INT J CURR SCI 2016, 19(4): E 86-101

REVIEW ARTICLE ISSN 2250-1770

Development of Synthetic Seed Technology in Plants and its Applications: A Review

Potshangbam Nongdam

Department of Biotechnology, Manipur University, Imphal-795003, Manipur, India

Department of Biotechnology, Manipur University, Canchipur, Imphal-795003, Manipur, India

Corresponding author: nongpuren@gmail.com

Abstract

Artificial/ Synseed/ Synthetic seeds are produced by encapsulation of plant micropropagules (somatic embryos, shoot

buds/shoot tips, calli, nodal segments, embryogenic masses, protocorms and protocorm like bodies) with specific coating

materials. The outer coating matrix provides protection and nutrition to the encapsulated plant tissues. Calcium alginate gel

is the most preferred among other available protective coverings as it enhances capsule formation and provides sufficient

firmness to alginate beads to withstand mechanical injury to propagules. Nutrients, growth regulators, antibiotics and other

adjuvants are incorporated into coating matrix to facilitate normal development of plant propagules leading to synseed

germination and healthy plant formation. Somatic embryos are the commonly used explant propagule for synseed

development because of the availability of well-established in vitro culture protocols for somatic embryogenesis. Synthetic

seed technology has important application in large scale multiplication of commercially valuable plants which are difficult to

propagate through conventional vegetative methods. Another significant aspect of synthetic seeds is the possibility of

conserving germplasm of rare and endangered economically important plants through short term cold storage and

cryopreservation. Inspite of its bright prospect, synseed technology is besotted with several drawbacks due to limited

availability of appropriate micropropagules in large scale, immature and asynchronous somatic embryo development and

very low conversion rate of synseeds into normal plants. Refinement of technology is the need of hour to make its wide

applications at commercially level. The review aims to stress on various aspects of synseed technology development in

several commercially important plants using different explant propagules. The various applications and limitations of the

technology in plant science research have also been emphasized in the present article.

Keywords: synthetic seeds, desiccation, protocorms, somatic embryos, callus

Received: 13th September 2016; Revised: 28thSeptember; Accepted: 06th October; © IJCS New Liberty Group 2016

Introduction

Plants are generally propagated mostly through

seeds in nature. In some crops, propagation through seeds

has not achieved success because of seed heterozygosity,

minute size, and absence of endosperms and absolute

necessity of fungal infection for germination (Saiprasad,

2001). Some plants can be vegetatively propagated but

conventional methods are time consuming, expensive

and cannot produce plants at larger scale. Production of

artificial seeds/synseeds using synthetic seed technology

can play an important role as alternative to other

conventional methods for large scale propagation and

long term germplasm storage of useful crop varieties.

The typical plant seeds have embryos with one or two

cotyledons which are associated with endosperms

containing food reserves for developing embryos. The

whole structure is enclosed by rigid covering called testa

which protects the inner delicate structure from injury

and desiccation and also helps in maintaining embryo

viability till its germination. In synthetic seeds, artificial

coating materials like sodium alginate, agar, gelrite and

sodium pectate replace the function of seed coat by

encapsulating somatic embryos (SEs), shoot buds, nodal

segments or any other plant tissues (Gantait et al., 2015).

The naked somatic embryos or shoot buds when exposed

to natural environment will not survive as they are highly

sensitive to desiccation and other infectious pathogens.

The encapsulating agents give protection to enclosed

explant propagules apart from giving nutrition for their

proper development. The derived artificial seeds/

synseeds when sowed should possess the ability to

convert into plants under in vitro and ex vitro conditions

(Bapat and Mhatre, 2005). The plant micropropagules for

synthetic seed production are obtained through somatic

embryogenesis, organogenesis, axillary bud, protocorm

and protocorm like bodies (PLBs) proliferation systems

using in vitro culture methods. This encapsulation

technology has been applied to produce synthetic seeds

of a number of plants belonging to angiosperm and

gymnosperm. The present review aims to highlight the

past and present status of synthetic seed development,

use of different explant propagules for synseed

preparation along with limitations and applications of the

technology in the field of agriculture and forestry.

Synthetic seed production

The first synthetic seeds were produced by

encapsulated carrot somatic embryos with

polyoxyethylene followed by desiccation (Kitto and

Janick, 1982). The encapsulated somatic embryos and

calli of carrot were dried for several hours on telfon

surface under the lamina flow cabinet followed by

rehydration after inoculating them in freshly prepared

medium. The polyoxyethylene was favorable for use as

coating materials for synthetic seeds as it was non-toxic,

readily soluble in water, could be easily dried to form

thin film and did not support the growth of

microorganism. Redenbaugh et al. (1984) developed a

technique for encapsulation of single, hydrated somatic

embryo of alfalfa plant and since then encapsulation of

somatic embryos of several plant species in hydrogel

have been successfully performed by several workers.

The production of synthetic seeds was primarily based on

encapsulation of somatic embryos before it was extended

to other plant tissues generated under in vitro culture

condition. The somatic embryos were employed for

synthetic seed production mainly due to presence of

radicle and plumule which ultimately differentiated into

roots and shoots (Gray et al., 1993; Dodeman and et al.,

1997). They were developed from somatic cells in in

vitro condition unlike zygotic embryos which were

obtained from fusion of male and female gametes. The

normal embryos have protective seed coat and

endosperms which provide protection and nutrition to

developing embryos. The somatic embryos on other hand

lack both endosperms and protective covering which

make them inconvenient to store and handle normally.

