Introduction

Production of seed occurs as an outcome of a sexual procedure; on that account, in cross-pollinating species the naturally produced seeds are genetically different to individual parent (Senaratna 1992). A distinctive feature of seeds is its potential to secure dormancy by undergoing intense self-drying which allows it to be preserved for a long period of time. Nevertheless, several tropical and subtropical plants with pharmaceutical value consist of certain distinctiveness, thus making it complicated to conserve through conventional methods. A large number of medicinal plant species bear desiccation-sensitive or recalcitrant seeds that confine the storage duration only up to few weeks or months. Apart from that, high metabolic activities and high incidence of pathogenic infestation play significant role in reducing the storage span. Furthermore, the vulnerability of germplasm, while collecting from field gene banks, increases since they are subjected to attacks by pests and pathogens or to natural catastrophes. It is due to the vegetative nature of the germplasm that distribution and exchange of the same become inconvenient and a substantial threat of disease transmission prevails (Chaudhury and Malik 2003). Recently, an escalating interest has shown to be perceived on the usage of synthetic seed produced via encapsulation technology (Fig. 1) that is quite an impeccable route for safer conservation and exchange of the species. For the preservation and large-scale micropropagation of exclusive rare hybrids, choice genotypes and sterile unsteady genotypes, as well as genetically modified plants with non-availability of seeds or that which demands a mycorrhizal–fungal liaison for their germination, synthetic seed technology has opened new direction with huge opportunity. Of late, the interest of researchers has been focused on the encapsulation technology for the delivery of germplasm and for other assorted analytical studies (Ara et al. 2000). Although, the primary account on the formation of artificial seed was reported by Kitto and Janick (1982), the concept of the same was specified by Murashige (1977). The innovation of desiccated synthetic seeds through application of water-soluble resin coat and polyoxyethylene glycol over carrot somatic embryo (SE) was reported by Kitto and Janick (1982). Later on, Redenbaugh et al. (1984) successfully produced synthetic seeds for alfalfa through encapsulation of SEs with alginate hydrogel. Ever since, quite a number of research groups have been reported to progress work upon synthetic seeds for various plants counting cereals, fruits, ornamentals, medicinal plants, vegetables, trees and orchids (reviewed by Rai et al. 2009).

Fig. 1
figure 1

Different stages of synthetic seed development and its regeneration in Rauvolfia serpentina, an important medicinal plant. a Effect of sodium alginate level (in 100 mM calcium chloride) on size, shape and consistency of beads (bar 5 mm), b in vitro-regenerated multiple shoots serving as a source of explants for encapsulation (bar 3 mm), c polymerization of encapsulated explants (bar 5 mm), d encapsulated shoot tip, microshoot, multiple shoot and nodal segment in 3 % (w/v) sodium alginate and 100 mM calcium chloride (bar 3 mm), e germinating synthetic seed developed from encapsulation of nodal segment (bar 2 mm), f germinated synthetic seed with well developed shootlets (on plant growth regulator-free MS medium) (bar 5 mm) (source: unpublished photos of S. Gantait and S. Kundu)

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In spite of the fact that the technique for artificial seed production has been reported for more than three decades, yet the same has not been developed amply for the medicinal plants. Rare and threatened medicinal plants may be safeguarded from various adversities by the implementation of encapsulated shoot tip (ST) and nodal segment (NS). The key imperfections in conventional propagation of a few species with medicinal value are: diminished endosperm, low germination rate and seedless varieties (Saiprasad 2001). Quite a number of taxa are desiccation-sensitive or possess recalcitrant seeds for which they only can be stored for a limited time (Rai et al. 2008). The exigency for the production of artificial seeds as a technique, which blends the benefits of clonal propagation with those of seed propagation and storage, can be inferred from the above-mentioned reasons. To have an edge over all these issues, considerable attention has been paid to apply encapsulated SEs in clonal propagation and short-term storage (reviewed by Kikowska and Thiem 2011).

However, there are several factors that substantially influence the success of encapsulation of plant materials to produce synthetic seeds, their storage and regeneration. The factors range from initial choice of explants or plant materials, encapsulating agent and matrix. This review gives a complete illustration of existing scenario of synthetic seed development in several plants with medicinal importance as influenced by the above-mentioned factors and their prospects in conservation and germplasm exchange. The details of the factors those play critical role in synthetic seed development and regeneration of medicinal plants are summarized in Table 1.

