.54 M NAA
(for Source A) MS
medium + 3%
sucrose and 100 mg/l
myo-inositol
(for sprouting, source
B) MS
medium + 0.54 M NAA
(rooting medium)
-MS medium
88% (after
30 days of
storage at 22
2 C)
48.2%
Hibiscus moscheutos
(hardy hibiscus)
DKW medium + 3%
sucrose + 1 10-7 M TDZ
NS
35 mm
synseed production
Propagule
size
after treatment
used for
(as required)
encapsulation
Ananus comosus
(pineapple)
MS medium + 0.55 mM
myo-inositol + 88 mM
sucrose + 0.22 mM
BA + 0.003 mM IAA
MS
1.5
2.0 mm
Pinus patula
(Mexican weeping pine)
DCR basal
medium + 120 mM
maltose + 2 g/l Gellan
gum + 2 M
2,4-D + 2.5 M
NAA + 1 M BA
SE
NA
Arnebia euchroma
(zicao; arnebia root)
Dendranthema grandiora
(chrysanthemum)
MS medium + 2.5 M
SE
IBA + 2.5 M BA
Modied MS salts (mac- NS
ro-nutrient content reduced by half) + 2.5 mg/l
thiamine HCl + 0.2 mg/l
pyridoxine-HCl + 0.2 mg/
l biotin + 100 mg/l + 8 g/
l + 30 g/l
sucrose + 0.5 mg/l BA
MS medium + 1.0 mg/l
SB, PC
BA + 0.5 mg/l IBA
56 mm
Morus sp.
(mulberry variety-S54)
LSBM medium + 8.88 M

BA + 2 M TIBA
Punica granatum
(pomegranate)
Vanilla planifolia (vanilla)
45 mm
3% SA + -MS (except
CaCl2) + 0.49 M
IBA + 11.7 M silver
nitrate
Beads were exposed to light 2.75% SA
for at least 2 weeks prior to
planting in the greenhouse
30
*50 mM
30
*50 M
30
Medium grade
vermiculite placed
under intermittent
mist (bead planted
Divakaran et
al. (2006)
Kavyashree et
al. (2006)
75% (spouting
only for
source A) 81%
(sprouting for
source B) 21%
conversion only
from source B
87% rooting
(source A) 90%
rooting
(source B)
Naik and
Chand (2006)
95%
Nishitha et al.
(2006)
Preece and
West (2006)
Encapsulating
matrix
composition
Plant species
(common name)
synseed production
Phyllanthus amarus
(bahupatra)
Populus tremula
P. tremuloides
(hybrid aspen)
Hibiscus moscheutos
(hardy hibiscus)
Acca sellowiana
(pineapple guava, feijoa)
Propagule used for

encapsulation

BA
NA
DKW medium + 3%
sucrose + 10-7 M TDZ
LPm medium + 8 mM
Glu + 20 M
2,4-D + 0.1 mg/l
myo-inositol
Tylophora indica (antamul) Explant taken
from in vivo source
Protonema cultured
Splachnum ampullaceum
on Heller's medium
(purple-vased stink
+ 3% sucrose
moss, bryophyte)
OMM
Citrus nobilis C. deliciosa

(Kinnow mandarin)
For somatic
embryogenesis,
ovules were
cultured on MS
medium + 9.29 M
Kn + 3% sucrose
Coleus forskohlii (makandi) MS medium
+ 2.22 M BA
Pogonatherum panicum
(golden hair grass)
MS medium + 2 mg/l
BA + 0.2 mg/l NAA
Pretreatment or
after treatment
(as required)
Encapsulating
matrix
composition

regrowth
or CaCl2
medium for
synseed
1 cm deep and not
covered in greenhouse)
MS medium
% Conversion
References
(complete
plantlet recovery
in
vitro)
ST
NA
3% SA + MS medium
75 mM
20
ST
0.5
0.7 cm
*1.4%
MS medium + 1.3 M
BA + 3% sucrose
100%
NS
4 mm
4% SA + MS
medium + 1.3 M
BA + 3% sucrose
2.75% SA
50 mM
30
SE
NA
1% SA + LPm medium
*50 mM
15
DKW medium + 3%
sucrose + 10-7 M TDZ
LPm medium
80% (after
1.5 year storage)
70.3% (after
60 days of
culture)
NS
35 mm
3% SA + MS medium
100 mM
30
91%
Gametophyte
bud
1% SA
*100 mM
20 min
MS medium + 2.5 M
BA + 0.5 M NAA
Heller's medium + 3%
sucrose + B5 vitamins
NS
34 mm
2.5%
SA + -OMM + 1 mg/l
Zea + 50 g l-1 sucrose
*1.1%
35
OMM
SE
NA
Two pretreatment
a) sprouting induction
(micro-cuttings were
placed in 50 ml sealed
glass vessel containing
15 ml of 1 mg/l
GA3 + 30 g/l
sucrose + vessel put
on a rotator shaker
(100 rpm for 24 h)
in growth chamber
under darkness at
23 2 C b) sprouting
initiation treatment
(after sprouting induction,
micro-cuttings were placed
on lter paper which placed
on vessel containing -MS
proliferation medium + 0.7% agar, kept in
growth chamber at
23 2 C under darkness
for 6 days)
For maturation embryos
allowed to grow on same
medium as used for
induction
4% SA + MS medium
75 mM
45
MS medium + 9.29 M
Kn
81.94%
Singh et al.
(2007)
ST, NS
3% SA + MS + 2%
sucrose
1.36 g/
150 ml
NA
92% (shoot
multiplication)
75% (complete
plantlet
recovery)
Swaroopa
et al. (2007)
SB
35 mm
3% SA + MS
medium + 1% AC
*2% with 0.3% 20

bavistin
MS medium + 2.22 M
BA + 3% sucrose (for
multiplication) MS medium + 3% sucrose (for
complete recovery of
plantlet)
-MS medium + 2%
sucrose
>90%
Wang et al.
(2007)
90%
Singh et al.
(2006)
Tsvetkov et al.
(2006)
West et al.
(2006)
CangahualaInocente
et al. (2007)
Faisal and
Anis (2007)
100% (after short Malln et al.
to medium-term (2007)
storage) 60%
(after 2 years of
storage) 50%
(after 2.5 years
of storage)
Data was not in Micheli et al.
(2007)
terms of %
conversion (1.8
and 1.9 shoots/
capsule after
30 days of
storage at 18 and
4 C)
193
Olea europaea (olive)
Propagule
size
194
Table 1 (continued)
Propagule
size
after treatment
used for
(as required)
encapsulation
Plant species
(common name)
synseed production
Psidium guajava (guava)
SE
Somatic embryogenesis
induced from zygotic
embryos on MS
medium + 4.52 M 2,
4-D + 5% sucrose. After
8 days of treatment with
2,4-D explants transferred
to PGR-free MS medium
+5% sucrose
Rai et al. 2007
ST
Psidium guajava (guava)
Encapsulating
matrix
composition

regrowth
medium for
or CaCl2
synseed
References
% Conversion
(complete
plantlet recovery
in
vitro)
Globular and cotyledonary

stage embryos transferred
to MS medium + 1.0 mg/l
ABA or 10% sucrose for
maturation
2% SA
100 mM
2030
MS medium + 3%
sucrose
91.6%
Rai et al.
(2008a)
35 mm
3% SA
*100 mM
2030
Liquid MS medium
97.2%
Anderson
medium + 7 mg/l
2-iP + 100 mg/l
PVP + 100 mg/l
ascorbic acid + 10 mg/l
citric acid
R4 medium
96%
Rai et al.
(2008b)
Singh (2008)
Rhododendron maddeni
(rhododendron)
Anderson
medium + 7 mg/l
2-iP + 100 mg/l
PVP + 100 mg/l ascorbic
acid + 10 mg/l citric acid
ST
NA
3% SA + Anderson
medium + 3% sucrose
60 mM
30
Spartina alterniora
(smooth cordgrass)
Calli proliferated on
modied R4 medium
followed by suspension
culture in liquid medium
modied by adding
FeSO4.7H2O, myo-inositol,
thiamine HCl, pyridoxine
HCl and casein acid
hydrolysate
SE, MP
NA
2% SA
*100 mM
30
SE and
zygotic
embryos
34 mm
(SEs)
*15 g/l (for

SEs) * 5.5 g/l
(for zygotic
embryos)
10 (for SEs) 30 Macro and

micronutrients of MS
(for zygotic
medium
embryos)
50 mM
30
MS medium + 0.5 M
TDZ + 0.075% PPM
100%
Lata et al.
(2009)
100 mM
-MS medium + 3%
sucrose + 10% coconut
water
-MS medium
68% (shoot
development)
Nagesh et al.
(2009)
>90% (after
3 week of
storage at 4 C)
87.8%
Sharma et al.
(2009a)
80%
Siddique and
Anis (2009)
Srivastava et
al. (2009)
Cannabis sativa
(marijuana)
MS medium + 0.5 M TDZ NS
NA
Curculigo orchioides
(black musali)
MS medium + 1 mg/l
BA + 1 mg/l Kn
Shoot bud
56 mm
4% SA (for SEs) or 3%
SA (for zygotic
embryos) + MS
medium + 0.5 mg/l
IAA + 0.5 mg/l
NAA + 2 mg/l
BA + 30 g/l sucrose
5% SA + MS + 0.5 M
TDZ + 2.5 M IBA
+0.30.5% PPM
2.5% SA + MS medium
Spilanthes mauritiana
(toothache plant)
MS medium + 1.0 M
BA + 0.5 M IAA
NS
34 mm
4% SA + MS medium
100 mM
2025
Spilanthes acmella
(toothache plant)
In vitro seedling grow on

