Biotechnology Advances 27 (2009) 671–679

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Research review paper

The encapsulation technology in fruit plants—A review

Manoj K. Rai a,⁎, Pooja Asthana a, Shashi Kant Singh b, V.S. Jaiswal a, U. Jaiswal a

a Lab of Morphogenesis, Department of Botany, Banaras Hindu University, Varanasi—221005, U.P., Indiab Department of Life Sciences, Institute of plant Biology, National Taiwan University, Taipei, Taiwan, ROC

⁎ Corresponding author.E-mail address: mkraibhu@gmail.com (M.K. Rai).

0734-9750/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2009.04.025

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 December 2008Received in revised form 24 April 2009Accepted 24 April 2009Available online 3 May 2009

Keywords:ConservationCryopreservationEncapsulationFruit cropsIn vitro propagationSomatic embryogenesisSynthetic seed

Encapsulation technology is an exciting and rapidly growing area of biotechnological research. This hasdrawn tremendous attention in recent years because of its wide use in conservation and delivery of tissuecultured plants of commercial and economic importance. Production of synthetic seeds by encapsulatingsomatic embryos, shoot buds or any other meristmatic tissue helps in minimizing the cost ofmicropropagated plantlets for commercialization and final delivery. In most of fruit crops, seed propagationhas not been successful because of heterozygosity of seeds, minute seed size, presence of reducedendosperm, low germination rate, and also some are having seedless varieties. Many species havedesiccation-sensitive intermediate or recalcitrant seeds and can be stored for only a few weeks or months.Under these circumstances, increasing interest has been shown recently to use encapsulation technology forpropagation and conservation. Many fruit plants are studied worldwide for breeding, genetic engineering,propagation, and pharmaceutical purposes. In this context, synthetic seeds would be more applicable inexchange of elite and axenic plant material between laboratories and extension centers due to small beadsize and ease in handling. Due to these advantages, interest in using encapsulation technology hascontinuously been increasing in several fruit plant species. The purpose of this review is to focus uponcurrent information on development of synthetic seeds in several fruit crops.

© 2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6712. Synthetic seed: concept, process and application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672

2.1. Advantages of synthetic seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6723. Development of synthetic seeds in some important fruit plant species: case studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673

3.1. Apple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6733.2. Banana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6743.3. Citrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6753.4. Grapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6753.5. Guava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6753.6. Mango . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6753.7. Mulberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6763.8. Papaya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6773.9. Pine apple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6773.10. Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677

4. Concluding remarks and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678

l rights reserved.

1. Introduction

Fruit crops are being cultivated since olden times as fruits are veryimportant dietary components. Fruits have essential roles in themaintenance of life due to their high nutritive value and are important

672 M.K. Rai et al. / Biotechnology Advances 27 (2009) 671–679

in running the economy of many peoples due to their commercialvalue. However, several biotic (diseases, insects, pests etc.) and abiotic(salt, drought, heat, cold etc.) stresses are constant factors that limitthe production of fruit plants. As many of the fruit plants are largetrees and have a prolonged juvenile phase, their improvementthrough traditional breeding methods is not possible (Jaiswal,2003). In most of the fruit crops, seed propagation has also not beensuccessful. This may be due to heterozygosity of seeds, minute seedsize, presence of reduced endosperm, low germination rate, and alsobecause some are having seedless varieties (Saiprasad, 2001).Conventional propagation methods such as grafting, air layering,stooling etc. for improving many fruit trees already exist but extendedjuvenility has made these techniques time consuming and cumber-some. Plant tissue culture offers an effective solution of such problemsof propagation of fruit crops. For improvement of fruit crops throughseveral biotechnological approaches, highly efficient regeneration is aprerequisite (Litz and Jaiswal, 1991).

Many fruit species particularly tropical and subtropical fruit plantshave characteristics that make it difficult to conserve them by usingtraditional methods. Many species have desiccation-sensitive inter-mediate (i.e. papaya, banana, citrus etc.) or recalcitrant seeds (i.e.mango, avocado, litchi, jackfruits etc.) and can be stored only for fromweeks to fewmonths. This is mainly because of their high metabolismand high incidence of fungal infection. In addition, collections ofgermplasm from field gene banks are exposed to natural disasters,attacks by pests and pathogens and labour costs are very high.Distribution and exchange from field gene banks are difficult becauseof vegetative nature of the material and greater risk of disease transfer(Chaudhury and Malik, 2003). To circumvent these problems,increasing interest has been shown recently to use encapsulationtechnology for conservation and germplasm exchange of thesespecies. This write up presents a brief overview of current status ofdevelopment of synthetic seeds in several fruit crops and their role inconservation and germplasm exchange.