Table 1 Artificial seed production at their optimal conditions for different medicinal plant species (in chronological order and alphabetical order within each year)
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Selection of plant materials

The technique for production of artificial seed of medicinal plants includes encapsulation of plant material (approximately 3–5 mm long). As explants, a variety of unipolar vegetative propagules, microcuttings, differentiating aggregates or bipolar vegetative propagules such as SE and protocorm-like body (PLB) were successfully exploited, for the development of synthetic seeds. At the commencement, for artificial seed production, SEs were exploited as plant material. Substantial endeavors have achieved encapsulation of both embryogenic and non-embryogenic in vitro-derived plant material (Gantait et al. 2012; Gantait and Sinniah 2013), in general, over the past several years (as reviewed by Ara et al. 2000; Lambardi et al. 2006; Sharma et al. 2013). However, the present review gives updated accounts chiefly since last two decades on selection of planting materials and their influence on encapsulation of typical medicinal plants, in particular (Table 1).

Somatic embryos

The mechanism by virtue of whichever haploid or diploid cells turn to plants, experiencing distinctive embryonic phases, devoid of the union of gametes, is stated as somatic embryogenesis (Williams and Maheswaran 1986). The procedure may possibly be direct with the direct development of embryogenic cells from explanted cells, or else indirect with a transitional callogenesis period (Merkle et al. 1990). Since an SE has the competence to possess shoot and root poles simultaneously, it is considered to be a bipolar structure (Standardi and Piccioni 1998). It is because of its bipolar nature that amidst a range of assorted propagules, SE has been triumphant in establishing the fact that it is the best suited entity for synthetic seed production. Since, SEs possess the efficiency to be formed clonally in bulk, that makes them desirable items for encapsulation. Synthetic seeds, comprising SE surrounded in a defensive covering, was suggested as a low-cost, high-capacity propagation technique (Redenbaugh 1990). Keeping in view the remarkable advantages that SEs possess over auxiliary propagules the same was fruitfully exercised for synthetic seed development in scores of plant species. The prospective and employment of SE in development of synthetic seeds has been reviewed and presented in Table 1. SE was effectively utilized for synthetic seed production in several medicinal plant species, for instance, Musa spp. (Ganapathi et al. 2001), Quercus robur (Prewein and Wilhelm 2003), Rotula aquatic (Chithra et al. 2005); Pinus patula (Malabadi and van Staden 2005), Arnebia euchroma (Manjkhola et al. 2005); Citrus nobilis × C. deliciosa (Singh et al. 2007), Vitis vinifera (Nirala et al. 2010), Dalbergia sissoo (Singh and Chand 2010), Clitoria ternatea (Kumar and Thomas 2012), Catharanthus roseus (Maqsood et al. 2012), Hemidesmus indicus (Cheruvathur et al. 2013a), Rhinacanthus nasutus (Cheruvathur et al. 2013b), Artemisia vulgaris L. (Sudarshana et al. 2013), Anethum graveolens (Dhir et al. 2014), Swertia chirayita (Kumar and Chandra 2014).

It was evident that there were two major hurdles to overcome in the way of producing artificial seeds using SE as explants. In the procedure involving utilization of SE for synthetic seed production, the major impediment is a metachronus and delayed development of the embryonic terminal (Castellanos et al. 2004). Even though the encapsulation phase was achieved fruitfully, yet the regeneration of encapsulated SE was not successful in comparison to the other explants. The regeneration was around 50 % in majority of the medicinal plants taken into account. It is significant to mention, there was only 26 % regeneration in case of Quercus robur synthetic seeds cultured on P24 medium fortified with 0.9 μM BA and 0.1 μM IBA (Prewein and Wilhelm 2003). There was only one instance where synthetic seeds developed from SE resulted in 100 % regeneration in MS with 2 μM BA plus 0.5 μM IBA (in Hemidesmus indicus by Cheruvathur et al. 2013a). Interestingly, both of these extreme results were achieved in comparable media formulations which explain the species specificity of SE in terms of its regeneration potency. Hence, to improve plantlet conversion from synthetic seeds, an adept embryogenic system is indispensible. In terms of synthetic seeds, empirical use of somatic embryogenesis has not yet evolved accordingly. Within a species, embryogenic competency is frequently in a small number of genotypes, or is attained either by employing zygotic embryos or immature explants. This restricts its exercise for propagating preferred cultivars (Standardi and Piccioni 1998). Furthermore, in spite of expensive production procedure, yet there prevails a low success on encapsulation potency of SE, buds, shoots or other meristematic tissue that can develop into a complete plant and thus can be considered as ‘artificial seeds’ (Pond and Cameron 2003).