-MS medium + 0.5 M
GA3
Explants taken from in
vivo source
NA
NS
34 mm
4% SA + MS medium
100 mM
2025
MS medium + 1.0 M
BA + 0.5 M NAA
NS
NA
3% SA + MS medium
75 mM
20
ST, NS
35 mm
3% SA + 1.5% sucrose
*3%
2030
-MS + 5 M
BA + 0.5 M IAA
MS medium
5 mm
4% SA + MS
medium + 2% sucrose
*100 mM
30
Nothofagus alpina
(rauli-beech)
Ocimum basilicum
(sweet basil)
Cineraria maritima (dusty
miller or silver dust)
Pogostemon cablin
(patchouli)
MS medium + 2.22 M BA NS
MS medium + 2.22 M
BA
>90%
Utomo et al.
(2008)
Cartes et al.
44% (for SEs)
100% (for zygotic (2009)
embryos)
11.76% (after
6 month of
storage at 25
2 C)
91.1%
Sharma et al.
(2009b)
Swamy et al.
(2009)
23 mm
synseed production
Propagule used for

encapsulation
Propagule
size
Pretreatment or
after treatment
(as required)
Encapsulating
matrix
composition

regrowth
or CaCl2
medium for
synseed
Vitex negundo
(ve-leaved chaste tree)
Simmondsia chinensis
(jojoba)
MS medium + 5.0 M
BA + 0.5 M NAA
BA + 0.5 mg/l NAA
NS
3 mm
3% SA + MS medium
*100 mM
30
ST
35 mm
3% SA
100 mM
2030
Vitis vinifera
(grape)
Eclipta alba
(yerba de tago)
Das et al. (2002)
SEs
57 mm
2% SA
*100 mM
4050
MS medium + 8.8 M BA
ST, NS
3% SA
*1.11%
3045
Vanda coerulea
(orchid)
IY medium+20 g/l+TDZ
(concentration not
mentioned)+NAA
(concentration not
mentioned )
MS medium + 4.4 M BA
PLB
3 mm
3% SA + IY
medium + 20 g/l
sucrose
100 mM
30
NA
NS
35 mm
3% SA + MS medium
100 mM
2030
100%
-MS medium + 2%
sucrose
SE
NA
2.5% SA + -MS
medium + 2% sucrose
43.3%
20
Zingiber ofcinale
(ginger)
Cucumis sativus
(cucumber)

BA + 3% sucrose
For embryogenesis,
hypocotyl pieces cultured
on MS medium + 2 M
2,4-D + 0.5 BA + 3% sucrose followed by cell
suspension in liquid MS
medium + 5 M
NAA + 1 M BA
MSs
35 mm
For proper maturation

cotyledonary stage SEs
transferred to -MS
medium + 10% sucrose for
2 weeks prior to
encapsulation
MS medium + 4.4 M
BA
-MS medium + 2%
sucrose
4% SA + MS medium
100 mM
30
66%
Cell
suspension
having SEs
NA
3% SA
*100 mM
30
Citrus sinensis
Poncirius trifoliate
(carrizo citrange)
MS salts + vitamin
mixture + 30 g/l
sucrose + 1 mg/l
NAA + 10 mg/l BA
NS
34 mm
2.5% SA + -MS
medium + 50 g/l
sucrose
*1.1%
35
MS medium without
PGR
100% (sprouting)
Only 17% of
rooting after
encapsulation
German
et al. (2011)
Khaya senegalensis
(mahogany)
MS medium + 3%
sucrose + 4.4 M BA
ST, NS
46 mm
3% SA + MS
medium + 4.4 M
BA + 3% sucrose
100 mM
30
-MS medium + 3%
sucrose + 4.4 M BA
52-98%
Hung and
Trueman
(2011)
Picrorhiza kurrooa
(katuka)
NA
ST, NS
35 mm
For synchronization, all the

cell passed through 150 M
sieve after every 10 day of
subculturing and then
incubated on gyratory
shaker, shifted to modied
suspension media
containing CH+ NAA for
23 days. Finally
maturation was done on
MS basal medium.
Micro-cuttings were
immersed for 3 days in the
dark and kept at 23 1 C
in 50 ml glass jars (10
micro-cuttings per jar)
containing 15 ml of rooting
solution (5 mg/l IBA and
15 g/l sucrose (root induction pretreatment)
Excised shoot tips were
pre-treated by culturing on
agar-solidied 1/2-MS medium containing 245 M
IBA + 2% sucrose for 24 h in
darkness
MS medium + 3%
sucrose
Neutral gel formed by
dissolving phytagel in
autoclaved tap water
3% SA + -MS
medium + 1.5%
sucrose
*3%
30
MS basal medium
Mishra et al.
(2011)
Flickingeria nodosa
(orchid)
Burgeff's N3F basal

medium + 2% sucrose
PLBs
NA
2% SA + Burgeff's N3F
medium + 2% sucrose
100 mM
30
21.43% (after
3 month of
storage in a mist
environment at
25 2 C)
95%
92.6%
66.6%
(sprouting) and
70% (rooting)
36%
94.3% (after
4 week of
storage)
94.9%
Ahmad and
Anis (2010)
Kumar et al.
(2010)
Nirala et al.
(2010)
Ray and
Bhattacharya
(2010)
Sarmah et al.
(2010)
Singh et al.
(2010)
Singh and
Chand (2010)
43.3%
57% (after
10 weeks of
storage at 4 C)
Sundararaj et
al. (2010)
Tabassum et
al. (2010)
Eclipta alba
(yerba de tago)
Dalbergia sissoo
(sissoo)
MS medium + 2.5 M
Kn + 1.0 M NAA
MS medium (for
sprouting) and MS
medium + 1 mg/l IBA
(for rooting)
Quarter strength B5
macrosalts.
MS medium + 3%
sucrose
% Conversion
References
(complete
plantlet recovery
in
vitro)
Nagananda
et al. (2011)
195
196
Table 1 (continued)
Plant species
(common name)
synseed production
Propagule
size
after treatment
used for
(as required)
encapsulation
Encapsulating
matrix
composition

regrowth
medium for
or CaCl2
synseed
212
300 m
2% SA
*15 g/l
30
NS
35 mm
3% SA + WPM
100 mM
30
ST, NS
35 mm
100 mM
1520
NS
35 mm
Synseed were treated with 3% SA + MS medium

200 mM KNO3 for 5 min
followed by rising with
sterile DDW and 1.0 mg/l
GA for 2 min (self-breaking
treatment)
3% SA + WPM
100 mM
30

Aranda Wan Chark Kuan
TDZ
Blue Vanda coerulea
Grifft. Ex. Lindl. (orchid)
PLBs
4 mm
3% SA + -MS medium 75 mM
Corymbia torellina
MS medium + 3%
C. citriodora (eucalyptus) sucrose + 2.2 M BA
ST, NS
46 mm
Pretreatment with 19.6 M

IBA
3% SA + MS
medium + 2.2 M BA
100 mM
SE
MS medium + 2 mg/l
BA + 0.2 mg/l
NAA + 3 mg/l ABA or 4%
sucrose
MS medium + 0.5 M TDZ NS
34 mm
35 mm
MS medium + 4.52 M
2,4-D
SE
NA
4% SA + MS
medium + 3%
sucrose + 1 mg/l
BA + 0.2 mg/l NAA
5% SA + MS
medium + 0.5 M
TDZ + 2.5 M
IBA + 0.30.55 PPM
2.5% SA + 3% sucrose
In vitro raised seedlings
NS
34 mm
4% SA + MS medium
Rauvola tetraphylla
(be still tree)
Stevia rebaudiana
(stevia)
Rauvola serpentina
(Indian snakeroot
or Sarpagandha)
Clitoria ternatea
(buttery pea)
Cannabis sativa
(marijuana)
Catharanthus roseus
(Madagascar
periwinkle)
Decalepis hamiltoni
i (swallow root)
Liquid MS
medium + 2 mg/l
Kn + 1 mg/l IBA
WPM + 7.5 M
BA + 2.5 M NAA
NA
WPM + 5.0 M
BA + 1.0 M NAA
97%
Rihan et al.
(2011)
90.3%
Alatar and
Faisal (2012)
Ali et al.
(2012)
100%
WPM + 5.0 M
BA + 1.0 M NAA (for
sprouting) and -liquid
MS medium + 0.5 M
IAA
MS medium
80% (after
4 weeks of
storage at 4 C)
Faisal et al.
(2012)
96.2%
Gantait et al.
(2012)
30
MS medium + 3%
sucrose + 2.2 M BA
62100%
100 mM
45
92%
50 mM
30
MS medium
supplemented with
2 mg/l BA + 0.5 mg/l
NAA
MS medium + 0.5 M
TDZ
Hung and
Trueman
(2012a,
2012b)
Kumar and
Thomas
(2012)
100 mM
15
MS medium + 1.34 M
NAA + 1.1 M BA
84.33%
Maqsood
et al. (2012)
100 mM
2025
MS medium + 5 M
BA + 0.5 M
IAA + 30 M ADS
77%
Sharma and
Shahzad
(2012)
20
Lata et al.
60% (after
(2012)
24 weeks of
storage at 15 C)
Abbreviations: ABAabscisic acid, ACactivated charcoal, ADSadenine sulphate, Anderson medium (Anderson, 1975), B5Gamborg medium (Gamborg et al., 1968), BA6-benzyladenine, DCRGupta and Durzan basal medium (Gupta and Durzan, 1985),
DKW mediumDriver and Kuniyuki medium (Driver and Kuniyuki, 1984), 2,4-D2, 4-dichlorophenoxyacetic acid, GA3gibberellic acid, Gluglutamic acid, Heller's mediumHeller (1953), IBAindole-3-butyric acid, 2-iP(2-isopentenyl) adenine, IY
mediumIchihashi and Yamashita's medium (Ichihashi and Yamashita, 1977), Knkinetin, LPm mediumvon Arnold and Eriksson (1981), LSBMLinsmaier and Skoog's basal medium (Linsmaier and Skoog, 1965), MCmicrocutting, MPmicroplantlet,
MSmicroshoot, MS mediumMurashige and Skoog's medium (Murashige and Skoog, 1962), NAA-naphthalene acetic acid, NAinformation not available, Nitsch mediumNitsch (1951), NSnodal segment, OMMolive modied medium
(Mencuccini et al., 1997), P24 medium(Teasdale, 1992), PCprotocorm, PGphloroglucinol, PGRplant growth regulator, PPMPlant Preservative Mixture, PVPpolyvinylpyrrolidone, QLQuoirin and Lepoivre medium (Quoirin and Lepoivre,
1977), R4 mediumChaleff and Stolarz (1981), SAsodium alginate, SBshoot bud, SEsomatic embryo, STshoot tip, TDZthidiazuron, TIBA2,3,5-triidobenzene acid, W mediumWhite's medium (White, 1943) medium, WPMwoody plant medium
(Lloyd and McCown, 1980), Zeazeatin.
MS
Brassica oleracea var