The naked somatic embryos are generally enclosed

in protective coating materials to produce synthetic

seeds. The coating materials however should not produce

damaging effect to the embryos, be mild enough to allow

germination, be sufficiently durable for rough handling

during manufacture, storage, transportation and planting

and also give nutrition to embryos for germination

(Sharma et al., 2012). The synthetic seeds successfully

prepared should be transplantable using existing farm

machinery. Several gels like agar, alginate,

carboxymethyl cellulose, gelrite, guargum, sodium

pectate etc. have been tested for encapsulation of plant

propagules for synthetic seed development (Redenbaugh

et al., 1987). However, calcium alginate encapsulation is

the most favorable for providing protective covering as it

enhances capsule formation and provides sufficient

firmness to alginate beads to withstand mechanical injury

(Saiprasad, 2001). Also its moderate viscosity and

reduced toxicity to somatic embryos with low cost and

bio-compatibility characteristics make it the most sought

after hydrogel for synseed encapsulation (Khor and Loh,

2005; Kikowska and Thiem, 2011). The encapsulating

matrix is provided with nutrients and other growth

regulators which play the role of artificial endosperms

(Germana et al., 1998). Seed germination efficiency and

somatic embryo viability are enhanced with the

incorporation of nutrients and growth regulators into

coating materials. Other adjuvants like fungicides,

pesticides, antibiotics and microorganisms (eg. Rhizobia)

may be included into the encapsulation matrix to instill

different properties to synthetic seeds to suit to prevailing

conditions (Kikowska and Thiem, 2011). Addition of

antibiotics may prevent bacterial contamination of

encapsulated plant propagules (Bekheet, 2006). Khor and

Loh (2005) reported improvement of conversion

potential and vigor of synseeds when activated charcoal

was supplemented in coating gel. Charcoal can break up

the alginate and helps in increasing the respiration of

somatic embryos inside the coat. Charcoal is also

presumed to play a role in retaining nutrients within the

hydrogel capsule and also helps in releasing them slowly

to developing embryos. Absorption of harmful

polyphenolic exudates released by encapsulated explant

propagules in vitro is also facilitated by activated charcoal

(Ganapathi et al., 1992).

The primary goal of synthetic seed technology is to

produce somatic embryos that resemble more closely to

normal seed embryos in terms of storage and handling

capabilities by providing nutritive covering. The

synthetic seeds can later be used for large scale clonal

propagation and germplasm conservation of rare and

commercially useful crop varieties. Apart from using

popular somatic embryos, other unipolar structures like

apical shoot tips, axillary shoot bud, embryogenic calli,

protocorm and protocorm like structures (PLBs) are

widely utilized for development of synseeds.

Redenbaugh et al. (1991) first treated shoot tip and

axillary bud micropropagules for root induction before

they were encapsulated. Root induction treatment with

auxins was avoided for alginate encapsulation of shoot

tips of mulberry and banana to produce synseeds and

their successful conversation to plantlets (Bapat and Rao,

1990). The procedure for synthetic seed production

starting from different plant micropropagules are

diagrammatically represented in figure 1.

Different types of synthetic seeds

The plant propagules obtained from different

sources are encapsulated with coating materials which

act as artificial endosperms by proving nutrition to

embryos apart from their protective function. Synthetic

seeds are broadly classified into desiccated and hydrated

seeds depending on different approaches undertaken for

their development as per the requirement.

Desiccated synthetic seeds

Encapsulation of somatic embryos is performed

using polyoxyethylene followed by desiccation under

controlled conditions. Desiccation can be performed

slowly or rapidly depending upon the requirement. It

takes one or two week times to gradually desiccate the

encapsulated seeds in chamber of decreasing humidity

while instant desiccation involves the opening of sealed

petridish containing synseeds and leaving directly open

overnight for quick drying (Ara et al., 2000). The

desiccated synthetic seeds can be developed for those

plant species which have somatic embryos resistant

against desiccation. The carrot SEs can be encapsulated

with polyox by mixing equal volumes of embryonic

suspension and a 5% (w/v) solution of polyox to give a

final concentration of 2.5% polyox. The suspension was

dispensed as 0.2 ml drops from the pipette on to telfon

sheets and dried to wafers in a laminar flow hood. The

drying time was based on the ability of water to separate

from teflon plate and it usually took about 5 hours. The

embryo survival and conversion of seeds were

determined by redissolving the wafers in freshly prepared

embryogenic medium and culturing the rehydrated

embryos. High conversion frequency of somatic embryos

of celery could be obtained by incorporating abscisic acid

(ABA) and mannitol to maturation medium (Halal,

2011). The synthetic seeds with desiccation to 10-15%

can be stored at room temperature for about one year

without being lost in cell viability and germination

potential of seeds. More healthier seedlings could be

derived from dried somatic embryos when compared to

seedlings obtained from undried embryos (Senaratna

et al., 1990).

Hydrated synthetic seeds

Hydrated artificial seeds consist of somatic

embryos or suitable plant tissues enclosed by a hydrogel.

Many substances like potassium alginate, agar, gelrite,

sodium pectate have been examined but calcium alginate

was found to be most effective coating material for

hydrated synthetic seeds (Redenbaugh et al., 1987).

Alginate is a straight chain, hydrophilic, colloidal

polyuronic acid composed primarily of hydro-b-D

mannuronic acid residues with 1-4 linkages. For

production of hydrated seeds, the plant materials are

mixed with sodium alginate gel (0.5-5.0 % w/v) which is

followed by dropping into the calcium chloride solution

(30-100 mM) using pipette. Round and firm beads of

calcium alginate containing somatic embryos are formed

as the ion exchange occurs resulting in the replacement

of sodium ions with calcium ions (Reddy et al., 2012).