2. Synthetic seed: concept, process and application

The concept of synthetic seed was given by Murashige (1977), butfirst report on the development of synthetic seeds was published byKitto and Janick (1982). They reported the production of desiccatedsynthetic seeds by coating a mixture of carrot somatic embryo in awater-soluble resin, polyoxyethylene glycol (Polyox). Later, Redenbaughet al. (1984) were successful in producing synthetic seeds for alfalfa byencapsulating somatic embryos with alginate hydrogel. Since thenseveral research groups have been working on synthetic seeds withdifferent plant species including cereals, fruits, vegetables, ornamentals,medicinal plants, forest trees and orchids (Bapat et al., 1987; Bapat andRao,1988;Mathur et al.,1989; Ganapathi et al.,1992; Corrie and Tandon,1993; Sharma et al.,1994;Maruyama et al.,1997; Sarkar and Naik,1998;Ara et al., 1999; Mandal et al., 2000; Sicurani et al., 2001; Rout et al.,2001; Nyende et al., 2003; Chand and Singh, 2004; Singh et al., 2006a,b;Naik and Chand, 2006; Micheli et al., 2007; Faisal and Anis, 2007; Raiet al., 2008a,b; Singh et al., 2009).

A synthetic seed or artificial seed is referred to as artificiallyencapsulated somatic embryo, shoot bud or any other meristmatictissue that can be used as functionally mimic seed for sowing andpossesses the ability to convert into a plant under in vitro or ex vitroconditions and that can retain this potential even after storage(Capuano et al., 1998; Ara et al., 2000). Earlier, the concept of syntheticseeds was based only on the encapsulation of somatic embryos thatcould be handled like a real seed for transport, storage and sowing,but, in recent years, the encapsulation of non-embryogenic vegetativepropagules like apical shoot buds, axillary buds, nodal segments, etc.have also been employed as a suitable alternative to somatic embryos(Sarkar and Naik, 1998; Standardi and Piccioni, 1998; Ara et al., 2000;Danso and Ford-Lloyd, 2003; Bapat and Mhatre, 2005; Rai et al.,

2008b). The main advantage of these non-embryogenic vegetativepropagules would be in those crops where either somatic embryo-genesis is not well established or do not produce uniform qualityembryos. In such cases synthetic seed system may be useful forpropagation and delivery of tissue cultured plants (Rao et al., 1998).

Based on technology established, there are two types of syntheticseeds: hydrated and desiccated. Although, the most studied methodinvolves the encapsulation of propagules in hydrogel for syntheticseed production (Redenbaugh andWalker, 1990). A number of coatingagents such as sodium alginate, potassium alginate, carrageenan,sodium alginate with gelatin, sodium pectate, carboxymethyl cellu-lose etc. are used for encapsulation and among these substancessodium alginate has been extensively used (Redenbaugh et al., 1987;Rao et al., 1998; Ara et al., 2000). Due to absence of a nutritive tissuelike the endosperm of the natural seed, synthetic seeds have lowconversion ability in some cases (Arun Kumar et al., 2005). Addition ofnutrients, carbon sources, growth regulators and antimicrobial agentssuch as antibiotics, fungicides etc. in the gel matrix which apparentlyserved as a synthetic endosperm, facilitated growth and survival ofencapsulated propagules (Redenbaugh et al., 1987; Gray, 1990;Bapat and Mhatre, 2005). Such additives should be non-toxic topropagules and allow the development of plants without any variation(Redenbaugh and Ruzin, 1989; Bapat andMhatre, 2005). Hindrance ofthe gel capsule for the emergence of the root and shoot fromencapsulated propagule is another mechanical problem in encapsula-tion technology, although, adopting the self-breaking alginate gelbeads technology could overcome this shortcoming (Onishi et al.,1994). Calcium alginate capsule pretreated with potassium nitratebecomes soften and allow the easily emergence of shoot and root fromalginate beads (Onishi et al., 1994). Application of potassium nitrate inthe breaking of alginate capsule has also been reported in a few plantspecies (Guerra et al., 2001; Arun Kumar et al., 2005).

To produce hydrated synthetic seeds, the propagules (somaticembryos, shoot buds, nodal segments etc.) are carefully isolated fromin vitro cultures and mixed with encapsulation mixture [sodiumalginate (0.5–5.0% w/v) prepared either in double distilled water orliquid nutrient medium] and dropped into a complexing agent such ascalcium chloride or calcium nitrate solution (30–150 mM). After 30–40min incubation in calcium chloride solution, alginate beads becomehard. An ion-exchange process takes place during this period resultingin the replacement of sodium ions by calcium ions forming calciumalginate (Redenbaugh and Walker, 1990; Ara et al., 2000). Hardeningof calcium alginate bead is affected by the concentration of sodiumalginate and calcium chloride and it may vary with differentpropagules as well as with different plant species. After washingwith sterile double distilled water such encapsulated propagules arecultured on nutrient medium or different substrates like wet filterpaper, cotton or soilrites with medium or double distilled water forplantlet conversion.