botrytis (cauliower)
Burgeff's N3F
medium + 2 mg/l
AdSO4 + 1 mg/l NAA
Perlite + MS
medium + 2 mg/l Kn
and 2 mg/l NAA
WPM + 7.5 M
BA + 2.5 M NAA
BA
References
% Conversion
(complete
plantlet recovery
in
vitro)
Gelation from outside to inside

Polyelectrolyte solution (e.g. Na-alginate)
+ Somatic embryo
Cross-linking solution (CaCl2)
Ca++
Ca++
Ca++
Ca++
Inotropic gel (Ca-alginate)
Fig. 2. Simple bead (Single layered synseed) formation [redrawn from the report of
Patel et al. (2000) with permission].
material, the requirements of denite ingredients of the hydrogel

matrix (viz., inorganic nutrients, organic nutrients, plant growth regulators (PGRs), carbon sources, etc.) are species-specic. Preparation
of a gel matrix in nutrient medium improves the re-growth of encapsulated plant tissue (Ahmad and Anis, 2010; Chand and Singh, 2004;
Sundararaj et al., 2010). Various PGRs and growth additives were also
added to the gel matrix to further enhance synseed conversion. In
Adhatoda vasica (vasaka), a comparison was made between the additive effect of PGRs in gel matrix and conversion media (Anand and
Bansal, 2002). They reported that a synseed prepared in Gamborg's
medium (B5, Gamborg et al., 1968) supplemented with kinetin (Kn,
4.65 M) and phloroglucinol (PG, 50.0 mg/l) resulted in highest
synseed conversion (66.28%) when inoculated on basal B5 medium.
However, such beads showed reduced conversion frequency when
planted on B5 medium supplemented with a similar combination of
Kn and PG as added to the gel matrix. Similarly, seeds prepared in a
B5 matrix exhibited reduced conversion frequency (54.58%) on B5
medium supplemented with Kn (4.65 M) and PG (50.0 mg/l).
Thus, their study revealed that the presence of nutrients and PGRs
in the gel matrix was more crucial than in the sowing medium for
A. vasica synseed. Tsvetkov et al. (2006) reported that 3% sucrose in
the gel matrix had a more pronounced effect on synseed conversion
than PGRs in hybrid aspen (Populus tremula P. tremuloides).
Synseeds are reported to be highly susceptible to bacterial, fungal
and other microbial infections in the greenhouse (Vij and Kaur,
1994). To reduce microbial contamination, various antimicrobial
agents such as bavistin (Pattnaik et al., 1995), vitrofural G-1 (Nieves
et al., 2003), plant preservative medium (PPM) (Micheli et al.,
2002) can be added to the gel matrix. However, such chemicals
impair convertibility which can be successfully alleviated by adding
PGRs to the gel matrix. When activated charcoal (AC) is incorporated
into synseeds, it also improves the conversion and vigor of the encapsulated propagules of tropical forest trees. AC not only stimulates the
diffusion of gases and nutrients, but also helps in breaking down
alginate which in turn facilitates enhanced respiration of propagules,
thus preventing the loss of vigor that extends the storage period
signicantly (Saiprasad, 2001). Furthermore, AC absorbs unwanted
exudates such as 5-hydroxymethylfurfural (a toxic breakdown
product of sucrose formed during autoclaving) and other harmful
phenolic products (Wang et al., 2007). In addition, AC retains nutrients within the hydrogel capsule and releases them slowly, thus
197
providing an uninterrupted, long-term supply of nutrients to the

growing tissue. Inclusion of 1.25% AC also improved the conversion
frequency of encapsulated somatic embryos in Oryza sativa (rice)
(Arun Kumar et al., 2005). Besides this, natural PGRs like coconut
water and tomato juice have also been successfully used for synseed
conversion. Being inexpensive, natural additives provide a cheaper
substitution for expensive, sometimes synthetic, PGRs (Swamy et
al., 2009).
Another mechanical problem in encapsulation technology is the hindered emergence of the root or shoot of the encapsulated propagule by
the gel capsule, although adopting self-breaking alginate gel bead technology could overcome this problem (Onishi et al., 1994). In this technology, synseeds are pretreated with potassium nitrate (KNO3). This
self-breaking synseed technology was adapted to Feijoa sellowiana
(goiabeira serrana), O. sativa (hybrid rice) and Stevia rebaudiana
(stevia) (Ali et al., 2012; Arun Kumar et al., 2005; Guerra et al., 2001).
O. sativa synseeds with a synthetic endosperm were applied a
self-breaking treatment by dipping them in 200 mM KNO3 solution
for 60 min and rising in sterile tap water for 40 min or more until the
beads became swollen (Arun Kumar et al., 2005) while F. sellowiana
and S. rebaudiana synseeds were dipped in 100 mM and 200 mM
KNO3 solution for 20 and 5 min, respectively (Ali et al., 2012; Guerra
et al., 2001). During pretreatment with KNO3, the K + ions replace the
Ca2+ ions of the calcium alginate capsule thus allowing the synseeds
to soften and open the subsequent conversion to plantlets (Onishi et
al., 1994). In F. sellowiana, Guerra et al. (2001) reported 81.2% opening
of the capsule vs. 0% in the treatment with water while in O. sativa,
Arun Kumar et al. (2005) observed a 47% conversion frequency relative
to normal synseeds (without a self-breaking coat).
Hydrated synseeds are sticky and difcult to handle on a large
scale and dry rapidly in the open air. This problem can be resolved
by coating the beads with Elvax 4260 (ethylene vinyl acetate acrylic
acid terpolymer; Dupont, USA) (Redenbaugh and Walker, 1990).
3.2. Double-layered synseed
To prepare double-layered synseed, a single-layered bead is further
coated with a similar concentration of sodium alginate solution and
dropped into calcium chloride solution for 30 min then washed with
sterile DDW. These double-encapsulated synseeds have all the merits
of a single-layered synseed, with the added advantage of double encapsulation for better protection. Pinker and Abdel-Rahman (2005) tested
the feasibility of forming a second layer in Dendranthema grandiora
(chrysanthemum) synseeds. They observed that if the second layer
was prepared in MS medium, the infection rate was higher, while the
second layer prepared by Ca-alginate dissolved in water or mannitol reduced contamination considerably. Similarly, double-layered synseeds
were prepared for Malus pumila (M.26 apple rootstock) using in
vitro-derived apical buds (Micheli et al., 2002).
3.3. Hollow beads
In conventional single-layered synseeds, propagules are usually
located near the surface of beads which does not ensure their complete protection in contrast to a natural seed where the seed coat provides better protection. Hollow beads seem to be a promising tool in
the realization of true synseeds by providing complete protection to
the propagule. Hollow beads have various advantages over singleand double-layered synseeds, although the process of hollow bead
formation is labor-intensive and costlier (Table 2).
Considering various potentials of hollow beads over single- and
double-layered synseeds, Patel et al. (2000) adopted this approach
for the encapsulation of embryogenic calli of D. carota (carrot), as
well as calli and shoot tips of Solanum tuberosum (potato). In this
technique, propagules were suspended into a solution containing
carboxymethyl cellulose (CMC) and calcium chloride and then
198
Table 2
Advantages of hollow bead encapsulation [modied from Patel et al. (2000)].
Advantages over natural seeds
Advantages over naked somatic embryos
Advantages over encapsulated somatic embryos
Rapid clonal propagation
Easy handling (storage, sowing)
Economical propagation of plants which

cannot be easily propagated via natural
seeds (i.e. hybrids, transgenic plants, trees)
Economic production of virus-free plants
Easy maintenance of genetic resources
Protection from adverse environmental

conditions (temperature, dryness)
Closer to natural seeds because of complete

protection of somatic embryos in a sterile core
Continued development of somatic embryo
within the hollow bead
Protection against pathogens