The hardness and rigidity of capsules mainly depend

upon the number of sodium ions exchanged with calcium

ions. So, hardness of calcium alginate gel can be

modulated by changing the concentration of sodium

alginate and calcium chloride solution along with change

in duration of complexing. In most cases 2% of sodium

alginate gel when complexed with 100 mM Calcium

chloride solution produced desirable quality synthetic

seeds for many plant species (Redenbaugh et al., 1987;

Oceania et al., 2015). Ca-alginate capsules are difficult to

handle as they are very wet and tend to stick together

slightly. Moreover, the Ca-alginate loses water rapidly

and dries very fast to form hard pellet within few hours

when the beads are exposed to normal atmosphere.

However, these problems can be solved by coating the

capsules with Elvax 4260. Antibiotic mixture containing

rifampicin, cefatoxine and tetracycline-HCl can also be

added to the matrix to avoid bacterial contamination

(Bekheet, 2006). When there is encapsulation of plant

materials exuding high phenolic compounds, activated

charcoal (0.1-0.4%) may be incorporated to the matrix to

absorb the phenolic exudes (Ganapathi et al., 1992).

Propagules for synseed production

The somatic embryos were earlier employed as the

only explant for synthetic seed development in several

plants. But subsequent reports by different workers

showed the use of varieties of plant micropropagules like

unipolar apical shoot tips and buds, nodal segments,

embryogenic masses and calli along with hosts of other

explants like protocorms or protocorm like bodies, bulb,

bulblets, hairy roots and microtubers (Reddy et al., 2012;

Gantait and Sinniah, 2013)..

Somatic embryos

Embryos which are obtained asexually from

somatic cells without the union of gametes are called

somatic embryos. In vitro development of plant somatic

embryos was first reported by Reinert (1958) and

Steward et al. (1958) independently. The direct SEs

develop directly from explanted cells while indirect SEs

derive from explant tissues through intervening callus

phase (William and Maheswaran, 1986). The SEs

undergo globular, heart, torpedo and cotelydonous stages

like zygotic embryos to germinate and develop into

fertile plants. SEs however do not go through desiccation

and dormancy unlike normal embryos and enter the

germinating phase as soon as they are fully formed

(Zimmerman, 1993). They are the most favorable

propagules use for production of synthetic seeds due to

presence of radicle and plumule which develop in single

step into roots and shoots respectively without subjecting

to any specific treatment (Standardi and Piccioni, 1998).

With the advancement of plant tissue culture technology,

somatic embryos have been induced successfully in

number of plants making somatic embryos more

preferable for artificial seed production as they can be

more easily available. The SEs can be preserved in viable

state for longer duration if the moisture content can be

maintained at 10% like normal seeds by drying (Ara

et al., 2000).

Attempts have been made to desiccate somatic

embryos before encapsulation to exploit this potential.

Desiccation tolerance was induced in somatic embryos of

Alfalfa plant by exogenous application of abscisic acid

(Senaratna et al., 1989). When the somatic embryos after

encapsulation were dried to 10-15% and stored in dry

state for about 4 weeks, 65% of somatic embryos

survived and germinated like true seeds. Sometimes

emergence of shoot and root meristem during synthetic

seed germination was prevented by nutritive outer

covering. In order to avoid this scenario, gel capsules had

been prepared which were self-breaking under humid

conditions. This involved the rinsing of beads thoroughly

in running tap water which was followed by immersion

of beads in a 200 mM solution for 60 mins and finally

desalting them by rinsing them in running tap water for

40 minutes (Onishi et al., 1994). The synthetic seeds

showed 50% conversion in two weeks when sowed in

green house conditions. The use of somatic embryos as

explant propagules have been demonstrated in several

plants like Armoracia rusticana (Shigeta and Sato,

1994); Asparagus officinalis (Mamiya and Sakamoto,

2001); Oryza sativa (Suprasanna et al., 2002); Solanum

melongena (Huda and Bari, 2007); Zea mays

(Thobunluepop et al., 2009); Oryza nivara (Jaseela et al.,

2009); Vitis vinefera (Nirala et al., 2010); Tylophora

indica (Devendra et al., 2011); Rhinacanthus nasutus

(Cheravathur et al., 2013); Asethum graveolens (Dhir et

al., 2014); Apple rootstock MM.106 (Farahani et al.,

2015); Litchi (Das et al., 2016); Citrus (Micheli and

Standardi, 2016).

Protocorms and protocorm-like bodies

The miniature exalbuminous orchid seeds when

inoculated in culture medium in vitro started swelling in

one or two weeks indicating successful germination due

to imbibition of water and nutrients (Nongdam and

Chongtham, 2011). The embryos underwent several

division to develop into irregularly shaped

parenchymatus cell mass called spherules (Nongdam and

Tikendra, 2014). The hairy globular spherules developed

into protocorms which are oval, elongated, branched and

spindle shaped bodies considered to be an intermediate

structure between embryos and plants (Fig. 1a). The

protocorms directly differentiated into complete

seedlings after undergoing morphogenetic changes (Fig.

1b). Protocorm-like bodies are similar to protocorms in

their function and morphology but are developed from

plant parts other than seeds of orchids under in vitro

conditions. In different orchids like Cymbidium

giganteum, Dendrobium wardianum and Spathoglottis

plicata, synthetic seeds have been produced by

encapsulating protocorms or protocorm-like bodies with

calcium alginate gel (Sharma et al., 1992; Corrie and

Tandon, 1993; Nagananda et al., 2011). The encapsulated

protocorms of C. giganteum developed into healthy

plantlets when grown on nutrient medium in vitro or in

sterile soil and sand mixture under greenhouse

conditions. The frequency of synthetic seed conversion

was higher in vitro as compared to conversion of

germinated seeds in sand and soil mixture. Mohanty et al.