Shoot tips

Amid various other non-embryogenic materials, ST explants have proven to be the most amenable on account of their mitotic activity in the meristem (Ballester et al. 1997). STs possessing apical buds were utilized for the encapsulation of some medicinal plant species (Table 1). The most interesting part of using shoot buds as explants is the requirement of space and cost of mass propagation via synthetic seed production in comparison to conventional ST culture in vitro. The amount of space and culture media needed for multiple shoot formation and its proliferation is 20 times higher than what is required for micropropagation through synthetic seeds (Fig. 1b, c), using shoot buds as explants. Therefore, encapsulation of shoot buds (apical or axial) provides for an effortless transportation of considerably bulk number of propagules even in limited space (Fig. 1c). Apical and axial shoot buds can easily be regenerated into plants, as long as there is an ample supply of nutrients and rooting does not pose to be problematic; sometimes these regenerate spontaneously from synthetic seeds (Fig. 1d, e). Moreover, further investigation is necessary to determine the procedure by which the growth of apical and axial buds, in an encapsulation matrix, can be suspended until desirable span of time.

STs were effectively utilized for synthetic seed production (Fig. 1d) in several medicinal plant species, such as, Adhatoda vasica (Anand and Bansal 2002), Chonemorpha grandiflora (Nishitha et al. 2006), Phyllanthus amarus, Withania somnifera (Singh et al. 2006a, b), Populus tremula × P. tremuloides (Tsvetkov et al. 2006), Coleus forskohlii (Swaroopa et al. 2007), Rhododendron maddeni (Singh 2008), Curculigo orchioides (Nagesh et al. 2009), Cineraria maritime (Srivastava et al. 2009), Simmondsia chinensis (Kumar et al. 2010), Eclipta alba (Ray and Bhattacharya 2010), Khaya senegalensis (Hung and Trueman 2011), Picrorhiza kurrooa (Mishra et al. 2011), Stevia rebaudiana (Ali et al. 2012), Corymbia torellina × C. citriodora (Hung and Trueman 2012a, b), Mentha arvensis (Islam and Bari 2012), Beta vulgaris (Rizkalla et al. 2012), Ceropegia spiralis, C. pusilla (Murthy et al. 2013), Centaurium erythraea (Piątczak and Wysokińska 2013), Cucumis sativus (Adhikari et al. 2014), Terminalia arjuna (Gupta et al. 2014), Stevia rebaudiana (Nower 2014).