Longer shelf life
No early emergence
Entrapment and coating realized in one step
dripped into a constantly-stirred sodium alginate solution (Fig. 3). In

initial experiments with carrot, it was found that after 14 days of culture, 100% of hollow beads encapsulated in calcium alginate converted in
the liquid core, 13% of which protruded out by bursting from the capsule.
Embryogenic callus, encapsulated inside hollow beads, induced somatic
embryogenesis while callus encapsulated in conventional calcium alginate beads detached from the beads early in development and no somatic embryogenesis occurred in any of the media. With potato, callus
developed in 50% of hollow beads, whereas 81% of shoot tips encapsulated in hollow beads sprouted and grew out of the capsules. In contrast to
this, in cyclamen (Cyclamen persicum), lower conversion of somatic embryos was reported from hollow beads than from conventional alginate
beads (Winkelmann et al., 2004).
This approach was not found to be suitable also for encapsulation of
moss bud (S. ampullaceum) (Pourjavadi et al., 2006); regeneration was
not satisfactory, for the following reasons: a) the strong net developed
by the highly viscose alginate impeded a steady exchange of nutrients
with the growth medium. That could be avoided by mechanically
breaking the beads with tweezers, as is often done. b) Excessive uptake
of water by beads during cold storage (5 1 C) led to a loss of sphericity, as well as loss of insulation and subsequent damage to the plant material in a relatively short period of time. These occurrences may have
been due to the inclusion of CMC in the bead, which has a high capacity
to absorb water. This may have led to the opening-up of large pores in
the bead matrix wall and to the loss of protection of the plant material.
4. Propagules used for synseed production
A synseed acts as an alternative seed for those species in which zygotic seeds are either not produced or not viable. A variety of
Gelation from inside to outside
Crosslinking solution (e.g. CaCl2)
+ Polymer + somatic embryo
Polymer solution (Na-alginate)
Core (sol or gel) + somatic

embryo
micropropagules have been successfully utilized for synseed production. These encapsulating units can be categorized into two types, as
described next:
4.1. Bipolar propagules
A somatic embryo is regarded as a bipolar structure as it possesses
shoot and root poles at the same time (Standardi and Piccioni, 1998).
Among various propagules, somatic embryos have been found to be
the best suited entity for synseed production, and being bipolar in nature (each with a radicle and plumule), they are able to develop roots
and shoots in a single step. Considering the advantages of somatic embryos over other propagules, these have been successfully exploited for
synseed production in a number of plant species: C. persicum (Jalali et
al., 2012; Winkelmann et al., 2004), Arnebia euchroma (Manjkhola et
al., 2005), Rotula aquatica (Chithra et al., 2005), P. guajava (Rai et al.,
2008a), Spartina alterniora (Utomo et al., 2008), O. sativa (Roy and
Mandal, 2008), Nothofagus alpina (Cartes et al., 2009), Vitis vinifera
(Nirala et al., 2010), Catharanthus roseus (Maqsood et al., 2012). Somatic
embryos have been utilized in both dried and hydrated state with encapsulation for synseed production. The process used for desiccating somatic embryos has already been described in subsection 2.1. The basic
problem in the exploitation of somatic embryos for synseed production
is an asynchronous and late maturation of the embryonic pole
(Castellanos et al., 2004). Maturation has been hampered in many
woody plant species due to precocious conversion, spontaneous repetitive embryogenesis, and embryo dormancy (Teixeira da Silva and
Malabadi, in press). Thus, a well-tuned embryogenic system is required
to improve plantlet conversion from synseeds. To address this need,
various osmoticants have been used to enhance embryo maturation in
cork oak (Garcia-Martin et al., 2001, 2005). Embryo weight increment
is an indicator of embryo quality and a pre-requisite for successful conversion (Garcia-Martin et al., 2001, 2005). Nowadays, mathematical
models have been developed with the aim of monitoring the
large-scale uniform production of microplants (Konan et al., 2006).
Combining these two pre-requisites, a technique was suggested for
monitoring the growth of cork oak somatic embryos inside Petri dishes
with a standard system of image capture and a digital system of image
analysis which simplied the problem of quantifying and comparing
growth in different treatments and culture media (Pintos et al., 2008).
This method permitted growth assessment without the risk of contamination and opened up the possibility of automated control of culture
growth for scaling-up plant production.
The practical use of somatic embryogenesis, in terms of synseeds,
has not evolved accordingly. Since embryogenic ability is often found
only in a few genotypes within a species or is achieved by only using
zygotic embryos or juvenile plant material as initial explants, this
limits its use for cloning selected superior varieties (Standardi and
Piccioni, 1998).
4.2. Unipolar propagules
Ionotropic gel (Ca-alginate)