2.1. Advantages of synthetic seeds

Encapsulation technology is an exciting and rapidly growing areaof seed biotechnological research. It has considerable impact onconservation and delivery of tissue cultured plants in a moreeconomical and convenient way (Rao et al., 1998). The scope ofsynthetic seeds is presented in Fig. 1. It is an excellent technique forpropagation of rare hybrids, elite genotypes, genetically engineeredplant, and rare and endangered plants for which the seeds are eithervery expensive or are not available (Mandal et al., 2000). As theproduction costs for hybrid seeds (in vegetable, forage, cereals orcommercially important plants) or other conventional propagationmethods are very high, synthetic seeds should offer a low costalternative (Rao et al., 1998). Some other potential advantages ofencapsulation technology include ease in handling (due to small sizeof capsule), genetic uniformity of plants and direct delivery to the field

Fig. 1. Scope of synthetic seeds.

673M.K. Rai et al. / Biotechnology Advances 27 (2009) 671–679

or green house (Maruyama et al., 1997). Synthetic seeds can be madeavailable throughout the year whereas most of tree plants produceseeds only in certain months of the year (Bapat and Mhatre, 2005).

Many commercially important plants particularly cereals, fruits,medicinally important plants are studied worldwide for breeding,genetic engineering, propagation, and pharmaceutical purposes. Inthis context, the most important application of synthetic seeds forthese plants could be in exchange of elite and axenic plant materialbetween laboratories due to small bead size and relative ease ofhandling these structures (Hasan and Takagi, 1995; Danso and Ford-Lloyd, 2003; Naik and Chand, 2006; Rai et al., 2008b).

Conservation is an important aspect of encapsulation technology.In vitro conservation involves the maintenance of explants in apathogen-free environment for short to medium or long-term(Engleman et al., 2003). For short- and medium term storage, theaim is to increase the interval between subcultures by reducinggrowth. This is achieved by modifying the environmental conditionsand/or the culture medium. This method is also referred to as slow-growth conservation (Engelmann, 1997). Various approaches havebeen applied for slow-growth maintenance of cultures (Gupta andMandal, 2003). These include

– Maintenance under reduced temperature and/or reduced lightintensity

– Use of growth retardants such as ABA (Abscisic acid)– Use of minimal growth medium (restrict the growth of cultures by

alteration of mineral content and/or sucrose in medium)– Use of osmoticum (i.e. mannitol, sorbitol, high sucrose concentra-

tions etc.)– Reduction in oxygen concentration– Combination of more than one treatment.

Long-term storage of synthetic seeds can be achieved throughstorage at ultra-low temperature, termed as cryopreservation, isusually carried out by using liquid nitrogen (−196 °C). At thistemperature, all cellular division and metabolic processes aresuspended, hence, plant material can be stored for unlimited period(Engelmann, 1997). In recent years, several new cryopreservationtechniques have been developed. Among these, encapsulation–dehydration and encapsulation–vitrification procedures are basedon the technology developed for the production of synthetic seeds. In

encapsulation–dehydration method, explants are encapsulated inalginate beads, pregrown in liquid medium enriched with sucrose for1–7 days, partially desiccated in the air current of a laminar air flowcabinet or with silica gel, then frozen rapidly (Engleman, 2003). Thistechnique has been applied in several plant species such as sugarcane(Paulet et al., 1993), grapevine (Wang et al., 2000), apple (Paul et al.,2000), citrus (Gonzalez-Arnao et al., 2003), Strawberry (Clavero-Ramirez et al., 2005). Encapsulation–vitrification is a combination ofencapsulation–dehydration and vitrification, in which explants areencapsulated in alginate beads, treated with cryoprotective sub-stances, followed by dehydrationwith vitrification solutions, and thensubjected to freezing (Engleman, 2003). This technique has also beenapplied in strawberry (Hirai et al., 1998), potato (Hirai and Sakai,1999), cassava (Charoensub et al., 2004). In recent years, cryopre-servation is routinely employed for long-term conservation of plantgenetic resources in a number of cases, however, the possibility ofcultures losing their genetic integrity through somaclonal variation isan important drawback (Engleman et al., 2003).

3. Development of synthetic seeds in some important fruit plantspecies: case studies

During last three decades, large numbers of reports werepublished regarding the development of synthetic seeds and theirapplication in propagation and conservation of several fruit crops(Tables 1 and 2).

3.1. Apple

Apple (Malus sp., family - Rosaceae) is an important temperatefruit crop and it is second in production among temperate fruit cropsgrown worldwide. A large number of currently grown commercialapple cultivars are hybrid selection and have been derived as chanceseedling (Korban and Chen, 1992). Encapsulation technology wouldbe providing a possibility of propagation and conservation of selectedrootstocks of apple for further breeding programs.

In order to be considered usable as synthetic seeds in Applerootstock M.26 (Malus pumila), different explants were encapsulatedin alginate beads (Piccioni and Standardi, 1995; Piccioni, 1997;Capuano et al., 1998; Sicurani et al., 2001; Brischia et al., 2002). As

Table 1Development of synthetic seed in some fruit plant species.