Nodal segments

Most commonly used propagules, denoted to as microcuttings (viz. NS), are used for synthetic seed production, most likely because of the fact that these explants are produced with such relative simplicity, after the micropropagation technique has been set up (Piccioni and Standardi 1995). In terms of storage potential, the period sufficient for exchange between laboratories and the post-storage proliferation with rooting abilities, encapsulated microcuttings proved to be efficient without the risk of physiological variation (Micheli et al. 2007). NSs with axillary buds (ABs) were effectively utilized for synthetic seed production (Fig. 1d) in several medicinal plant species, such as, Morus sp. (Pattnaik and Chand 2000), Dalbergia sissoo (Chand and Singh 2004), Ananus comosus (Gangopadhyay et al. 2005), Dendranthema × grandiflora (Pinker and Abdel-Rahman 2005), Punica granatum (Naik and Chand 2006) Tylophora indica (Faisal and Anis 2007), Olea europaea (Micheli et al. 2007), Coleus forskohlii (Swaroopa et al. 2007), Cannabis sativa (Lata et al. 2009), Spilanthes mauritiana (Sharma et al. 2009a), Spilanthes acmella (Sharma et al. 2009b), Ocimum basilicum (Siddique and Anis 2009), Cineraria maritime (Srivastava et al. 2009), Pogostemon cablin (Swamy et al. 2009), Vitex negundo (Ahmad and Anis 2010), Eclipta alba (Ray and Bhattacharya 2010), Eclipta alba (Singh et al. 2010), Citrus sinensis × Poncirius trifoliate (Germanà et al. 2011), Khaya senegalensis (Hung and Trueman 2011), Picrorhiza kurrooa (Mishra et al. 2011), Rauvolfia tetraphylla (Alatar and Faisal 2012), Stevia rebaudiana (Ali et al. 2012), Rauvolfia serpentine (Faisal et al. 2012), Corymbia torellina × C. citriodora (Hung and Trueman 2012a, b), Cannabis sativa (Lata et al. 2012), Decalepis hamiltonii (Sharma and Shahzad 2012), Ruta graveolens (Ahmad et al. 2012), Curcuma amada (Banerjee et al. 2012), Mentha arvensis (Islam and Bari 2012), Ceropegia bulbosa (Dhir and Shekhawat 2013), Rauvolfia tetraphylla (Faisal et al. 2013) Withania somnifera (Fatima et al. 2013), Stevia rebaudiana (Khan et al. 2013), Ceropegia spiralis, C. pusilla (Murthy et al. 2013), Centella asiatica (Prasad et al. 2014), Salvia splendens (Sharma et al. 2014), Sterculia urens (Subhashini Devi et al. 2014), Phyllanthus fraternus (Upadhyay et al. 2014), Balanites aegyptiaca (Varshney and Anis 2014). It was observed from these studies that there was emergence of multiple shoots frequenting from the synthetic seeds (Fig. 1f), thus proving the encapsulation of NSs as a proficient approach and signifying the occurrence of multiple axillary primordia on the explants.

Protocorm-like bodies

With an exception in atypical instances (Ilan et al. 1995), the term ‘protocorm-like body’ remained limited to in vitro culture of orchids (Ishii et al. 1998). The structures of PLBs were differentiated from typical embryos by the absence of a distinct embryonic alignment (Norstog 1979). Alternatively they are formed with several meristematic centers that modify into regular embryos, shoots and roots (da Silva et al. 2000). Generally, orchid breeders sprout thousands of seeds in tiny containers, within sterile environment. The resultant protocorms have a tendency to develop into bunches of seedlings that is required to be detached physically; a process which is both tiresome and expensive. The breakthrough achieved by the innovation of synthetic seed system wherein single seed or protocorm were encapsulated in an apposite matrix, would evidently minimize the intricacy of seedling sorting and planting. Likewise, for the purpose of encapsulation, PLBs could be employed. So far, the only study involving PLBs as explants to produce synthetic seeds of medicinal plants was carried out by Mohanty et al. (2013) in Dendrobium nobile, a typical orchid with pharmaceutical importance. Utilizing the direct regeneration potential of PLBs, the same achieved near 80 % conversion of synthetic seeds. PLBs are proliferated asexually and the synthetic seeds prepared thereof can ideally be termed as clonal seeds, provided they retain their genetic fidelity.

Hairy roots

Inoculating the plants with Agrobacterium rhizogenes triggers the transfer-DNA comprised in the plasmid to be introduced in plant genome and simultaneously promotes the induction of ‘hairy roots’ that promptly advances rapid growth of root cultures. Such hairy root cultures are regarded as valuable resources of secondary metabolites and enzymes. Circumventing the extensive use of conventional unipolar and bipolar explants, Piątczak and Wysokińska (2013) and Rawat et al. (2013) employed hairy root fragments as a novel approach in synthetic seed development of medicinal plants Centaurium erythraea and Picrorhiza kurrooa with 86 and 73 % regeneration, respectively. Eventually they extended the conception of synthetic seeds by introducing fragments of hairy roots for the first time in medicinal plants.