Fig. 3. Hollow bead formation [redrawn from the report of Patel et al. (2000) with
permission].
Plant propagules with only a shoot or root pole are referred as unipolar propagules. A new perspective in synseed technology was initiated with the use of non-embryogenic unipolar plant propagules
199
2011), Brassica oleracea var botrytis (Rihan et al., 2011), Carrizo citrange
(German et al., 2011), K. senegalensis (Hung and Trueman, 2011), C.
torellianaC. citriodora (Hung and Trueman, 2012a,b) and Decalepis
hamiltonii (Sharma and Shahzad, 2012). Gangopadhyay et al. (2009) developed an encapsulation-based antibiotic selection technique for encapsulated microshoots of pineapple var. Queen. In most reports, a 35 mm
long single nodal segment or shoot tip explant, possessing one or two axillary/apical buds were used for synseed production (Ahmad and Anis,
2010; German et al., 2011; Hung and Trueman, 2012a,b), although
Rihan et al. (2011) excised a 0.2-0.3 mm size microshoot for synseed production in B. oleracea var botrytis (cauliower) while Chand and Singh
(2004) dissected 10-mm long nodal segments for synseed preparation
in D. sissoo.
A highly effective protocol was developed for shoot tip encapsulation in four clones (K61, K522, K584 and K686) of K. senegalensis, a medicinally important and timber-yielding plant (Hung and Trueman,
2011). The capsules were prepared in sodium alginate solution
containing full-strength MS medium with 4.4 M 6-benzyladenine
(BA) and 3% sucrose followed by polymerization in calcium chloride.
Shoot regrowth from capsules was evaluated on agar-solidied and liquid MS medium with or without 4.4 M BA. Shoots emerged from 92 to
100% of capsules after 6 weeks of incubation. Only medium containing
BA stimulated multiple shoots, which were longer than those from
their corresponding agar-solidied or liquid hormone-free media in
clones K522 and K686. Liquid medium with BA provided more shoots
than agar-solidied medium in the presence of BA for clones K522
and K686. Shoots produced on BA-containing culture medium were
sturdy and each excised shoot produced multiple shoots or a plantlet
upon transfer to the same medium or an indole-3-butyric acid
(IBA)-containing medium, respectively. Thus, culture media supplemented with BA was considered to be optimal for subsequent
shoot proliferation from K. senegalensis-encapsulated explants. They
whose potential advantages are easy to understand. It would be possible to exploit the natural ability of the species to produce vegetative
propagules or, when nothing else is available, to use easy-to-obtain
somatic fragments of the plant directly, to reduce the difculties
and generalize the application of synseed technology to as many genotypes and species as possible. Also, the risks of somaclonal variation would be reducedrelative to somatic embryosby using
unipolar propagules (Standardi and Piccioni, 1998). The scientic literature reports several examples of unipolar propagules used as
synseed. Standardi and Piccioni (1998) divided unipolar propagules
into three groups according to the type of propagule:
4.2.1. Nodes with apical or axillary buds and microshoots
These types of propagules, also referred to as microcuttings, are one of
the most frequently considered for synseed production, probably because
of the relative ease with which these explants are produced once the
micropropagation system has been established (Piccioni and Standardi,
1995). Shoot tips and nodal segments have been successfully exploited
for synseed development in a number of plant species as they assure
a high degree of genetic stability without encountering somaclonal variations (Piccioni, 1997). Non-embryogenic plant propagules have been
widely used in synseed technology for a range of plant species: Morus
spp. (Pattnaik and Chand, 2000; Pattnaik et al., 1995), Eucalyptus grandis
(Watt et al., 2000), A. vasica (Anand and Bansal, 2002),
Dendranthemagrandiora (Teixeira da Silva, 2003), Dalbergia sissoo
(Chand and Singh, 2004), Ananas comosus (Gangopadhyay et al., 2005),
C. grandiora (Nishitha et al., 2006), Punica granatum (Naik and Chand,
2006), Gerbera jamesonii (Taha et al., 2009), Cineraria maritima
(Srivastava et al., 2009), Musa sp. (Sandoval-Yugar et al., 2009), Cannabis
sativa (Lata et al., 2009), S. mauritiana (Sharma et al., 2009a; Fig. 4),
S. acmella (Sharma et al., 2009b), Z. ofcinale (Sundararaj et al., 2010),
Vitex negundo (Ahmad and Anis, 2010), Picrorhiza kurrooa (Mishra et al.,
E
A
D
Fig. 4. Different stages of plantlet recovery from encapsulated nodal segments of Spilanthes mauritiana. (A) Encapsulated nodal segments in 3% sodium alginate and 100 mM calcium chloride. (B) Sprouted synseeds after 2 weeks on MS medium supplemented with BA (1.0 M) and IAA (0.5 M). (C) Complete plantlets (shoot and root development) after
4 weeks of culture. (D) An acclimatized plantlet. (E) A twig showing owering.
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performed a similar synseed experiment with shoot tips and nodal segments of four highly proliferating clones (A80, C46, C48 and C79) of
C. torelliana C. citriodora, a eucalypt (Hung and Trueman, 2012a).
They found higher growth frequencies (78100% and 76100% for
encapsulated shoot tips and nodal segments, respectively) and length
of regrowing shoots (2.75.7 and 2.56.0 nodes from encapsulated
shoot tips and nodal segments, respectively) on PGR-free half- and
full-strength MS media. However, mean number of shoots per capsule
was higher (1.44.4 shoots/capsule) on full-strength MS media
supplemented with 2.2 M BA with or without 0.3 M -naphthalene
acetic acid (NAA) relative to PGR-free re-growth media. Thus, the optimal medium for K. senegalensis and Corymbia shoot re-growth
depended upon the purpose of the capsules. BA-free sowing media
would be preferable when a germplasm collection of a few capsules
from many clones was transferred between laboratories and where it
would be important to maximize the probability of at least one shoot
emerging from each clone. Sowing medium containing BA may be preferable when many capsules of only a few clones are transferred, although rapid shoot proliferation is required from each clone. In
contrast to this, maximum shoot development (25 shoots/bud) from
encapsulated axillary buds of Morus species (mulberry) was possible
on MS medium containing 4.4 M BA (Pattnaik and Chand, 2000).
Among six species of Morus (M. alba, M. australis, M. cathyana,
M. nigra and M. bombycis), one step regeneration i.e., both shoot and
root formation was recorded in M. alba, M. bombycis and M. latifolia although the shoots that were recovered from encapsulated buds of
M. australis, M. cathyana and M. nigra failed to root on re-growth
media and needed an additional in vitro root induction step.
Naik and Chand (2006) reported encapsulation of nodal segments
of P. granatum (pomegranate) for germplasm distribution and
exchange. Two types of explant source i.e., source A and source B
were used for collection of nodal segments followed by hydrogel encapsulation. Source A was established using mature nodal segments
while source B was derived from cotyledonary nodes. In both cases,
encapsulated nodal segments sprouted best on MS medium supplemented with 100 mg/l myo-inositol, 4.44 M BA and 0.54 M NAA.
A maximum of 68% (source A) and 81% (source B) sprouting was noticed with this combination of PGRs and 23 shoots were recovered
from each encapsulated nodal segment. However, a one-step conversion, i.e. simultaneous shoot and root formation, was possible only
with encapsulated nodal segments of source B. About 21% of the encapsulated nodal segments cultured on PGR-free full-strength MS
medium supplemented with 100 mg/l myo-inositol showed conversion within 40 days which was the maximum among planting
media tested while no rooting was noticed with source A on any of
the planting media tested. Thus, similar to Morus species, a separate
experiment was needed for efcient root induction of capsulederived P. granatum microshoots (Naik and Chand, 2006).
Although nodal segments are the most suitable (among various unipolar propagules) for encapsulation studies as they possess a preexisting axillary meristem (mostly two axillary buds/node relative to
single apical buds in shoot tip explants), three sets of problems can be
highlighted. The rst is related to the nature of the organ used, i.e., a
vegetative plant segment, devoid of any storage tissues and strongly
adapted to in vitro culture conditions. The second is related to the lack
of a root apex and the inability of explants to spontaneously form
roots. The third is the overall additional cost of the micropropagation
system. Among all, in vitro root induction is the major obstacle, especially encountered for recalcitrant woody plant species, although this can
now be more easily overcome with CO2-enrichment in aerated vessels
(see, for example, Teixeira da Silva et al., 2006). It is assumed that encapsulation inhibits oxygen supply to the propagule and suppresses
root induction (Piccioni, 1997). However, in some species, such as
C. grandiora, Coleus forskohlii, C. maritima, Eclipta alba and P. kurrooa,
encapsulated nodal segments with apical or axillary buds demonstrated
a high adventitious rooting capacity after sowing on PGR-free nutrient
media (Mishra et al., 2011; Nishitha et al., 2006; Ray and Bhattacharya,
2010; Srivastava et al., 2009; Swaroopa et al., 2007). On the other hand,
in many other species, an appropriate root induction protocol was
suggested to be integrated with the encapsulation protocols. In the literature, the following methods listed next have been tested for root induction of synseed-derived propagules with varying degrees of success.
4.2.1.1. Pretreatment of explants prior to encapsulation. Piccioni (1997)
suggested a method of incubating explants in the dark for inducing
root primordia followed by the addition of PGRs in the gel matrix
for higher conversion from encapsulated beads. Pretreatment of
explants with cytokinin(s) and auxin(s) has also been used to enhance the conversion frequency of synseeds i.e., complete plantlet development (with a healthy shoot and root system) (Chand and Singh,
2004; German et al., 2011; Pattnaik et al., 1995; Soneji et al., 2002)
(Table 1). Recently, Hung and Trueman (2012a) reported that the
conversion of C. torelliana C. citriodora (four clones viz., A80, C46,
C48 and C79) synseed depended almost entirely on IBA pretreatment,
particularly for nodal segments which rarely (04%) produced roots
without IBA. Pretreatment with 19.6 M IBA consistently resulted in
optimal conversion (62100%) for all four Corymbia clones and two
explant types (shoot tips and nodal segments). The use of 4.9 M
IBA afforded a high conversion from encapsulated shoot tips but in
three of the four clones, this concentration resulted in a lower conversion (4456%) from encapsulated nodes than when 19.6 M IBA was
used. Similarly, pretreatment with 245 M IBA consistently provided
one of the highest conversion percentages for each K. senegalensis
clone (K61, K522, K584 and K686) (52100%) (Hung and Trueman,
2011). This auxin level was much higher than 10.8 M NAA plus
39.4 M IBA (79.17% conversion) and 4.90 M (85% conversion)
used for A. comosus (Soneji et al., 2002) and D. sissoo (Chand and
Singh, 2004), respectively. This was very close to the optimal concentration (260 M IBA) used for rooting K. senegalensis microshoots
(Danthu et al., 2003). The concentrations (4.939.4 M IBA) used
for A. comosus, D. sissoo and M. pumila synseeds resulted in similar
conversion frequencies (58100%) (Chand and Singh, 2004; Piccioni,
1997; Soneji et al., 2002).
4.2.1.2. Incorporation of PGRs to the gel matrix. The addition of PGRs to
the gel matrix improves the efciency of synthetic endosperm around
the vegetative propagules and thus provides a simpler method for the
successful recovery of complete plantlets. Pinker and Abdel-Rahman
(2005) emphasized that the addition of 5.71 M indole-3-acetic acid
(IAA) to the gel matrix (prepared in modied MS) resulted in 100%
root formation from encapsulated nodal segments of C. grandiora.
Nishitha et al. (2006) suggested the addition of 11.7 M silver nitrate
(AgNO3) along with 0.49 M IBA to enhance the conversion frequency in synseeds of C. grandiora.
4.2.1.3. Incorporation of PGRs to the regrowth media. Supplementation
of PGRs to the germination medium has been found to eliminate the
requirement of an additional in vitro root induction step prior to acclimatization. Ahmad and Anis (2010) found that the addition of 2.5 M
kinetin (Kn) and 1.0 M NAA to MS basal medium induced a mean of
2.8 roots/shoot from encapsulated nodal segments of V. negundo.
Sharma et al. (2009b) reported a maximum of 87.8% conversion frequency on MS medium supplemented with 1.0 M BA and 0.5 M
NAA for S. acmella synseeds. For Tylophora indica synseeds, optimum
conversion (91%) was achieved on MS medium supplemented with
2.5 M BA and 0.5 M NAA (Faisal and Anis, 2007). In Ocimum
basilicum, maximum conversion frequency (80%) was obtained on
half-strength MS medium supplemented with 5.0 M BA and 0.5 M
IAA (Siddique and Anis, 2009). However, Alatar and Faisal (2012)
reported 90.3% complete plantlet recovery when Rauvola tetraphylla
synseeds were placed on woody plant medium (WPM, Lloyd and
McCown, 1980) augmented with 7.5 M BA and 2.5 M NAA.
4.2.1.4. Re-culturing of capsule derived microshoots on rooting media.

Gangopadhyay et al. (2005) devised a two-step method to achieve
maximum bead conversion in A. comosus (pineapple). In the rst
step, shoots were retrieved from encapsulated beads and in the
second step, these microshoots were rooted in liquid medium
(supplemented with 0.01 mM IBA and 0.002 mM Kn) supported
with Luffa-sponge. In contrast, Pattnaik and Chand (2000) used
half-strength MS medium supplemented with different concentrations of IAA, IBA and indole-3-propionic acid (IPA) for root initiation
in capsule-derived microshoots of Morus species (mulberry). In M.
australis and M. cathyana, optimum rooting was achieved on
half-strength MS medium containing 5.7 M IAA, 4.9 M IBA and
5.3 M IPA while that of M. nigra required only 4.9 M IBA. In contrast, shoots of M. alba, M. bombycis, M. latifolia and M. nigra rooted
best on half-strength MS medium augmented with 4.9 M IBA. In
P. granatum, rooting was possible by re-culturing the capsulederived microshoots on half-strength MS medium supplemented
with 50 mg/l myo-inositol, 1.5% sucrose and NAA (0.0545.37 M)
or IBA (0.0494.9 M). Among various treatments, maximum rooting
efciency (8790%) was obtained with 0.54 M NAA (Naik and
Chand, 2006). In contrast, Singh (2008) reported successful rooting
in capsule-derived microshoots of Rhododendron maddeni using liquid
Anderson medium (Anderson, 1978) supplemented with 1.0% AC and
0.20 mg/l IBA. In contrast, Swamy et al. (2009) reported rooting on
PGR-free half-strength MS basal medium for microshoots retrieved
from encapsulated beads of P. cablin. Srivastava et al. (2009) reported
healthy root formation in capsule-derived microshoots of C. maritima
following a 2-week transfer to half-strength MS medium containing
1.0 mg/l NAA.
4.2.2. Microbulbs, microtubers, rhizomes and corms
These explants are generally naturally ready to convert, since they
are natural propagules that have been differentiated by evolution for
vegetative multiplication of individuals and often also include storage
tissues. Encapsulation of microbulbs, rhizomes and protocorms has
been reported for a number of plant species (Datta et al., 1999;
Martin, 2003; Saiprasad and Polisetty, 2003; Standardi and Piccioni,
1998). In Ipsea malabarica (Malabar daffodil orchid), encapsulated
bulbs cultured either on PGR-free half-strength MS or 6.97 M
Kn-supplemented medium facilitated 100% conversion (Martin,
2003). Although conversion was independent from culture medium,
the number of shoots developed varied. A maximum of 7.2 shoots/
capsule formed on half-strength MS medium supplemented with
6.97 M Kn while 4.6 shoots/capsule were induced on PGR-free
half-strength MS medium. Moreover, the growth of shoots on
Kn-enriched medium was faster than that on medium devoid of Kn.
Bekheet (2006) established a synseed protocol for the conservation
of garlic by encapsulating in vitro-proliferated bulblets of garlic. Maximum shoot recovery (6.30), shoot length (6.00 cm) as well as shoot
fresh mass (3.80 g) were obtained on MS medium containing 2 mg/l
BA and 2 mg/l NAA.
Synseed production in orchids is of special signicance in view of
the tiny and non-endospermic seeds they produce. Corrie and Tandon
(1993) used protocorms for synseed production in Cymbidium
giganteum and induced healthy plantlets upon transfer either to
nutrient medium or directly to sterile sand and soil. The conversion
frequency was comparable under both in vitro (100%) and in vivo
(88% in sand, 64% in sand and soil mixture) conditions. These observations have made it feasible to transplant aseptically-grown
protocorms directly to soil, and to cut the cost of raising in vitro plantlets and their subsequent acclimatization. Encapsulation of PLBs is
well documented in many orchids such as C. giganteum, Dendrobium
wardianum, Dendrobium densiorum, Phaius tonkervillae, Oncidium,
Cattleya and Spathoglottis plicata (Ara et al., 2000; Saiprasad and
Polisetty, 2003; Vij et al., 2001). Saiprasad and Polisetty (2003)
optimized the most suitable stage of PLBs for encapsulation in three
201
orchid genera (Dendrobium, Oncidium and Cattleya). They found that

