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Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 11 (2017) pp. 662-674 Journal homepage: http://www.ijcmas.com Review Article https://doi.org/10.20546/ijcmas.2017.611.079 Synthetic Seed Technology M. Mudasir Magray*, K.P. Wani, M.A. Chatto and H.M. Ummyiah Division of vegetable sciences, SKUAST-K, Srinagar, India *Corresponding author ABSTRACT Keywords Synthetic seed. Micro propagules, Sporogenesis Article Info Accepted: 07 September 2017 Available Online: 10 November 2017 Synthetic seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability to convert into a plant under in vitro or ex vitro conditions and that retains this potential also after storage. Earlier, synthetic seeds were referred only to the somatic embryos that were of economic use in crop production and plant delivery to the field or greenhouses (Gray, et al., 1991). Implementation of synthetic seed technology requires manipulation of in vitro culture systems for large scale production of viable materials that are able to convert into plants, for encapsulation, somatic embryogenesis, organogenesis and enhanced auxiliary bud proliferation systems are the efficient techniques for rapid and large scale in vitro multiplication of elite and desirable plant species. Introduction In general there are two types of seeds which can be used for propagation of plants and thus help in the maintaining the survival of plants in nature: utilizes during the process of its germination. There substances are frequently found in the endosperm. Thus endosperm may contain variety of stored materials such as starch, oils, proteins etc. In some plants, however, the reserve food material is present in cotyledons. Natural Seed Artificial Seed Importance of natural seed Natural Seed The seed provides an expedient living unit for the study of wholeness that is a complex of biological factors which can be considered simultaneously. The seed occupies that sector of an organism life cycle form mega sporogenesis (genetic) to the formation of seedling (ecological). However, a seed is not truly a reproductive structure, but rather an adaptive mechanism to facilitate suspending The seed stage of seed plants represents a unique developmental phase of the spermatophyte life-cycle, and as such involves structures, not characteristic of other stages of development. The essential structure of seed is defined as a ripened ovule consisting of an embryo and its coat. The normal seed contains materials which it 662 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 growth and interrupting the coutinum of homeostasis in the life cycle. to natural environment, it is envisaged that for large scale mechanical planting and to improve the success of plant (in vitro derived) delivery to the field or greenhouse, the somatic embryos or even the other micropropagules useful in synthetic seed production would necessarily require some protective coatings. Encapsulation is expected to be the best method to provide protection and to convert to in vitro derived propagules into synthetic seeds of a number of plant species belonging to angiosperms and gymnosperms (Table 1). Nevertheless, their number is quite small in comparison to the total number of plant species in which in vitro regeneration system has been established. Seeds are the corner stone of agriculture because when seeds are planted in the soil and given water, nutrients, light and some protection from pests would reproduce plant and seeds identical to that planted and also produce number of seeds which could be used for food or feed. Synthetic seeds Synthetic seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability to convert into a plant under in vitro or ex vitro conditions and that retains this potential also after storage. Technology Basic hindrance to synthetic seed technology was primarily based on the fact that the somatic embryos lack important accessory tissues, i.e., endosperm and protection coatings, that make them in convenient to store and handle (Renden baugh, et al., 1993). Furthermore, they are generally regarded to lack a quiescent resting phase and to be incapable of undergoing dehydration. The primary goal of synthetic seed research was, therefore, to produce somatic embryos that resemble more closely the seed embryos in storage and handling characteristics so that they can be utilized as a unit for clonal plant. Propagation and germplasm conservation. In achieving such a goal the technology of encapsultation has evolved as the first major step of production of synthetic seeds. Later it was thought that the encapsulation synthetic seeds should also contain growth nutrients, plant growth promoting micro-organisms (e.g., micorrhizae), and other biological components necessary for optimal embryo-toplant development. The choice of coating material for making synthetic seeds is also an important aspect for synthetic seed production. Earlier, synthetic seeds were referred only to the somatic embryos that were of economic use in crop production and plant delivery to the field or greenhouses (Gray et al., 1991). In the recent past, however, other micropropagules like shoot buds, shoot tips, organogenic or embroyogenic etc. Implementation of synthetic seed technology requires manipulation of in vitro culture systems for large scale production of viable materials that are able to convert into plants, for encapsulation, somatic embryogenesis, organogenesis and enhanced auxiliary bud proliferation systems are the efficient techniques for rapid and large scale in vitro multiplication of elite and desirable plant species. Through these systems a large number of