Calli

Among a wide array of propagules, calli is the least exploited in terms of synthetic seed production of medicinal plants, based on its intricate use during encapsulation and subsequent regeneration. The undifferentiated nature of calli and the requirement of successful differentiation potential presumably restrict its acceptance as an explant for synthetic seed production. Kim and Park (2002), and later on Zych et al. (2005) were the only researchers who successfully encapsulated calli of Allium sativum and Rhodiola kirilowii, respectively, and attained as high as 95–100 % regeneration. However, in a more recent study on Cucumis sativus, Tabassum et al. (2010) employed cell suspension derived from friable callus and ensured 57 % regeneration of synthetic seeds.

Encapsulating agent and matrix

Since the matrix of capsules is largely accountable for the direct surroundings of the plant material and thus proves that the same has a significant impact on the eventual sustainability of the synthetic seed. It is absolutely necessary that the artificial seed coat ought to shield the explants, possess the efficiency to include nutrients as well as other growth and biological factors, protect the developed artificial seed throughout storage and handling, subsume a mechanism for activating ‘germination’, be innocuous, maintain a good affinity with the biological and chemical systems; and lastly, should rather be biodegradable as well (Khor and Loh 2005). The soft hydrogel protects the explant by making sure that the least amount of pressure be put on the same, thus assuring a much lesser damage to the plant material. In the perspective of hydrogel, the explants are commingled with a polymeric solution that when dropped in the other liquid comprising divalent metal ions, commenced a cross-linking effect, giving rise to the hydrogel. The resultant encapsulation beads held the explants firmly and continued to provide sufficient resistance to exterior mechanical stress, for easy handling.

It is quite evident, notwithstanding the fact, that the selection of plant material apposite for propagation into a plant is the nucleus of the synthetic seed conception; the selection of associated matrix constituents employed in combination with the biological substances is equally vital. Quite a few factors are responsible for the successful production of synthetic seeds, counting the crucial ones such as the level and type of gel required for encapsulation and the extent of exposure of encapsulated seeds to CaCl2·H2O, as well (Redenbaugh et al. 1991, 1993). Gels such as agar, alginate, carboxy methyl cellulose, carrageenan, gelrite, sodium pectate, etc., were exploited for artificial seed development, of which alginate encapsulation was proved to be appropriate for the same. Several gel types are exploited for encapsulation; however, sodium alginate established itself to be the most frequently used matrix because of low cost, gelling properties, and its nontoxic nature (Cheruvathur et al. 2013a). Mainly, the hardness of the hydrogels is contingent on the number of Na+ ions (in sodium alginate solution), exchanged with Ca2+ ions (in CaCl2·H2O solution), consequently ensuing in the creation of insoluble calcium alginate (Daud et al. 2008). Usage of agar as gel matrix is not preferred because of its inferior nature when compared to alginate relating to long-standing storage. Alginate is the preferred choice since it aids to ameliorate capsule development and additionally, the firmness of alginate beads assures a much-improved protection (in comparison to agar) to the encased somatic embryos from mechanical damage (Saiprasad and Polisetty 2003).