fractionated PLBs, 1315 days after culture, at the leaf primordia stage
were most suitable for encapsulation. An encapsulation matrix prepared with MS medium (-strength) supplemented with 0.44 M BA
and 0.54 M NAA gave 100% conversion of encapsulated PLBs when
cultured on MS medium supplemented with 0.44 M BA and 0.54 M
NAA (Dendrobium) or MS medium supplemented with 2.69 M NAA
(Oncidium) or MS medium supplemented with 5.38 M NAA (Cattleya).
Sarmah et al. (2010) produced synseeds in Vanda coerulea, an endangered monopodial orchid by encapsulating PLBs regenerated from the
leaf base. A maximum of 94.9% conversion frequency was noticed
when encapsulated PLBs were inoculated immediately on IY medium
(Ichihashi and Yamashita, 1977). Nagananda et al. (2011) encapsulated
the PLBs of Flickingeria nodosa and achieved 95% conversion on Burgeff's
medium (Withner, 1955) supplemented with 2% sucrose, 2 mg/l adenine sulphate (AdSO4) and 1 mg/l IAA, after 3 months' storage at 4 C.
Recently, Gantait et al. (2012) reported alginate-encapsulation of
Aranda Vanda PLBs. Individual PLBs (4 mm long) were best encapsulated with 3% sodium alginate and 75 mM calcium chloride. The highest
percentage conversion (96.4%) was obtained on PGR-free half
strength-MS medium.
4.2.3. Meristemoids, cell aggregates and primordia
These propagules are more complex to examine since they are not
homogenous groups like microcuttings. In fact, differentiating propagules might eventually develop into one of the previous groups, i.e.,
evolve into buds or shoots (Yoshida, 1996), or form a natural propagule, such as a corm (Sharma et al., 1992; Tandon et al., 1994). The
characteristic feature of the reports concerning this type of propagule
is that the explants are encapsulated before the end of the differentiation phase, for example encapsulation of proliferating callus. Proliferating embryogenic callus clumps are also known to regenerate
easily and show great potential for genetic transformation and can
therefore be used for synseed production with promising results.
Kim and Park (2002) reported a single-layer encapsulation of
dehydrated embryogenic garlic callus using alginate gel. Encapsulated
callus with a water loss of less than 50% showed 93% conversion
frequency on half-strength MS medium. The microshoots that regenerated from encapsulated calli were much longer than those recovered from naked calli. Patel et al. (2000) successfully encapsulated
the embryogenic callus of D. carota and S. tuberosum using a hollow
bead approach.
5. Possible modes of synseed utilization
Synseed technology has unraveled new vistas in the eld of plant
biotechnology. Synseeds can be employed in different ways for the
management of plant germplasm and some modes of their utilization
are described next.
5.1. In vitro plant production
Encapsulation provides a convenient and reliable means of
producing plants in vitro. Synseeds can be efciently planted in vitro either on semi-solid culture medium or planting substrate (perlite,
vermiculite, vermicompost, soilrite, soil, sand, gravel) for conversion
into complete plantlets (Mandal et al., 2000; Pinker and AbdelRahman, 2005). Generally, nutrient-rich media are more effective
than nutrient-decient substrates for successful recovery of plantlets
(Mandal et al., 2000), but in Dendranthema grandiora, synseeds
germinated more efciently on perlite moistened with MS medium
than on solidied nutrient-rich medium (Pinker and Abdel-Rahman,
2005). The PGR requirement in nutrient medium varies from plant to
plant. For instance, encapsulated axillary buds of Ocimum spp. exhibited
maximum conversion (9599%) on MS basal medium supplemented
with 1.1 M BA (O. americanum), 2.2 M BA (O. gratissimum) or
202
4.4 M BA (O. basilicum and O. sanctum) (Mandal et al., 2000). However,

for encapsulated embryos of O. sativa, MS medium supplemented with
1 mg/l BA, 1 mg/l Kn and 0.5 mg/l NAA was critical for synseed conversion (maximum = 87.5%) (Roy and Mandal, 2008). In contrast, encapsulated nodal segments of D. sissoo (Chand and Singh, 2004) and
Z. ofcinale (Sundararaj et al., 2010) showed maximum conversion
response on PGR-free half- and full-strength MS media, while
Kavyashree et al. (2006) reported multiple shoot formation from
synseeds of mulberry var. S54 on Linsmaier and Skoog (LS, Linsmaier
and Skoog, 1965) medium augmented with 8.88 M BA and 2 M 2, 3,
5-tri iodo benzoic acid (TIBA). Gelling agent is also an important factor
for in vitro synseed conversion. Generally, agar is used as a gelling agent
for synseed conversion medium (Cameron, 2008). In contrast, Singh
(2008) reported better conversion of synseed on phytagel compared
to agar.
5.2. Direct sowing

Sowing synseeds ex vitro provides a commercially important and
cost-effective technique for direct recovery of plantlets. This technology will greatly impact the large-scale production of commercially
important plant species at a reduced cost. Although a large number
of plants can be produced in tissue culture through embryogenesis
and/or direct or indirect organogenesis, in some cases, delivery is
cumbersome. Direct sowing of synseeds to soil or other substrates
helps to avoid the acclimatization procedure required for tissue
culture-raised plantlets. Synseeds are also an ideal delivery system
enabling ease and exibility of handling and transport of propagules
compared to voluminous parcels of seedlings or tissue-cultured
plantlets. Conversion of synseeds on nutrient-free substrate is a prerequisite for sowing under non-sterile conditions. Mandal et al.
(2000) suggested that the successful conversion of synseeds into
plantlets on a simple planting substrate such as sand, soil, soilrite or
vermi-compost is necessary for their use in commercial-scale
propagation. Despite this, successful conversion of encapsulated
tissues on various planting substrates has been reported only for a
few plant species either in a controlled culture room environment or
greenhouse conditions (Chand and Singh, 2004; Faisal and Anis, 2007;
Kavyashree et al., 2006; Lata et al., 2009; Sharma et al., 2009b; Singh
et al., 2006). Moderate conversion (40%) of encapsulated somatic embryos was found on sterile sand (Roy and Mandal, 2008). The major limiting factor reducing conversion is the low-nutrient availability.
Therefore, it is necessary to build up a nutrient reservoir for the encapsulated plant tissue, either endogenously or exogenously. Singh et al.
(2006), Faisal and Anis (2007) and Sharma et al. (2009b) supplied
half- or quarter-strength MS nutrients to soilrite for effective in vivo
conversion of synseeds and reported 62, 43 and 63% conversion in
Phyllanthus amarus, T. indica and S. acmella, respectively. Similarly,
Chand and Singh (2004) reported 45% conversion on peat moss moistened with half-strength MS medium, after 30 days of ex vitro sowing of
Dalbergia sissoo synseeds. Kavyashree et al. (2006) exogenously supplied half-strength LS nutrients in horticultural grade soilrite mix
(peat: perlite: vermiculite, 1:1:1 (v/v)) for ex vitro conversion (45.5%)
of mulberry synseeds with healthy shoots and roots. After sowing for
30 days, they reported a maximum of 11.6 shoots/synseed with 7.7
roots/synseed on this planting substrate. The feasibility of encapsulated
cauliower microshoots in commercial substrates (compost, vermiculite, perlite and sand) irrigated with different solution mixtures
including sterilized DDW, MS medium with or without different concentrations of Kn and NAA was evaluated by Rihan et al. (2011). The
use of MS medium supplemented with 2 mg/l Kn and 2 mg/l NAA
gave an optimal response with both perlite and compost. This study
showed the high growth capacity of cauliower synseed in commercial
substrate which is considered to be a promising step for their direct use
in vivo.
During direct sowing, contamination by microorganisms is one of the