(2013) produced synseeds in Dendrobium nobile using

PLBs and observed conversion of synseeds significantly

high at 80% as PLBs being highly potential for direct

plantlet generation. Mohanraj et al. (2009) encapsulated

PLBs of Coelogyne breviscapa with 3% sodium alginate

matrix and stored for 60 days before being germinated to

seedling on MS medium supplemented with different

growth regulators. The germination percentage of

synthetic seeds decreased gradually with increase in

storage time.

Embryogenic masses and Calli: Regenerative and stable

embryogenic masses can be used for production of clonal

plants and for studies of genetic transformation. But

maintaining them for longer duration in bioreactors and

culture vessels is difficult due to frequent subculturings

(Ara et al., 2000). The laborious and expensive

subculturing procedure can be overcome by

encapsulating the embryogenic masses with sodium

alginate and store them at 40oC after 6-benzyl amino

purine (BAP) treatment (Redenbaugh et al., 1991). The

synthetic seeds can be stored for around 2 months

without losing its viability and original proliferative

capacity. But more research needs to be done to

understand whether the storage period of synthetic seeds

can be extended and efficiency and proliferative nature of

embryogenic masses decrease with the increase in the

storage period. Regenerative embryogenic masses have

been utilized for synthetic seed production in few plants

like Anthurium andreanum (Nhut et al., 2004) and

Arnebi euchroma (Manjkhola et al., 2005). Plant callus is

unorganized mass of proliferative cells produced by

isolated cells, tissues or organs under the influence of in

vitro culture conditions (Fig. 1c). Callus formation is

associated with the development of progressively more

random planes of cell division, lower cell specializations

and loss of organized structures (Wagley et al., 1987).

The acceptance of calli as explant propagules for synseed

preparation is limited due to their undifferentiated nature

and low differentiation potential (Gantait et al., 2015).

The use of calli for synseed development was

successfully observed for the first time in Allium sativum

by Kim and Park (2002) showing high conversion and

regeneration rate of synseeds to plants. Zych et al. (2005)

and Reedy et al. (2005) also attempted successfully to

formulate synseeds from callus derived from in vitro

culture of Rhodiola kirilowii and Rauvolfia serpentina

respectively.

Apical shoot tips/ shoot buds and nodal segment

The shoot buds and apical shoot tips are unipolar

structure without root meristem. The conventional shoot

tip culture in vitro requires higher space and culture

media as compared to micropropagation of shoot tip/buds

encapsulated synseeds (Gantait et al., 2015). The

requirement of smaller space ensures easy transportation

of plant propagules from one center to another. The shoot

tip explants were induced to root before encapsulation by

exposing to indole-3-butyric acid (IBA) treatment for 3-6

days. Successful conversion to plantlets from synseeds

developed by encapsulating banana and mulberry buds

without auxin treatment was also reported (Bapat and

Rao, 1990; Ganapathi et al., 1992). Figure 1(d) represents

synseeds developed by enclosing in vitro derived shoot

tips with calcium alginate covering. The use of apical

shoot tips/ buds as explants for synseeds development

have been successfully reported in different plant such as

Catalpa ovate (Wysokinska et al., 2002); Stevia

rebaudiana (Andlib et al., 2011); Glycyrrhiza glabra

(Mehrotra et al., 2012); Terminalia aryria (Gupta et al.,

2014); Cucumis sativus (Adhikari et al., 2014) and

Solanum tuberosum (Ghanbarali et al., 2016). The nodal

explants derived either from natural or in vitro

regenerated plants can be encapsulated for synseed

production (Fig. 1e). The plantlet conversion frequency

of in vitro derived nodal explants would be significantly

higher as compared to that of mature nodal tissues

obtained from field grown plants due to higher

meristematic and organogenetic potential. Nodal explants

have been employed for the production of synseeds in

number of plants like Hibiscus moschentus (Prrece and

West, 2006); Pogostemon cablin (Swamy et al., 2009);

Eclipta alba (Singh et al., 2010); Vitex negundo (Ahmad

and Anis, 2010); Picrorhiza kurrooa (Mishra et al.,

2011); Stevia rebandiana (Khan et al., 2013);

Phullanthus fraternus (Upadhya et al., 2014); Blackberry

(Jadan et al., 2015); Physalis peruviana (Yucesan et al.,

2015) and Tomato (Oceania et al., 2015).

Applications of synthetic seeds

There are increasing numbers of medicinal plants

whose population are declining in an alarming rate and

are in the verse of extinction. Rampant deforestation,

rapid expansion of cities and industries in the expense of

natural forested areas, excessive exploitation of rare and

medicinally important plants for economic gains are the

main reasons for increasing number of plants becoming

rare and endangered in their natural habitats. Large scale

propagation for medicinal plants through conventional

methods has limitations as majority of plants are either

seedless varieties, or have reduced endosperms and lower

germination rate (Rai et al., 2009).

Fig.1. The procedure of synthetic seed production using different plant propagules

Fig. 2 (a-e). (a) In vitro protocorm formation in orchid (bar 4 mm), (b) Protocorms directly differentiated into seedlings (bar

6 mm), (c) In vitro callus formation in pea (bar 6 mm), (d) Synthetic seeds with shoot tip explant propagules (bar 5 mm) and

(e) Synseeds developed by encapsulating nodal segments (bar 6 mm).

(b) (a)

(c) (d) (e)

Many of them are also desiccation-sensitive or have

recalcitrant seeds because of which they cannot be stored

for longer period. The synthetic seed technology can be

employed for mass propagation and conservation of rare

and threatened medicinal plants by encapsulating somatic

embryos and meristematic vegetative propagules with

suitable coating materials. Artificial synseeds have been

successfully developed using different plant propagules

in several medicinally important plants like Allium

sativum, Cannabis sativa, Catalpa ovata, Rauwolfia

serpentine and Slevia rebaudiana (Wysokinska et al.,

2002; Belkeet, 2006; Ray and Battarcharya, 2008; Lata

et al., 2009; Andlib et al., 2011).