Plant species Propagules encapsulated References

Actinidia deliciosa (Kiwifruit) Apical and axillary buds Adriani et al. (2000), Romay Alvarez et al. (2002)Ananas comosus (Pine apple) Axillary buds Soneji et al. (2002), Gangopadhyay et al. (2005)Carica papaya (Papaya) Somatic embryos Castillo et al. (1998)Citrus sp. Somatic embryos Antonietta et al. (1999, 2007)Malus pumila (Apple rootstock M.26) Apical and axillary buds, Nodes, Shoot tips Piccioni and Standardi (1995), Piccioni (1997), Capuano et al. (1998),

Sicurani et al. (2001), Brischia et al. (2002)Mangifera indica (Mango) Somatic embryos Ara et al. (1999)Morus indica (Mulberry) Axillary buds Bapat et al. (1987), Bapat and Rao (1990), Machii (1992), Pattnaik et al. (1995),

Pattnaik and Chand (2000), Kavyashree et al. (2006)Musa sp. (Banana) Shoot tips, Shoot apices, Somatic embryos Ganapathi et al. (1992), Rao et al. (1993), Suprasanna et al. (2001),

Ganapathi et al. (2001)Pistacia vera (Pistachio) Somatic embryos and embryogenic mass Onay et al. (1996)Psidium guajava (Guava) Somatic embryos, Shoot tips, Nodal segments Akhtar (1997), Rai and Jaiswal (2008), Rai et al. (2008a,b,c)Punica granatum (Pomegranate) Nodal segments Naik and Chand (2006)Pyrus communis (Pear) Shoot tips Nower et al. (2007)Rubus idaeus (Raspberry) Shoot buds, In vitro derived microcutting Piccioni and Standardi (1995), Romay Alvarez et al. (2003)Vitis vinifera (Grape) Somatic embryos Das et al. (2006)

674 M.K. Rai et al. / Biotechnology Advances 27 (2009) 671–679

compared to non-encapsulated M.26 apple rootstock, single nodesencapsulated in alginate beads immediately after a 24-hour treatmentwith IBA (indole-3-butyric acid) and sucrose in dark produced muchlower percentage of plantlet conversion (Piccioni, 1997). However,addition of growth regulators to the artificial endosperm and cultureof the single nodes for root primordia initiation for different days indarkness before encapsulation allowed production of comparativelyhigher percentage of plantlet conversion (Piccioni, 1997). In order toachieve a time and hand-labor saving, cost effective procedure andselection of suitable explants for encapsulation, Sicurani et al. (2001)and Brischia et al. (2002) compared the performance of encapsulatedorganogenetic explants of M.26 apple rootstock that were prepared byhand and mechanical manipulation. Conversion of synseed intoplantlets was 25% from encapsulated hand-cut uninodal microcut-tings and 11% from machine-ground explants (Brischia et al., 2002).These type of research provided useful information to devise amechanical protocol for the production of synthetic seed through

Table 2Reports on synthetic seed based in vitro conservation of some fruit plants.

Plant species Plant material Conservation method (Slow

Actinidia deliciosa (Kiwifruit) ST Encapsulation–dehydrationAnanas comosus (Pine apple) SBs Low temp. storage

ST Encapsulation–dehydrationSA Encapsulation–vitrification

Citrus sp. SEs Low temp. storageSEs Encapsulation–dehydrationEA Encapsulation–dehydrationSEs Low-temp. storage

Fragaria ananassa (Strawberry) M Encapsulation–vitrificationST Low-temp. storage

Malus domestica ST Encapsulation–dehydrationMalus sp. ST Encapsulation–dehydrationMalus pumila (Apple rootstock M.26) ST Encapsulation–dehydrationMangifera indica (Mango) SEs Encapsulation–dehydrationMorus indica (Mulberry) ABs Low-temp. storage

ST Encapsulation–dehydrationPsidium guajava (Guava) SEs, ST Use of growth retardent (AB

growth medium, Low-tempPunica granatum (Pomegranate) NS Low-temp. storagePyrus communis (Pear) ST Encapsulation–dehydrationRubus idaeus (Raspberry) ST Low-temp. storageVitis vinifera (Grape) ST Encapsulation–dehydration

ABs Encapsulation–dehydrationECS Encapsulation–dehydration,SEs Use of growth retardent (AB

ABs—axillary buds, EA—embryonic axes, ECS—embryogenic cell suspension, M—meristem, NStips.

encapsulation of differentiating propagules inwoody species (Sicuraniet al., 2001).

3.2. Banana

Banana (Musa sp., family-Musaceae) is one of the most importantfruit crops of the world. It is mostly propagated vegetatively bysuckers but the number of suckers produced in a year is limited.Hence, tissue culture based propagation of banana using shoot tipexplants has become a practice all over the world (Ganapathi et al.,2001). During past years, considerable efforts have been made forplant regeneration through synthetic seed in banana (Ganapathi et al.,1992, 2001; Suprasanna et al., 2001).