somatic embryos or shoot buds are produced which are used as efficient planting materials as they are plant regeneration either after having minor treatment or without any treatment with growth regulator(s). Because the naired micropropagules are sensitive to desiccation and / or pathogens when exposed 663 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 Based on the technology established so far, two types of synthetic seeds are known desiccated and hydrated. The desiccated synthetic seeds are produced from somatic embryos either naked or encapsulated in polyethylene glycol followed by their desiccation. Desiccation can be achieved either slowly over a period of one or two weeks sequentially using chambers of decreasing relative humidity or rapidly by unsealing the pier dishes and leaving then on the bench overnight to dry. Such types of synthetic seeds are produced only in plant species whose somatic embryos are desiccation tolerant. On the contrary, hydrated synthetic seeds are produced in those plants where the somatic embryos are recalcitrant and sensitive to desiccation. Hydrated synthetic seed are produced by encapsulating the somatic embryos are hydrogel capsules. million plants daily and competitive economically with the seed method (Mugashinge, 1977). Drew (1979) was active in developing methods to commercially propagate crops using somatic embryos. He suggested delivering carrot somatic embryos in a fluid drilling system, but was able to produce only three plants from carrot embryos on a carbohydrate free medium. He could not get success in producing many plants through this system. He faced a crucial problem and found the very slow rate of development of plantlets derived from culture. Kitto and Janick (1982) coated dumps of carrot embryos, roots and Cellus with polyongethylene. Some embryos survived the coating process as well as a desiccation step (Kitto and Janick, 1985a and 1985b). The early assessment of Murashigesirect (1977) on the difficulty of somatic embryogeny are still valid today. The quality and fidelity of somatic embryos are the limiting factors for development and scale up of artificial seeds. History of synthetic seeds The origin of the idea of an artificial seed is difficult to determine. Certainly, those who first produced somatic embryos may have considered such application (Steward, et al., 1958 and Reinert, 1958). The discovery of somatic embryogenesis in carrot in the year 1958 was almost simultaneously by F. C. Steward (USA) and J. Reinert (Germany). F. C. Steward a renowned plant physiologist at Cornell University in New York. However, it was not until the early 1970’s that the concept of using somatic embryos began to be presented as a potential propagation system for seed sown crops. Toshio Murashige gave a number of survivors in tissue culture propagation where he concluded with this concept. He formally presented his ideas on artificial seeds at the symposium on tissue culture for horticultural purposes in Belgium, September 6-9, 1977. His terse comments in the proceedings, however, were to be applicable, the cloning method must be extremely rapid, capable of generating several Interestingly, artificial seed prepared from shoot buds can also be used for plant propagation and this was reported by P. S. Rao’s group from BARC, Mumbai. Research on artificial seeds in rice is still in infancy and this technology through somatic embryogenesis, would offer a great scope for large scale propagation of superior, elite hybrids (Brar and Khush, 1994). Potential uses of artificial seeds Delivery system Reduced costs of transplants Direct greenhouse and field delivery of elite, select genotypes, hand pollinated hybrids, genetically engineered plants, sterile and unusable genotypes, large seed monocultures, mixed genotype plantations. 664 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 Carrier for adjuvant such as micro-organisms, plant growth regulators and pesticides protection of meiotically unstable elite genotypes. They are applicable for large scale monocultures as well as mixed genotype plantation. The synthetic seed provide us knowledge to understand the development, anatomical characteristics of endosperm and seed coat formation. Such seeds give the protection of meiotically unstable, elite genotype. Analytical tools Comparative aid for zygotic embryogeny Production of large numbers of identical embryos Comparative advantages of artificial seeds over classical as well as micro-propagation (with short tip culture). Determine role of endosperm in embryo development and germination The rapid and large scale multiplication minimal labour and low cost propagation. Study of seed coat formation The synthetic seeds so developed breed true. Artificial seeds can be directly delivered to the field. Thus eliminating transplantation and tissue hardening steps. There are potential advantages of artificial seed technology specially for tree genetic engineering. They can also provided with various kinds of adjuvants like plant growth regulators, useful micro-organism and pesticides to tailor a field specific. Plantable unit for a desired crop. However, genetic uniformity is maintained in all there propagation methods. Artificial seed technology can be very useful for the propagation of a variety of crop plants, especially crops for which true seeds are not used or readily available for multiplication (e.g Potato). The true seeds are expensive (e.g Cucumber and Geraniums) hybrid plants (e.g. Hybrid rice) and vegetatively propagated plants which are more prone to infections (e.g. day lily, garlic, potato, sugarcane, sweet potato, grape and mango) The artificial production of