major hurdles for the commercialization of encapsulation technology.
Nutrients, especially organic, released by the beads are responsible for
severe contamination (Abdel-Rahman, 2003; Nhut et al., 2005). The addition of fungicide (carbendazim or bavistin and benomyl, 50100 mg/l)
to the alginate matrix has been suggested for M. indica (Bapat and Rao,
1993) to counter this problem to a certain extent. German et al.
(2007) added another fungicide, 100 mg/l thiophanate-methyl to the
gel matrix of Citrus reticulata synseeds and reported 96.7 and 100% viability on lter paper and perlite, respectively. However, Lata et al.
(2009) used PPM in both the gel matrix and planting substrate used
for C. sativa. They reported 100% conversion on potting mixture of
fertilome with coco natural growth medium (1:1), moistened with
full-strength MS medium supplemented with 3% sucrose and 0.5% PPM.
Occasionally, fungicides can reduce the viability of encapsulated
propagules, thus to restrict contamination without affecting an
explant's viability while the use of double-layered synseeds was
suggested by Sharma et al. (1994) and Pinker and Abdel-Rahman
(2005) for Z. ofcinale and Dendranthema grandiora, respectively.
When the second layer was formed by Ca-alginate with water or
0.2 M mannitol, contamination was reduced considerably and only
6.7% of the beads were contaminated (Pinker and Abdel-Rahman,
2005). These ndings were similar to those of Kinoshita and Saito
(1992), who also used two-layered beads (with the second layer
containing water) for encapsulation of axillary buds from Betula
papyrifera (white birch) cultures but observed a low level of rooting.
Leakage of sucrose from beads to the substrate stimulated the proliferation of microorganisms and inhibited rooting. Rinsing away the
sucrose by spraying distilled water onto the perlite enhanced rooting
to 78% (Kinoshita and Saito, 1992). However, Nhut et al. (2005) developed a much easier method in which Cymbidium synseeds were
coated with a solution of a fungicide, chitosan.
Preece and West (2006) studied suitable conditions for ex vitro
synseed conversion in Hibiscus moscheutos cultivars, Lord Baltimore
and Southern Belle. After pre-treating with light providing a photon
ux of approximately 40 mol m 2 s 1 for a 16-h photoperiod at
25 C for 5 weeks, they planted synseeds in vermiculite and provided
intermittent mist during the course of incubation, resulting in 100%
germination. Rooting was best in vermiculite if the encapsulated
nodal segments were planted 1 cm deep and without any covering
as covering might block the penetration of light.
Ex vitro conversion of synseeds has been accomplished in sterile
(autoclaved) potting media (Chand and Singh, 2004; Faisal and Anis,
2007; Kavyashree et al., 2006; Lata et al., 2009; Rihan et al., 2011;
Sharma et al., 2009b; Singh et al., 2006). Ex vitro conversion avoids the
use of an in vitro culture passage, but practical application of this method
is restricted by the high cost of sterilizing potting media. Both problems
could be circumvented by pre-converting the synseeds in moistened
Petri dishes prior to transferring to non-sterile potting media as reported
in K. senegalensis (Hung and Trueman, 2011) and Corymbia spp. (Hung
and Trueman, 2012a). For pre-conversion of synseeds, shoot tips
and nodal segments were pre-treated with IBA (for induction of
root primordia), and maintained under light (16-h photoperiod at
100 mol m 2 s 1) in moistened Petri dishes for 4 weeks at 25 C.
Explants were pre-treated by culturing them on semi-solidied MS
medium supplemented with IBA (245 M for K. senegalensis and
19.6 M for Corymbia spp.) and 2% sucrose for 24 h in darkness. For
both plant species, organic compost was found to be most suitable
for direct conversion of synseeds under non-aseptic conditions. For
K. senegalensis, 4286% conversion was noticed for encapsulated shoot
tips while in Corymbia spp. 4690% conversion from shoot tips and 32
50% from nodal segments was recorded 4 weeks after sowing onto organic compost. Pre-conversion may enhance plantlet development in
non-sterile soils because of two factors. Firstly, microbial contamination
may be minimized due to a reduction in nutrient content of the synseed
matrix during pre-conversion and secondly, the shoots may be pre-
203
adapted to ex vitro conditions because of pre-acclimatization during the

pre-conversion period. Pre-conversion provides a simple and effective
technique for synseed distribution and direct transfer to nursery
conditions.
availability can be a limiting factor for the conversion ability of

Actinidia deliciosa (kiwi-fruit) synseeds.
5.3. Short-term germplasm conservation
Biochemical and physiological mechanisms of low temperature tolerance in higher plants have been intensively studied, leading to a comprehension of the mechanisms required for plant adaptation to low
temperatures. The attempt to conserve the plant propagules under
ultra low temperature for a long duration is termed cryopreservation
(Walter et al., 2006). Cryopreservation of biological tissues is useful
only if intracellular ice crystal formation is avoided as it causes irreversible damage to cell membranes, thus destroying their semi-permeability.
To date, various strategies have been employed for cryopreservation of
different plant tissues i.e., the two-step freezing, simple dessication,
encapsulation-dehydration, vitrication and encapsulation-vitrication.
The encapsulation-dehydration strategy originally developed by
Fabre and Dereuddre (1990) for Solanum shoot tips is more convenient
than others. It also avoids the use of a costly programmable freezer and
high concentrations of harmful cryoprotectants (Reinhoud et al., 2000).
This strategy is based on a successive osmotic and evaporative dehydration of plant cells which allows gradual extraction of water from encapsulated propagules in sucrose-rich medium. Sucrose concentration in
the beads is gradually increased by additional air-drying or desiccation
in a laminar air ow cabinet to reach the saturation point which results
in a glass transition during cooling to 196 C in LN, thus preventing
ice crystal formation (a cause of lethal damage to living cells) during exposure to ultra low temperature (Engelmann and Takagi, 2000).
Encapsulation-dehydration has been used for cryopreservation of
shoot tips in different genera of hardwood species including Malus,
Pyrus and Prunus. It is noteworthy that for 50% of the species
cryopreserved through this procedure, a survival rate of 80% or more
was achieved (Lambardi and De Carlo, 2003).
Encapsulation-dehydration and vitrication are simple and inexpensive techniques and maintain genetic stability while encapsulationvitrication is a hybrid of these two techniques that minimizes any
potential injury from vitrication (Moges et al., 2004) and offers various
advantages over encapsulation-dehydration in terms of greater recovery (Wang et al., 2004). Embryogenic callus of Dioscorea bulbifera (Yin
and Hong, 2010) and PLBs of Dendrobium candidum (Yin and Hong,
2009) were successfully cryopreserved using encapsulation-vitrication.
Synseed technology also acts as a tool for germplasm exchange between countries. For this purpose, synseed storage is a critical factor
that determines their successful conversion after transportation
abroad. Therefore, appropriate storage conditions and a denite
storage period are prerequisites to maintain synseed viability during
transportation that leads to successful commercialization of synseed
technology. Generally, 4 C has been found to be most suitable for
synseed storage (Faisal and Anis, 2007; Ikhlaq et al., 2010;
Kavyashree et al., 2006; Pintos et al., 2008; Saiprasad and Polisetty,
2003; Sharma et al., 2009a,b; Singh et al., 2007; Tabassum et al.,
2010). Gangopadhyay et al. (2005) stored encapsulated microshoots
of A. comosus in different racks of a refrigerator over a range of temperatures (4, 8, 12 and 16 C) for 60 days. Beads stored at 8 C
showed maximum conversion frequency on MS medium supplemented with 0.022 mM BA and 0.003 mM IAA. Few investigations revealed the requirement of higher temperature (25 C) rather than
low temperature for amenable storage of synseeds in certain tropical
and sub-tropical crops. Sundararaj et al. (2010) observed 100%
re-growth of Z. ofcinale synseeds incubated at 25 C while no
re-growth was observed for synseeds stored at 4 C in the dark. Similarly, encapsulated microshoots of C. maritima (Srivastava et al.,
2009) and P. kurrooa (Mishra et al., 2011) were successfully stored
up to 6 and 3 months, respectively at 25 2 C in moist conditions.
A few previous studies on preservation of encapsulated explants of
eucalypt and mahogany have identied storage medium, irradiance or
storage temperature as important factors in maintaining their regenerative potential. Encapsulated axillary buds of E. grandis were stored successfully in water for 6 months at 10 C and 4 mol m-2 s1 irradiance
but could only be stored for 2 months at 28 C and 200 mol m 2 s 1
irradiance (Watt et al., 2000). Encapsulated shoot tips and cotyledonary
nodes of Cedrela ssilis were stored for 36 months in nutrient and
sugar-free media at 25 C and 2025 mol m -2 s -1 irradiance, with
higher regrowth capacity of encapsulated shoot tips on 0.4%
agar-solidied medium than on 1% agar-solidied medium (Nunes et
al., 2003). For Cedrela odorata, 020, 040 and 30-80% of encapsulated
shoot tips survived 12-month storage on 1% agar-solidied media at
20, 25 C (both at 35 mol m 2 s1 irradiance) or 12 C (in darkness),
respectively (Maruyama et al., 1997). Eucalypt and mahogany species
occur naturally in the tropics and subtropics and the temperatures
used for storage of their encapsulated explants parallel temperatures
typically used (1025 C) for maintaining in vitro cultures of tropical
and subtropical species (Trueman, 2006; Withers, 1991). Encapsulated
shoot tips of K. senegalensis survived longer at 25 C than at 4 C, with
7388% viability after 8 weeks (Hung and Trueman, 2011). They
noticed enhanced regrowth frequency of encapsulated K. senegalensis
explants up to 12 months when the storage conditions were 14 C
and darkness. Under these conditions, encapsulated Corymbia and
Khaya explants were preserved most effectively on half-strength MS
medium supplemented with 1% sucrose which provided very high frequencies of shoot regrowth (92100% for Corymbia and 7198% for
Khaya) and excellent shoot development after 12 months' storage
(Hung and Trueman, 2012b).
Ray and Bhattacharya (2010) optimized the best storage environment for E. alba synseed by changing in vitro physicochemical conditions. They extended the duration of storage up to 12 weeks by
decreasing sucrose concentration in an alginate matrix from 3 to 1
or 2%. Adriani et al. (2000) also analyzed the pronounced effect of
sucrose on re-growth ability of synseed and suggested that sucrose
5.4. Cryopreservation: an effective approach for long-term germplasm