The germplasm preservation of difficult to

propagate ornamental plants and recalcitrant species like

mango, cocoa and coconut can be performed by

cryopreserving their synseeds in liquid nitrogen (-1960C).

The metabolic function of cells during cryopreservation

is arrested and supplementation of sucrose, salicyclic,

mannitol and other nutrients to encapsulating matrix is

essential which may improve cell viability and tolerance

to dehydration and other abiotic stresses (Janda et al.,

2007; Katouzi et al., 2011). There are several potential

uses of synthetic seeds for important crops such as citrus,

grapes, mango etc. which are vegetatively propagated

and have long juvenile periods. The planting efficiency

can be increased by use of synthetic seeds instead of

cuttings. The synthetic seeds are considered to be highly

advantageous for germplasm conservation in grapes and

other similar crops. Banana is considered as one of the

most important crops because of its large consumption

and considerable export potential. They are normally

propagated by suckers as they do not produce viable

seeds (Matsumoto et al., 1995). Conventional method of

propagation does not guarantee large scale production of

banana, so synthetic seed technology can be used as

another option by encapsulating the shoot-tips excised

from tissue culture raised plants and somatic embryos

developed from callus tissues obtained from in vitro

culture of male flower buds (Ganapathi et al., 2001;

Hassanein et al., 2005; Sandavol-Yugar et al., 2009).

Bapat and Rao (1980) used undifferentiated callus

produced from stem segments of sandalwood - one of the

most commercially valuable forest trees to produce

synseeds. High genetic variations among the progenies

were observed when seeds were used for propagation of

sandalwood. But germination of synthetic seeds

produced by encapsulating the somatic embryos with

calcium alginate produced plants with genetic

uniformity. Mulberry plants are important components of

silk industry as their leaves are chief source of food for

silkworms. The multiplication of mulberry cultivars is

difficult due to restriction in rooting response though

cutting and grafting are used for vegetative propagation.

Moreover 30-40% cutting can only survive the time

period between pruning, transportation and final

transplantation (Bapat and Rao, 1990). The synthetic

seeds produced by encapsulation of in vitro raised

axillary buds could be easily packed in bottles and

transported which reduced space requirement, increases

seed viability and survival rate.

The synthetic seed technology has provided space

and equipment saving option for storage of plant

materials of different rare and commercially important

plants at low temperature. It has the potential for short

term and long term storage of plant germplasm in the

form of synseeds without losing cell viability by

cryopreserving them in liquid nitrogen (Arora et al.,

2010). Other advantages of synthetic seeds are easy

handling because of smaller size beads, higher scale up

capacity, possibility of automation of the whole

production process and direct delivery to field. The

artificial seeds with vegetative propagules enclosed

within them can be used for exchange of axenic plant

materials between the laboratories (Reddy et al., 2012).

The transport of synthetic seeds between the countries

does not require quarantine department permission. This

technology is also independent of seasonal variations as

they can be produced in controlled laboratory

environments.

Limitations of synthetic seeds

The synthetic seed technology inspite of having

bright prospects in large scale propagation and

germplasm conservation of rare and important plants is

associated with many limitations. Production of good

quality microprogagules in high number is prerequisite to

production of synthetic seeds. However, there are

limitations in the generation of viable micropropagules

for use in synseed production. Somatic embryos which

are commonly used explant propagules for synthetic seed

production should attain proper maturation to germinate

out of the coating materials to form healthy plantlets.

Improper maturation of somatic embryos reduces the

success rate of conversion of synthetic into normal

plants. Mature embryos will control germination and

conversion rate which are highly significant for

successful synseed production. However, asynchronous

development of embryos limits the production of normal

plants post synseed formation even after induction of

successful somatic embryogenesis (Ara et al., 2000).

Somatic embryos exhibited immense variation in their

morphology in response to prevailing conditions in

culture system. Long term treatment of ABA either

induced anomalous cotyledon formation in somatic

embryos or activated normal embryo development in in

vitro condition (Crouch and Sussex, 1981).

The conversion of synthetic seeds into plants after

successful germination is the most important aspect of

synthetic seed technology. Due to various reasons the

conversion rate is always very low which limits the

commercial application of this technology. Most of the

studies reported somatic embryogenesis of plants and

their subsequent encapsulation to produce synseeds with

very low success in conversion rate. The choice of

appropriate coating materials is one of important limiting

factors for synseed generation. Low success rate in the

use of synthetic seed technology may be attributed to

failure in the choice of effective encapsulating materials

which should be non-damaging, provide sufficient

protection and nutrition to developing embryos

(Rendenbaugh et al., 1991). Proper storage of synthetic

seeds is affected by lack of dormancy and stress

tolerance in somatic embryos. Also storage of synthetic

seeds at low temperature reduces significantly the

viability of synthetic seeds and its potential for

conversion to normal plants (Makowwczynska and

Andrezejewska-Golec, 2006).

Conclusion

The artificial seed technology is considered as an

alternative to slow and expensive conventional plant

propagation methods by generating several important

plants rapidly in large scale. Artificial seeds also offer

tremendous potential in micropropagation and

germplasm conservation of economically significant rare

and endangered plants. However, lack of production of

high quality micropropagules for synthetic seed

production coupled with low conversion rate to normal

plants has restricted the widespread use of synthetic seed

technology in the field of agriculture. Further detailed

investigations are required to refine this useful

technology for its wide applications at commercial scale.

References

Adhikari S, Bandyopadhyay TK, Ghosha P (2014).

Assessment of genetic stability of Cucumis

sativus L. regenerated from encapsulated shoot

tips. Sci Hortic 170: 115-122.