Several aspects come into play in moderating the environmental elements of temperature and humidity in order to safeguard the biological substances, and additionally offering a sizeable nutrient pool. Evidently, it can be said that matrix materials play a crucial role in the evaluation of the definitive sustainability of synthetic seed. Over the times, the conception of matrix materials has evolved into a moderately refined interaction that centers on the transformability of synthetic seeds. It was evident from the present review update that the majority of the optimum results in terms of nicely formed spherical beads were obtained with 100 mM CaCl2·H2O and 3 % sodium alginate, for example, in Punica granatum (Naik and Chand 2006), Tylophora indica (Faisal and Anis 2007), Vitex negundo (Ahmad and Anis 2010), Simmondsia chinensis (Kumar et al. 2010), Eclipta alba (Ray and Bhattacharya 2010, and Singh et al. 2010), Cucumis sativus (Tabassum et al. 2010), Khaya senegalensis (Hung and Trueman 2011), Rauvolfia tetraphylla (Alatar and Faisal 2012), Stevia rebaudiana (Ali et al. 2012), Rauvolfia serpentine (Faisal et al. 2012), Corymbia torellina × C. citriodora (Hung and Trueman 2012a, b), Ruta graveolens (Ahmad et al. 2012), Rhinacanthus nasutus (Cheruvathur et al. 2013b), Ceropegia bulbosa (Dhir and Shekhawat 2013), Rauvolfia tetraphylla (Faisal et al. 2013), Withania somnifera (Fatima et al. 2013), Dendrobium nobile (Mohanty et al. 2013), Begonia semperflorens (Sakhanokho et al. 2013), Cucumis sativus (Adhikari et al. 2014), Ochradenus baccatus (Al-Qurainy et al. 2014), Anethum graveolens (Dhir et al. 2014), Terminalia arjuna (Gupta et al. 2014), Phyllanthus fraternus (Upadhyay et al. 2014), and Balanites aegyptiaca (Varshney and Anis 2014), and the regeneration of synthetic seeds was around or more than 90 % in majority of the instances. Seemingly, 3 % solution of sodium alginate and 100 mM CaCl2 facilitated in the most advantageous ion exchange involving Na+ and Ca2+, producing compact, transparent, isodiametric beads. Lesser concentrations (1 and 2 %) of sodium alginate were not apposite in view of the fact that the capsules were of asymmetrical shape and being exceedingly fragile and squashy to hold; at the same time at elevated levels (4 and 5 % sodium alginate) the capsules were effectively firm (Fig. 1a) resulting in considerable impediment in conversion of synthetic seeds (in terms of emergence of propagule through breaking the alginate coat). The instances mentioned above were based on assessing the function of sodium alginate level, influencing the gel matrix or bead attribute and successive transformation of synthetic seeds. Reduced levels of sodium alginate may trigger lower endurance of beads formed. The lower endurance of beads exposed for a longer phase (usually for 30 min) to CaCl2 could adsorb large amounts of CaCl2 (Nagesh et al. 2009). Since adsorption is a surface phenomenon, higher accumulation of CaCl2 might restrain further progress, consequently initiating CaCl2-toxicity (Nagesh et al. 2009). On the contrary, beads with greater resistance formed because of higher level of sodium alginate (3 %) could adsorb fewer amounts of CaCl2 and thus ensuing lesser toxicity and ultimately resulting in high-frequency recuperation. Singh et al. (2010) also presented a report on the impact of sodium alginate and CaCl2 amalgamations on bead texture and uniformity, in Eclipta alba (L.) Hassk, one of the leading medicinal herbs. A comprehensive study has been carried out on sodium alginate. It is a biodegradable and biocompatible copolymer containing l-glucuronic and d-mannuronic acid units, and possesses the capability to produce hydrogels in presence of divalent cations. The taut structure and oversized pores of these water-insoluble gels make them proficient enough for encapsulation of live plant cells (Bajaj 1995). Moreover, these allow the exchange of elements back and forth from the adjoining medium.

Concluding remarks

From the available research literatures it is established that the explant-types and concentrations of encapsulation agent as well as matrix have noteworthy roles in production of synthetic seeds in medicinal plants. These three factors chiefly determine the fate of synthetic seeds escalating their conversion in a number of ways. Exploitation of unipolar explants such as shoot tip, nodal segment with axillary bud, microshoots, etc., with high morphogenic potential are the most responsive throughout the synthetic seed development procedure. However, the promoter role of these unipolar explants further depends on several other factors those influencing the regeneration prospective. Optimal levels of PGRs and their types in the conversion media regulate the germination of synthetic seeds and their subsequent development into complete plantlets. A coating of alginate, which provides ideal protection to the juvenile tissue inside and simultaneously facilitating the easy breakage during germination as and when required, is of utmost importance, apart from the standard media for conversion. Nonetheless, few negative reports exist as well. For instance, Tabassum et al. (2010) achieved a merely 57 % regeneration of synthetic seeds in Cucumis sativus using 3 % sodium alginate and 100 mM CaCl2·2H2O but they used cell suspension as the explant. From the existing literature it becomes further clear that in several cases the increased concentration (4 %) of sodium alginate and reduced level (75 mM) of CaCl2·2H2O resulted in significant conversion (95–99 %) of synthetic seeds that is comparable to that of the optimal combinations discussed earlier. Consequently, it is indispensable to focus on additional factors influencing the system of synthetic seeds formation directly or indirectly.

Author contribution statement

S. Gantait, S. Kundu—conceived the idea of the review; S. Gantait, S. Kundu—surveyed the literature and wrote the manuscript; S. Gantait, N. Ali, N. Sahu—scrutinized and corrected the manuscript.