Shoot tips isolated from multiple shoot cultures of banana cv.Basrai were encapsulated in 3% sodium alginate containing differentgel matrices (Ganapathi et al., 1992). The encapsulated shoot tipsregenerated in vitro on different media as well as substrates such as

-growth/cryopreservation) References

Suzuki et al. (1994), Bachiri et al. (2001)Soneji et al. (2002), Gangopadhyay et al. (2005)Gonzalez-Arnao et al. (1998b)Gamez-Pastrana et al. (2004)Antonietta et al. (1999, 2007)Gonzalez-Arnao et al. (1998a, 2003)Malik and Chaudhury (2006)Singh et al. (2007)Hirai et al. (1998)Lisek and Orlikowska (2004)Niino and Sakai (1992)Zhao et al. (1999)Paul et al. (2000)Wu et al. (2003)Bapat et al. (1987), Pattnaik et al. (1995),Pattnaik and Chand (2000), Kavyashree et al. (2006)Niino and Sakai (1992), Niino et al. (1992)

A), Use of minimal. storage,

Rai et al. (2008a,c)

Naik and Chand (2006)Niino and Sakai (1992)Lisek and Orlikowska (2004)Plessis et al. (1993)Zhao et al. (2001)

Encapsulation–vitrification Wang et al. (2002, 2004)A) Das et al. (2006)

—nodal segments, SA—shoot apices, SBs—shoot buds, SEs—somatic embryos, ST—shoot-

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filter paper, cotton and soilrite. Use of White's medium resulted inmaximum conversion of encapsulated shoot tips into plantlets.However, encapsulated shoot tips turned brown and severe bacterialinfection was also noted. Addition of 0.1% activated charcoal and theantibiotic mixture comprising of rifampicin, cefatoxime and tetra-cycline to the gel matrix circumvented this problem. Transportationand exchange of banana germplasm by encapsulated shoot tips ratherthan suckers would be easier, relatively inexpensive and safer.Suprasanna et al. (2001) also compared the eight banana and plantain(Musa sp.) cultivars for their regeneration through encapsulated shootapices. Cultivars (AAB, ABB and AB) displayed differences in theirregrowth after encapsulation and cultivars with the B genomesexhibited better response. Ganapathi et al. (2001) have been reportedfirst time encapsulation of somatic embryos of banana cv. Rasthali(AAB genomic group) to produce synthetic seeds. The frequency ofconversion of encapsulated embryos into plantlets varied consider-ably on different gel matrices and substrates used for plantdevelopment. Maximum conversion into plantlets from encapsulatedembryos was achieved when somatic embryos encapsulated in 5%sodium alginate and cultured on MS basal medium.

In vitro conservation of different banana cultivars using minimalgrowth medium (Banerjee and Langhe, 1985) and different methods ofcryopreservation (Abdelnour-Esquivel et al., 1992; Panis et al., 1990;Panis et al., 1996; Cote et al., 2000) have been reported, however,encapsulation technology based conservation of banana is not available.

3.3. Citrus

Citrus (family-Rutaceae) is most widely cultivated fruit crop oftropical and subtropical regions of the world. The juice of Citruscontains a high quantity of citric acid and are good sources of vitaminC and flavonoids.

Antonietta et al. (1999) attempted the production of synthetic seedsin Citrus reticulata Blanco through alginate-encapsulation of somaticembryos. As compared to non-encapsulated or encapsulated with agrowth-regulator free artificial endosperm, somatic embryos encapsu-latedwith an artificial endosperm containing GA3 (Gibberellic acid) hasgreater ability to plantlet conversion on agar medium. Encapsulationwith GA3 was also useful for storage of somatic embryos at 4 °C for onemonth without loss of their conversion ability. Singh et al. (2007)examined the effect of different storage conditions on conversion ofencapsulated and non-encapsulated somatic embryos of Kinnowmandarin. Different batches of encapsulated and non-encapsulatedembryoswere preserved at room temperature, 4 °C, in liquid nitrogen assuchandbyembedding in liquidparaffin. Plant recoveryof encapsulatedand non-encapsulated embryos was 58.33 and 51.38%, respectivelyfollowing storage for 7 months in liquid nitrogen. Recently, Antoniettaet al. (2007) evaluated the effect of the germicide PPMand the fungicideThiophanate-methyl on the performance of encapsulated somaticembryos during storage for different duration. They noted thatencapsulated somatic embryos can be stored at 4 °C, but not for aperiod longer than 60 days. The addition of Thiophanate-methyl in theartificial endosperm resulted in high levels of sprouting with promisingfrequencies of root development and conversion in to plantlets underin vitro and ex vitro conditions. In vitro conservation of several Citrusspecies using encapsulation–dehydration method of cryopreservationhas also been reported (González-Arnao et al., 1998a, 2003; Malik andChaudhury, 2006).

3.4. Grapes

Grapes (Vitis spp., family-Vitaceae) are one of the most economic-ally important fruit crops worldwide. Grape is heterozygous andexhibits pronounced inbreeding depression and have many seedlessvarieties (Gray andMeredith,1992). Therefore, an individual genotype

cannot be reproduced by seed. Thus encapsulation technology may bean appropriate method for propagation and conservation of grapes.