Ahmad N, Anis M (2010). Direct plant regeneration from

encapsulated nodal segments of Vitex negundo.

Biol Plt 748-752.

Andlib A, Verma RN, Batra A (2011). Synthetic seeds

an alternative source for quick regeneration of a

zero calorie herb-Stevia rebaudiana Bertoni. J

Parm Res 4(7): 2007-2009.

Ara H, Jaiswal U, Jaiswal VS (2000). Synthetic seed:

prospect and limitation. Curr Sci 78(12): 1438-

44.

Arora R, Mathur A, Mathur AK (2010). Emerging

trends in medicinal plant biotechnolgy. In: Arora

R, editor. Medicinal Plant Biotechnology.

Willingford; pp. 1-12.

Bapat VA, Mhatre M (2005). Bioencapsulation of

somatic embryos in woody plants. In: Jain SM,

Gupta PK, editors. Protocol for somatic

embryogenesis in woody plants. Springer; pp.

539-552.

Bapat VA, Rao PS (1988). Sandalwood plantlets from

synthetic seeds. Plt Cell Rep 7(6):434-436.

Bapat VA, Rao PS (1990). In vivo growth of

encapsulated axillary buds of mulberry (Morus

indica L.). Plt Cell Tiss Org Cult 20: 69-70.

Bekheet SA (2006). A synthetic seed method through

encapsulation of in vitro proliferated bulblets of

garlic (Allium sativum L.). Arab J Biotech 9(3).

Cheravathur MK, Kumar GK, Thomas TD (2013).

Somatic embryogenesis and synthetic seed

production in Rhinacanthus nasutus (L.) Kurz.

Plt Cell Tiss Org Cult 113: 63-71.

Corrie S, Tandon P (1993). Propagation of Cymbidium

giganteum Wall. through high frequency

conversion of encapsulated protocorms under in

vivo and in vitro conditions. Ind J Exp Biol 31:

61-64.

Crouch ML, Sussex IM (1981). Development and

storage-protein synthesis in Brassica napus L.

embryos in vivo and in vitro. Planta 153: 64-74.

Das DK, Rahman A, Kumara D, Kumari N (2016).

Synthetic seed preparation, germination and

plantlet regeneration of Litchi (Litchi chinensis

Sonn.). Amer J Plt Sci 7: 1395-1406.

Devendra BN, Srinivas N, Nail GR (2011). Direct

somatic embryogenesis and synthetic seed

production from Tylophora indica (Bum f.)

Merill, an endangered medicinally important

plant. Int J Bot 7: 216-222.

Dhir R, Shekhawat GS, Alam A (2014). Improved

protocol for somatic embryogenesis and calcium

alginate encapsulation in Anethum graveolens L.:

a medicinal herb. Appl Biochem Biotech 173:

2267-2278.

Dodeman VL, Ducreux G, Kreis M (1997). Zygotic

embryogenesis versus somatic embryogenesis. J

Expt Bot 48(313): 1493-1509.

Farahani F, Noormohamadi Z, Satari TN (2015).

Optimization of synthetic seed production in

MM.106 apple rootstocks from somatic embryos

and axillary buds. Bull Envn Phar Life Sci

4(6):68-78.

Ganapathi TR, Sriniva L, Suprasanna P, Bapat VA

(2001). Regeneration of plants from alginate-

encapsulated somatic embryos of banana cv.

Rasthali (Musa sp. AAB group). In Vitro Cell

Dev Biol Plt 37: 178-181.

Ganapathi TR, Suprasana P, Bapat VA, Rao PS (1992).

Propagation of banana through encapsulated

shoot tips. Plt Cell Rep 11:571-575.

Gantait S, Kundu S, Ali N, Sahu NH (2015). Synthetic

seed production of medicinal plants: a review on

influence of explants, encapsulation agent and

matrix. Acta Physio Plt 37: 98-110.

Gantait S, Sinniah UR (2013). Storability, post-storage

conversion and genetic stability assessment of

alginate-encapsulated shoot tips of monopodial

orchid hybrid Aranda Wan Chark Kuan Blue X

Vanda corrulea Grifft. Ex. Lindl. Plt Biotech Rep

7: 257-266.

Germana MA, Amanuele P, Alvaro S(1998). Effect of

encapsulation on citrus reticulate Balanco

somatic embryo conversion. Plt Cell Tiss Org

Cult 55:235-238.

Ghanbarali S, Abdollahi MR, Zolnorian H et al. (2016).

Optimization of the conditions for production of

synthetic seeds by encapsulation of axillary buds

derived from minituber sprouts in potato

(Solanum tuberosum). Plt Cell Tiss Org Cult

126(3): 449-458.

Gray DJ, Mc Colley DW, Compton ME (1993). High-

frequency Somatic Embryogenesis from

Quiescent Seed Cotyledons of Cucumis melo

Cultivars. J Amer Soc Hort Sci 118(3): 425-432.

Gupta AK, Harish M, Raj MP, Agarwal T, Shekhawat

NS (2014). In vitro propagation, encapsulation,

and genetic fidelity analysis of Terminalia

arjuna: a cardioprotective medicinal tree. Appl

Biochem Biotech 173: 1481-1494.

Halal NAS (2011). The green revolution via synthetic

(artificial) seeds: A review. Res J Agric Biol Sci

7: 464-477.

Hassanein AM, Ibrahiem IA, Galal AA, Salem JMM

(2005). Micropropagation factors essential for

mass propagation of banana. J Plt Biotech 7:175-

181.

Huda AKMN, Bari MA (2007). Production of

synthetic seeds by encapsulating asexual

embryo in Eggplant (Solanum melongena L.).

Int J Biotech 2:832-837.