storage
6. DNA marker technology in synseed experimentation

Although synseeds have been widely utilized for micropropagation
and conservation of various medicinal plant species (Anand and
Bansal, 2002; Ara et al., 2000; Ikhlaq et al., 2010; Lata et al., 2009; Nor
Asmah et al., 2011; Nyende et al., 2003; Ray and Bhattacharya, 2010;
Singh et al., 2006), the genetic stability of synseed-derived plantlets
has been less evaluated. In contrast, the assessment of genetic variability of cryopreserved materials via encapsulation/dehydration or vitrication methods has attracted more attention (Bekheet et al., 2007;
Hirai and Sakai, 2000; Scocchi et al., 2004). The increasing utilization
of synseed for germplasm conservation and propagation necessitates
the assessment of genetic stability of conserved propagules following
their conservation (Dehmer, 2005). In recent years, systematic sampling of germplasm and analysis of their molecular status through the
use of DNA marker technology has become common practice. Among
different DNA polymorphism detection techniques, random amplied
polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR)
analysis has widely been used to study clonal integrity, detect genetic
and somaclonal variations (Agnihotri et al., 2009; Borner, 2006; Jokipii
et al., 2004; Mandal et al., 2007) because of their simple genotyping,
easy handling, cost effectiveness, and wide availability. Bekheet
(2006), Srivastava et al. (2009) and Mishra et al. (2011) used RAPD
204
analysis to ascertain the genetic delity of synseed/capsule-derived

plantlets of Allium sativum (garlic), C. maritima and P. kurrooa, respectively. Srivastava et al. (2009) used 20 RAPD markers for this purpose.
Of 20 primers tested, 14 produced amplication products, and a total
of 69 bands with an average of 4.93 bands/primer were observed. Of
these 69 scorable bands, only 20% of bands were polymorphic. Cluster
analysis of the RAPD proles revealed an average similarity coefcient
of 0.944 thus conrming the molecular stability of C. maritima plants
derived from encapsulated microshoots following 6 months of storage.
Mishra et al. (2011) used 45 RAPD markers to ascertain the genetic delity of P. kurrooa plants growing after storage in encapsulated form.
Of 45 primers tested, 14 produced scorable amplied products. A total
of 68 bands were observed, 7.35% of which were polymorphic. They
reaveled an average coefcient of 0.966 thus conrming genetic stability of plants derived from encapsulated microshoots following
3 months of storage.
However, Gangopadhyay et al. (2005), Alatar and Faisal (2012)
and Faisal et al. (2012) used RAPD and ISSR to assess the genetic delity of A. comosus (pineapple), R. tetraphylla and Rauvola serpentina
plantlets, respectively growing after storage in encapsulated form.
Gangopadhyay et al. (2005) used 10 RAPD and 3 ISSR primers and observed identical proles in all the samples of A. comosus. Alatar and
Faisal (2012) used 20 RAPD and 5 ISSR primers for R. tetraphylla.
Out of 20 primers, 17 yielded clear and reproducible bands. The number of bands varied from 1 to 13. All the ISSR primers tested gave clear
and scorable bands. The number of bands for each primer ranged
from 11 to 15.
In contrast to these studies, Tabassum et al. (2010) employed amplied fragment length polymorphism (AFLP) analysis to detect the
level of variations among synseed derived regenerants of Cucumis
sativus (cucumber). For this study, they used ve EcoRI + MSeI primer
combinations. Using ve primer combinations, a total of 109 bands
were scored (an average of 21.8 primers or 35.77%). The number of
bands scored with each primer ranged between 13 and 28. The polymorphism ranged between 7.14% with primer E-AA + M-CTC to
15.38% with primer E-AG + M-CTC. The percentage polymorphism
obtained was almost negligible, depiciting genetic integrity among
regenerants.
non-sterile conditions, so as to make this technique feasible and practical for farmers and producers, is still a limitation (Jung et al., 2004).
Several factors are involved in this process such as poor survival,
which might be attributed to the lack of nutrients and oxygen supply,
but primarily the protection of synseed beads from attack by microorganisms (Bapat and Rao, 1993; Nhut et al., 2005). Further renement
of existing protocols and correct formulation of the gel matrix would
denitely improve ex vitro germination and enhance synseed germination, although this is species-specic and ideal conditions need to
be established on a case-by-case basis. Loss of tissue viability after
short- to long-term storage and occurrence of somaclonal variations
are other frequently encountered limitations of synseed technology
(Rai et al., 2009). One of the future applications of synseeds would
be in germplasm conservation through cryopreservation, thus further
experimentation is needed by using either hydrated calcium alginatebased or desiccated polyoxyethylene glycol-based encapsulation (Ara
et al., 2000).
Similar to all micropropagation techniques, conventional alginate
encapsulation is a very labor-intensive process. Each explant is handled multiple times, including excision, cutting to the appropriate
size, coating with sodium alginate, dipping in calcium chloride, washing in water and nally placement in a vessel for use or storage. In this
situation, bulk encapsulation has evolved as an efcient technique
with reduced labor (West and Preece, 2009). However, there are several problematic issues related to bulk alginate encapsulation including exposure of explants as a result of matrix shrinkage and reduced
shoot and root growth due to high sodium alginate concentration
(West and Preece, 2009). These problems need to be resolved in future research for proper utilization of this technique in long-term
conservation practices. Aside from the cost of developing encapsulating units, additional investments are necessary to develop methods
and machinery for handling synseed, both during production and
sowing. Although little progress has been made to demonstrate the
feasibility of synseeds, their successful implementation can only be
accomplished on a small scale while commercial use is still more of
a concept than a reality.
7. Problems, limitations and future prospects
Anwar Shahzad gratefully acknowledges the nancial support provided by the Council of Science and Technology, Uttar Pradesh (Project no.
CST/D3836), UGC (Project no. 39-369/2010) and DST-FIST Programme
2005 (Project no. SR/FST/LSI-085/2005). Shiwali Sharma is thankful to
UGC, for the award of BSR Fellowship in Science (1st April 2010) for
providing research assistance.
Synseed technology has drawn tremendous attention in recent

years because of its wide application in germplasm conservation and
exchange between countries. Despite various achievements, several
major problems still need to be resolved for its commercialization.
The rst requirement for the practical utility of synseed technology is
the large-scale production of high quality viable micropropagules in a
cost-effective manner which remains a major limiting factor (Ara et
al., 2000).
Although somatic embryos are the best suited entity for synseed
development, several bottlenecks such as loss of embryogenic potential with aging cultures, asynchronous development, precocious germination, structural anomalies and lack of desiccation tolerance
restrict their utilization (Ara et al., 2000). Thus, a judicious coupling
of synseed technology with mathematical modeling would allow
automated encapsulation and regulation of plant development to
take place (Pintos et al., 2008). This would tremendously increase
the efciency of encapsulation and generate homogenous high quality synseeds. Alternatively, the use of non-embryogenic propagules for
synseed production holds great promise to those species which are
recalcitrant to somatic embryogenesis. For most woody plant species,
a single rooting step is still the major obstacle for non-embryogenic
encapsulated beads and requires further research to improve their
conversion ability (Chand and Singh, 2004; Hung and Trueman,
2012a; Naik and Chand, 2006; Piccioni, 1997; Soneji et al., 2002).
Moreover, ex vitro or direct sowing of synseeds to soil under
Acknowledgment
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Biotechnology Advances 31 (2013) 186207

Contents lists available at SciVerse ScienceDirect

Research review paper

Synseed technologyA complete synthesis

Corresponding author. Tel.: +91 9837061683.

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

Since the formulation of the concept of synseed by Murashige

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

Cost-effective approach for ex

Comparative study aid for

Maintenance under reduced

Fig. 1. Potential uses of synseeds.

prevent the rehydration of embryos), it should be able to melt at a

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

success of the encapsulation method is affected by alginate and

Explant taken from in vivo Axillary bud

Morus alba, M. australis, M.

Musa spp. AAB group

Adhatoda vasica (vasaka)

Allium sativum (garlic)

Concentration Polymerization Composition of

2.5% SA + macro- and

i. Cold treatment was given

Kim and Park

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

Propagule used for

MS medium + 500 mg/l

Concentration Polymerization Composition of

Peat + perlite (1:1)

100% (for all

Nodal segments were

Embryogenic culture lines SE

Dalbergia sissoo (sissoo)

Explants taken from in

Boxus medium + 2.2 M

Self breaking treatment was

Semi-solid -MS medium

Oryza sativa (hybrid rice)

Rotula aquatic (takad)

(continued on next page)

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

2.5% SA + DCR basal

Perlite moistened with

Naik et al., 1999, 2000

LSBM medium + 8.88 M

Vanilla planifolia (vanilla)

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

Propagule used for

MS medium + 0.75 mg/l

Citrus nobilis C. deliciosa

Concentration Polymerization Composition of

*2% with 0.3% 20

(continued on next page)

S. Sharma et al. / Biotechnology Advances 31 (2013) 186207

Olea europaea (olive)

Psidium guajava (guava)

Psidium guajava (guava)

Concentration Polymerization Composition of

Globular and cotyledonary

*15 g/l (for

10 (for SEs) 30 Macro and

MS medium + 0.5 M TDZ NS

In vitro seedling grow on