Successful plantlet regeneration from encapsulated somaticembryos of grape (Vitis vinifera L.) was reported by Das et al. (2006).Cotyledonary-stage somatic embryos originating from leaf explantswere encapsulated individually in 2% alginate gel and encapsulatedsomatic embryos converted into plantlets successfully on 0.7% agarmedium containing B5 macrosalts (half strength), MS microsalts (fullstrength), 3% sucrose and 2.9 µM gibberellic acid. Transferring theembryos onto the full-strength B5 medium containing ABA (0.04 µM)for 4–6weeks prior to encapsulation resulted in extended storage of upto 90 days without loss of the conversion potential. In grapes, thepotential of using encapsulation–dehydration and encapsulation–vitrification method of cryopreservation for conservation have alsobeen explored by several workers (Plessis et al.,1993; Zhao et al., 2001;Wang et al., 2002, 2004).

3.5. Guava

Guava (Psidium guajava L., family-Myrtaceae) is a popular fruitcrop of tropical and subtropical regions of the world and important ininternational trade and domestic economy of several countries. Guavafruit is an excellent source of vitamin-C and traditionally, differentparts of plant are used in local medicine to treat gastroenteritis,diarrhoea, and dysentery (Jaiswal and Amin, 1992; Yadava, 1996; Raiet al., 2008a,b).

Synthetic seed production in guava has been reported by severalresearchers using somatic embryos, shoot tips and nodal segments(Akhtar, 1997; Biswas et al., 2007; Rai and Jaiswal, 2008; Rai et al.,2008a,b,c). Torpedo stage somatic embryos induced on zygoticembryos (Rai et al., 2007) were encapsulated in 2% sodium alginateand 100mMcalcium chloride and cultured on appropriatemedium forplant regeneration (Akhtar,1997). Maximumplantlet conversion fromencapsulated somatic embryos was observed on growth regulator freefull-strength MS medium. Recently, Rai et al. (2008a,b) have alsoemployed the encapsulation of vegetative propagules (shoot tips andnodal segments) for the development of synthetic seeds in guava.Superiority of liquid medium over semisolid medium in plantletconversion fromencapsulated shoot tips and nodal segments have alsobeen demonstrated (Rai et al., 2008a,b).

Although guava have orthodox seeds (Doijode, 2001), but guava ishighly heterozygous crop, hence, seed conservation is anun-appropriatemethod. Recently, Rai et al. (2008a,c) has been reported the short-termconservation of guava using encapsulation technology through slow-growthprocedure. AtABA (1mgl−1) orhigh sucrose (9%) containingMSmedium, most of encapsulated somatic embryos (about 90%) remainedin quiescent state similar to dormancy of zygotic embryos (Rai et al.,2008c). These encapsulated somatic embryos converted into plantletsafter transfer to medium containing 3% sucrose. The temporarysuppression in conversion of encapsulated somatic embryos by ABA orhigh sucrose offers a possibility of conservation of elite genotype ofguava for short-period. Rai et al. (2008a) compared the rate of retrievalof plantlets from encapsulated shoot tips stored at low temperature(4 °C) and room temperature under minimal growth medium (sucroselacking medium) for different days. They noted that storage ofencapsulated shoot tips under minimal growth medium was betterapproach than storage at low temperature (4 °C) for conservation ofguava. Encapsulation of individual somatic embryos and regeneration ofplantlets of guava from encapsulated shoot tips are presented in Figs. 2and 3.

3.6. Mango

Mango (Mangifera indica L., family-Anacardiaceae) is one of theimportant fruit crops of tropical and subtropical regions of the world.The juicy pulp of mango is a good source of provitamin-A (Ara et al.,

Fig. 2. Individual guava somatic embryos encapsulated in Ca-alginate beads ready forculture.

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2004). Due to its popularity and importance it is often referred to as‘the king of fruits’ in tropical world (Krishna and Singh, 2007).

Ara et al. (1999) reported complete plant regeneration fromencapsulated somatic embryos ofmango cv. Amrapali. Somatic embryos,originated from nucellus explants, were encapsulated in 2% sodiumalginate and 100 mM calcium chloride solution. The encapsulatedsomatic embryos converted into plantlets successfully on 0.6% agar-gelledmedium containing B5macrosalts (half strength),MS (Murashigeand Skoog, 1962) microsalts (full-strength), 3% sucrose and 2.9 mMgibberellic acid. Conversion of encapsulated somatic embryos intoplantlets was affected by addition of different strength of B5 macrosaltsin medium and maximum conversion was achieved on mediumcontaining full-strength B5 macrosalts. The percentage of conversion ofencapsulated somatic embryos was higher than that of naked somaticembryos of the same size on the same medium.