Jadan M, Ruiz R, Soria N, Mihai RA (2015). Synthetic

seeds production and the induction of

organogenesis in blackberry (Rubua glaucus

Benth). Rom Biotech Lett 20(1): 10143-10142.

Janda T, Horvath E, Szalai G, Paldi E (2007). Role of

salicyclic acid induction of abiotic stress

tolerance. In: Harat S, Ahmad A, editors.

Salicyclic acid-A plant hormone. Springer; pp.

91-150.

Jaseela F, Sumitha VR, Nair GM (2009). Somatic

embryogenesis and plantlet regeneration in an

agronomically important Wild rice species

Oryiza nivira. Asian J Biotech 1: 74-78.

Katouzi SSS, Majid A, Fallahian F, Bernard F (2011).

Encapsulation of shoot tips in alginate beads

containing salicylic acid for cold storage and

plant generation in sunflower (Helianthus annuus

L.) AJCS 11: 1469-1474.

Khan MK, Sharma T, Mishra P, Shukla PK, Singh Y,

Ramteke PW (2013). Production of plantlets on

different substrates from encapsulated in vitro

nodal explants of Stevia rebaudiana. Int J Recent

Sci Res 4: 211-215.

Khor E, Loh CS (2005). Artificial seeds. In: Nedovic V,

Willaert K editors. Application of Cell

Immobilization, Biotechnology, Springer; pp.

527-537.

Kikowska M, Thiem B (2011). Alginate-encapsulated

shoot tips and nodal segments in

micropropagation of medicinal plants: A review.

Kerba Polnca 57(4): 46-57.

Kim MA, Park JK (2002). High frequency plant

generation of garlic (Allium sativum L.) calli

immobilized in calcium aliginate gel. Biotech

Bioproc Eng 7: 206-211.

Kitto SK, Janick J (1982). Polyox as an artificial seed

coat for asexual embryos. Hort Sci 17: 488-490.

Lata H, Chandra S, Khan IA, Elsohly MA (2009).

Propagation through alginate encapsulation of

axillary buds of Cannabis sativus L- an important

medicinal plant. Physiol Mol Plt 15(1): 80-86.

Makowwczynska J, Andrezejewska-Golec E (2006).

Somatic seeds of Plantago asiatica L. Acta Soc

Bot Pol 75(1): 17-21.

Mamiya K, Sakamoto Y (2001). A method to produce

encapsulatable units for synthetic seeds in

Asparagus officinalis. Plt Cell Tiss Org Cult 64:

27-32.

Manjkhola S, Uppeandra D, Meena J (2005).

Organogenesis, embryogenesis and synthetis

seed production in Arnebia euchroma a critically

endangered medicinal plant of the Himalaya. In

Vitro Cell Dev Plt 41: 244-248.

Matsumoto K, Hirao C, Teixeira C (1995). In vitro

growth of encapsulated shoot tips in banana

(Musa sp.). Acta Hortic 370: 13-20.

Mehrotra S, Khaja O, Kukreja AK, Rahman L (2012).

ISSR and RAPD based evaluation of gentic

stability of encapsulated micro shoots of

Glycyrrhiza glabra following 6 months of

storage. Mol Biotech 52(3): 262-268.

Micheli M, Standardi A (2016). From somatic embryo to

synthetic seed in Citrus sp. through the

encapsulation technology. Methods Mol Biol

1359: 515-522.

Mishra J. Singh M, Palni LMS, Nandi SK (2011).

Assessment of genetic fidelity of encapsulated

microshoots of Picrorhiza kurrooa. Plt Cell Tiss

Org Cult 104: 181-186.

Mohanraj R, Ananthan R, Bai VN (2009). Production

and storage of synthetic seeds in Coelongyne

breviscapa Lindl. Asian J Biotech 11: 37-43.

Mohanty P, Nongkling P, Das MC, Kumaria S,Tandon P

(2013). Short-term storage of alginate-

ebcapsulated protocorm-like bodoes of

Dendrobium nobile Lindl: an endangered

medicinal orchid from northeast India. 3 Biotech

3: 235-239.

Nagananda GS, Satishchandra N, Rajath S (2011).

Regeneration of encapsulated protocorm like

bodies of medicinally important vulnerable

orchid Flickingeria (Dalz.) Seidenf. Int J Bot 7:

310-313.

Nhut DTN, Duy, Ha-vy HN, Khue CD, Khiem DV,

Hang NTT, Vinh DN (2004). Artificial seeds for

propagation of Anthurium TROPICAL. J Agric

Sci Technol 4: 73-78.

Nirala NK, Das DK, Reddy MK, Srivastava PS, Sopory

SK, Upadhyaya KC (2010). Encapsulated

somatic embryos of grape (Vitis vinifera L.) an

efficient way for storage, transport and

multiplication of pathogen free plant material.

Asia Pac J Mol Biol Biotech 18: 159-162.

Nongdam P, Chongtham N (2011). In vitro rapid

propagation of Cymbidium aloifolium (L) Sw.: A

medicinally important orchid via seed culture. J

Biol Sci 11: 254-260.

Nongdam P, Tikendra L (2014). Establishment of

efficient in vitro regeneration protocol for rapid

and mass propagation of Dendrobium

chrysotoxum Lindl. using seed culture. The

Scientific Wld J.

Oceania C, Doni T, Tikendra L, Nongdam P (2015).

Establishment of efficient in vitro culture and

plantlet generation of Tomato (Lycopersicon

esculentum Mill.) and development of synthetic

seeds. J Plt Sci 10: 14-24.

Onishi N, Sakamoto Y, Hirosawa T (1994). Synthetic

seeds as an application of mass production of

somatic embryos. Plt Cell Tiss Org Cult 39: 137.