Fig. 3. Plant regeneration from encapsulated shoot tips of guava. (A) Shoot tips encapsulated(C) Plantlet regeneration on agar-solidifiedMSmedium. (D) Plantlet regeneration in full-strength

Mango seeds are highly recalcitrant and cannot be stored forlonger duration. Tissue culture may be an ideal approach forconservation of germplasm of mango (Krishna and Singh, 2007).Supplementation of ABA (0.004 or 0.02 mM) in medium had nosignificant influence on conversion of encapsulated somatic embryosand plantlet development, but caused a 3-week delay in germination(Ara et al., 1999). Hence, by using this approach, somatic embryos ofmango could be conserved for short period. Wu et al. (2003)compared three cryopreservation protocols for embryogenic cultures:encapsulation–dehydration, pregrowth-dehydration and vitrification.Best result was obtained using vitrification method when about 94%recovery of embryogenic masses was achieved after cryopreservation.However, no recovery was acquired after cryopreservation using theencapsulation/dehydration technique, whereas, limited recovery(8.3%) was achieved with the pregrowth/dehydration technique.

3.7. Mulberry

Mulberry (Morus sp., family - Moraceae) plays a significant role inthe silk industry due to its foliage, which constitute the chief food forthe silkworm. Some species are valued for their sweet edible fruits oruseful timber. Conventionally mulberry is propagated through stemcuttings or by grafting. However, several cultivars of mulberry aredifficult to root and this impedes the propagation. Under thesecircumstances, encapsulation technology would be a potential toolfor a more efficient and cost-effective rapid clonal propagation system(Kavyashree et al., 2006). Since somatic embryogenesis has not yetbeen documented in mulberry, the possibility of using the non-embryogenic vegetative propagules for encapsulation have beenexplored by several workers (Bapat et al., 1987; Bapat and Rao, 1990;Pattnaik et al., 1995; Pattnaik and Chand, 2000; Kavyashree et al.,2006). Bapat et al. (1987) demonstrated the superiority of alginateover agar for the preparation of gel matrix. Addition of a fungicide to

in Ca-alginate beads. (B) Shoot and root emergence from alginate-encapsulated shoot tip.liquidMSmedium. (E)Well-developed plantlets regenerated from encapsulated shoot tips.

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the alginate beads prevents contamination of the buds and increasedsurvival of the buds when encapsulated in an alginate matrix undernon-aseptic conditions and sown in soil (Bapat and Rao, 1990).Pattnaik et al. (1995) attempted the production of synthetic seed indifferent varieties of mulberry through encapsulation of axillary budsobtained from mature plants in calcium alginate beads. Axillary budswere encapsulated in 4% sodium alginate and 75mMcalcium chloride.Maximum plantlet conversion was achieved on semi-solid MSmedium, especially when buds were pretreated with 1.0 mg l−1, BA(6-benzylaminopurine) for 36 h. The encapsulated buds could bestored for 60 days at 4 °C without loss of viability only when the gelmatrix contained MS nutrients, vitamins and sucrose. Morphogenicresponses of encapsulated axillary buds of six mulberries (Morus albaL.,M. australis Poir.,M. bombycis Koidz.,M. cathyana Hemsl.,M. latifoliaPoir., and M. nigra L.) on various planting media have also beenevaluated by Pattnaik and Chand (2000). Of the six Morus speciesstudied by Pattnaik and Chand (2000), the best shoot and rootdevelopment from encapsulated buds was achieved in M. alba.Encapsulated buds of M. bombycis, M. latifolia and M. nigra could bestored for 90 days, whereas M. alba, M. australis and M. cathyana onlyfor 60 days at 4 °C.

3.8. Papaya

Papaya (Carica papaya L., family-Caricaceae) is a common plant inthe gardens and dooryards of the lowland tropics, where it is popularbecause of its habit of bearing fruit continuously throughout year.Papaya is a good source of vitamin C and A (Manshardt, 1992). Papayais a cross-pollinated and typically seed-propagated crop (Castillo et al.,1998). Therefore it is particularly a suitable candidate for encapsula-tion technology.

Cotyledonary stage somatic embryos induced on zygotic embryoexplant were encapsulated in sodium alginate and calcium chloride toproduce synthetic seeds in papaya (Castillo et al., 1998). The frequencyof regeneration from encapsulated embryos was significantly affectedby the concentration of sodium alginate, the presence or absence ofnutrient salts in the capsule, and the duration of exposure to calciumchloride. A 2.5% sodium alginate prepared in liquid half-strength MSmedium and relatively short (10 min) exposure to calcium chloridewere optimum conditions for uniform encapsulation of embryos andconversion of encapsulated somatic embryos of papaya (Castillo et al.,1998). Encapsulation technology based conservation either usingcryopreservation or slow-growth procedure has not been reported inpapaya.

3.9. Pine apple

Pineapple (Ananus comosus L., family Bromeliaceae) is one of themost economically important tropical fruit crops. Conventionally,pineapple is propagated by propagules such as suckers, slips and crowns(Soneji et al., 2002). An effective means of propagating pineapplethrough synthetic seeds would be advantageous over conventional useof sucker, slips and crowns for germplasm maintenance, exchange andtransportation.