Prrece JE, West TP (2006). Greenhouse growth and

acclimatization of encapsulated Hibiscus

moschatus nodal segments. Plt Cell Tiss Org Cult

87: 127-138.

Rai MK, Asthana P, Singh SK, Jaiswal VS, Jaiswal U

(2009). The encapsulation technology in fruit

plants-A review. Biotech Adv 27: 671-679.

Ray A, Bhattarcharya S (2008). Storage and plant

regeneration from encapsulated shoot tips of

Rauvolfia serpentine-an effective way of

conservation and mass propagation. South Afr J

Bot 74: 776-779.

Reddy GS, Kumar DD, Babu RS, Madhav M (2005).

Callus culture and synthetic seed production in

Rauvolfia serpentina. Ind J Hort 62:102-103.

Reddy MC, Murthy KSR, Pullaiah T (2012). Synthetic

seeds: A review in agriculture and forestry.

African J Biotech 11(78): 14254-14275.

Redenbaugh K, Nichol J, Kossler ME, Paasch BD

(1984). Encapsulation of somatic embryos for

artificial seed production. In Vitro Cell Dev Biol

Plt 20: 256-257.

Redenbaugh K, Fujii J, Slade D, Viss P, Kossler M

(1991). Artificial seeds-encapsulated embryos.

Biotech Agri For 17:395-416.

Redenbaugh K, Slade D, Viss PR, Fujii J (1987).

Encapsulation of somatic embryos in synthetic

seed coats. Hort Sci 22: 803-809.

Reinert J (1958). Untersuchungen u¨ber die

morphogenese a Gewebekulturen. Ber Dtsch Bot

Ges 71:65.

Saiprasad GVS (2001). Artificial seeds and their

applications. Resonanace 6: 39-46.

Sandavol-Yugar EW, Vesco LDD, Steinmacher DA,

Stolf EC, Guerra MP (2009). Microshoots

encapsulation and plant conversion of Musa sp.

Cv. Grand naine. Ciencia Rural 39: 998-1004.

Senaratna T, Mckersie BD, Bowley SR (1990). Artificial

seeds of alfalfa (Medicago sativa L.) induction of

desiccation tolerance in somatic embryos. In

Vitro Cell Dev Biol Plt 26:85-90.

Senaratna T, Mckersie BD, Bowley SR (1989).

Desiccation tolerance of alfalfa (Medicago sativa

L.) somatic embryos, Influence of abscisic acid,

stress pretreatments and drying rates. Plt Sci

65:253-259.

Sharma S, Shahzad A, Silva JAT (2012). Synseed

technology-A complete synthesis. Biotech Adv

31:186-207.

Sharma A, Tandon P, Kumar A (1992).Regeneration

of Dendrobium wardianum Warner

(Orchidaceae) from synthetic seeds. Ind J Exp

Biol 30: 747-748.

Shigeta J, Sato S (1994). Plant regeneration and

encapsulation of somatic embryos of horseradish.

Plt Sci 102: 109-115.

Singh SK, Rai MK, Asthana P, Sahoo L (2010).

Alginate encapsulation of nodal segments for

propagation, short term storage and germplasm

exchange and distribution of Eclipta alba (L.).

Acta Physiol Plt 32: 607-10.

Standardi A, Piccioni E (1998). Recent perspective on

synthetic seed technology using nonembryogenic

in vitro-derived explants. Int J Plt Sci 159: 968-

978.

Steward EC, Mapes MO, Mears K (1958). Growth and

organized development of cultured cells. II:

organization in cultures grown from freely

suspended cells. Am J Bot 45: 705-708.

Suprasanna P, Bharati G, Ganapathi TR, Bapat TA

(2002). In vitro development of encapsulated

somatic embryos in rice. Trop Agric Res Ext

5:76-78.

Swamy MK, Balasubramanya S, Anuradha M (2009).

Germplasm conservation of Patchouli

(Pogostemon cablin Benth.) by encapsulation of

in vitro nodal segments. Int J Biodvers Conserv

1: 224-230.

Thobunluepop P, Pawelzik E, Vearasilp S (2009).

Possibility of sweet corn synthetic seed

production. Pak J Biol Sci 12: 1085-1089.

Upadhya R, Kashyap SP, Singh CS, Tiwari KN, Singh

K, Singh M (2014). Ex situ conservation of

Phyllanthus fraternus Webster and evaluation of

genetic fidelity in regenerates using DNA- based

molecular markers. Appl Biochem Biotech.

Wagley LM, Gladfelter HJ, Phillips GC (1987). De novo

shoot organogenesis of Pinus eldarica Medw. In

vitro. II. Macro-and micro-photographic evidence

of de novo regeneration. Plt Cell Rep 6: 167-171.

William EG, Maheswaran G (1986). Somatic

embryogenesis: factors influencing coordinated

behaviour of cells as an embryogenic group. Ann

Bot 57: 443-462.

Wysokinska H, Lisowska K, Floryanowicz-Czekalska K

(2002). Plantlets from encapsulated shoot buds of

Catalpa ovata G. Don. Acta Soc Bot Pol 71: 181.

Yucesan BB, Mohammed A, Arslan M, Gurel E (2015).

Clonal propagation and synthetic seed production

from nodal segments of Cape gooseberry

(Physalis peruviana L.) a tropical plant. Turk J

Agric For 39:797-806.

Zimmerman JL (1993). Somatic embryogenesis: A

model for early development in higher plants. Plt

Cell 5(10): 1411-1423.

Zych M, Furmanova M, Krajewska-Patan A, Lowicka

A, Dreger M, Mendlewska S (2005).

Micropropagation of Rhodiola kirilowii plants

using encapsulated axillary buds and callus. Acta

Biol Crac Ser Bot 47: 83-87.