Soneji et al. (2002) employed axillary buds of pineapple forencapsulation and production of synthetic seeds. Axillary buds isolatedfrom the tuft of multiple shoots were encapsulated in 3% sodiumalginate prepared using hormone-free MS basal medium, MS vitamins,0.56 mM myo-inositol and 0.06 M sucrose. Maximum conversion ofsynthetic seeds to plantlets occurred after pretreatment of shoots inliquidmediumcontaininggrowth regulators for 12h followedbycultureof synthetic seeds on an appropriate medium. Synthetic seeds stored at4 °C remained viable without sprouting for up to 45 days. Later,Gangopadhyay et al. (2005) also reported the encapsulation of micro-shoots in alginate beads for short-term storage of pineapple. They triedthe storage of synthetic seeds at four different temperatures (4, 8,12 and

16 °C) and found maximum percentage of shoot proliferation whenplaced again in MSmedium after storage at 8 °C. The rate of conversionwas satisfactoryevenbeyond45days of incubation. For the conservationof pineapple, encapsulation technology based cryopreservation (encap-sulation–dehydration and encapsulation–vitrification) have also beenimplemented by several researcher (Gonzalez-Arnao et al., 1998b;Gamez-Pastrana et al., 2004).

3.10. Pomegranate

Pomegranate (Punica granatum L., family - Lythraceae) is amedicinally important fruit tree of the tropics. The potential ofnutrient-alginate encapsulation of axenic nodal segments of pome-granate for encapsulation technology carried out by Naik and Chand(2006). Nodal segments from in vitro shoot cultures derived frommature nodal explants or axenic cotyledonary nodes were encapsu-lated in calcium alginate hydrogel containing MS medium supple-mented with 4.44 mM benzyladenine (BA) and 0.54 mM α-naphthalene acetic acid (NAA). Of various concentrations of sodiumalginate (1–6%) and the complexation solution of calcium chloride(50–125 mM), a combination of 3% sodium alginate and 100 mMcalcium chloride was most suitable for formation of ideal syntheticseeds. Of the different planting media evaluated, percent sprouting(shoot development) was highest in MS medium augmented with4.44mMBA and 0.54mMNAA. Encapsulated nodal segments could bestored up to 30 days at 4 °C.

Besides, development of synthetic seed have also been reported insome other fruit crops such as Actinidia deliciosa (Adriani et al., 2000;Romay Alvarez et al., 2002), Pistacia vera (Onay et al., 1996), Pyruscommunis (Nower et al., 2007), and Rubus idaeus (Piccioni andStandardi, 1995; Romay Alvarez et al., 2003) etc.

4. Concluding remarks and future prospects

Considerable progress has been made in the recent past of in vitropropagation via synthetic seeds in several fruit species. Encapsulationtechnology offers tremendous scope for the conservation and germ-plasm exchange of several fruit plants. Despite these advantages, directsowing of synthetic seeds in the field for commercial use remains alimitation of synthetic seed application due to low soil survival (Junget al., 2004). In many plant species either embryogenic system does notexist or produce low quality somatic embryos. Asynchrony of somaticembryos is an important hurdle to be overcomebefore advances leadingto extensive commercialization of synthetic seeds can occur (Saiprasad,2001). Therefore, refinements in protocols are necessary to get highquality somatic embryos to improve the propagation system throughsynthetic seeds. In those caseswhere synthetic seed has been developedfrom encapsulation of non-embryogenic vegetative propagules, for thedevelopment of simpler and cost-effective method of synthetic seed,explants should taken directly from field-grown plant because it isdesirable to raise cultures frommature plants with known features anddesired traits. However, inmost cases, in vitro raised plantlets were usedas the source of explant for encapsulation because explants frommaturetrees exhibit recalcitrance under aseptic conditions. In some plantspecies poor conversion of encapsulated propagules into plants isanother major problem and still remains one of the factors limitingcommercial application of this technology.Manipulation ofmediumandaddition of correct formulation of medium, growth regulators, carbohy-drate sources, antibiotics and fungicides in the synthetic endosperm canhelp to enhance the conversion frequency of encapsulated propagulesand requires detailed studies. Low conversion after conservation andoccurrence of somaclonal variations resulting fromcryopreservation arealso important features to be considered. Modification in slow-growthand cryopreservation protocols could resolve these bottlenecks.

Encapsulation technology is a promising technique for conserva-tion and transport of transgenic plants, non-seed producing plants,

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elite traits and plant lines with problems in seed propagation(Saiprasad, 2001), however, further detailed research is neededmainly for improvement in conversion of synthetic seeds andsubsequent plant growth in soil and above pointed all limitationsshould be taken into considerations while working on synthetic seedsparticularly in woody fruit trees.

Acknowledgement

Financial assistance provided by the Council of Scientific andIndustrial Research (CSIR), New Delhi, to the authors (MKR and PA), isgratefully acknowledged.

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