seeds has already been obtained successfully, in Zea mays, Apium gravelleus, Daucus carota, Lactuca sativa, Medicago sativa, Brassica spp, Gossypium hursutm, Santalum spp etc. The encapsulation of somatic embryo (hydrated or desiccated) provides a potential method to combine the advantages of clonal. Propagation with the low-cost high volume capabilities of seed propagation. These seeds can be produced within a short time (one month) whereas natural seeds are the end product of complex reproductive process and breeders have to wait for a longtime for development of new variety. Draw tissue culture principles The technique has developed around the concept that a cell is Totipotent concept that has the capacity and ability to develop into whole organism. The principles involved in plant tissue culture are very simple and primarily an attempt whereby an explain Artificial seeds can be produced at any time and in any season of a year. They are useful in preserving germplasm. 665 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 cannot be to same extent freed from interorgan inter-tissue and inter-cellular interactions and subjected to direct experimental unit. The most common culture in plant tissue is callus which is wound tissue composed of undifferentiated highly vacuolated and unorganized cells. generated in a sequential way, after adjustment of the hormonal conditions (Fig. 1). Somatic embryogenesis It is the process by which the somatic cells or tissue develop into differentiated embryo and each fully developed embryo is capable of developing into a plantlet (young or miniature plants). The concept of Totipotency of cells plant cells in vivo are not TiTopotent. Infact, with few exceptions, the only Totipotent cell is the fertilized egg. Some Tissues do not divide at all, other do so only occasionally. Meristems do divide but upon explanation are not capable of forming embryos. They are used, however in micro-propagation whereby new plants are generated via organogenesis. Some concept in science become inherently acceptable long before their practically is demonstrable. This was so in the concept of the totipotency of cells of higher plant. Even in the mid-twenties one encountered the Tact view that apart from inherent practical difficulties there was no theoretical reason why one e should not rear begonia plant from a single leaf hair cell. This view was traceable first to the then well recognized principles that as cells divide mitotically, they do equationally to produce daughter cells in Facsimile. Embryos can be obtained either directly from cultured explants (the organized structure, for example, leaf, hypocotyle, stem and other plants parts.) and anthers (or pollen) or indirectly from callus (unorganized mars of parenchymatious tissue derived from explants culture as a result of wound response) and isolated single cells in culture. The process of embryogenesis involves various stages of differentiation and development such as proembryo, globular, heart-shaped and torpedo embryo. Achievements and prospects of synthetic seed technology Somatic embryos In plants, the mature embryo consists of a bipolar structure carrying meristems at the terminal ends. These meristems, consisting of somatic cells, will contribute to morphogenesis by generating new organs such as shoos, leaves, and roots throughout the adult phase of the plants. In vitro somatic cells may regenerate an entire plant via of the two alternation path ways. Although various micro-propagules have been considered for synthetic seed production the somatic embryos have been largely favoured (Table 1) as these structures posses the radical and plumule that are able to develop into root and shoot in one step, usually without any specific treatment various types of artificial seeds have been prepared using somatic embryos which have been either dried or maintained fully hydrated, these may or may not be encapsulated (Kitto and Janick, 1999). Somatic embryogenesis, which reproduces the steps of Zygotic embryogenesis. Onishi sakmoto and Hirosoma have demonstrated a protocol for the production of synthetic seeds involving automation at the production and encapsulation stages. Organo-genisis, whereby under appropriate conditions (what matters is the auxin/cytokinin ratio) shoots and roots are 666 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 These authors have emphasized that high and uniform conversion of synthetic seeds under a practical sowing situation, such as nursery beds in a green house or in the field, is an essential revilement for their use a clonal propagation of plants. However, further research is needed to optimize protocols for production of viable synthetic seeds that could be stored for longer periods and could be commercially viable. In tree species like santalum album, pistacia vera and Mangifera indica also the somatic embryos have been encapsulated to produce synthetic seeds, reported by Onay et al., (1996), Bapat et al., (1992), etc. In many plant species (Table 1) the unipolar auxiliary shoot buds and / or apical shoot tips which do not have root meristems, have also been encapsulated to produce synthetic seeds. Auxiliary shoot buds and apical shoot tips 667 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 Procedure for production of artificial seeds Fig.1 Effect of auxin treatments on callus and somatic embryo of carrot 668 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 Fig.2 Germination efficiency of synthetic seeds of carrot on MS medium Table.1 List of plant species in which encapsulation technology has been applied to produce synthetic seeds Plant Propagule used for encapsulation Actinidia delicosa (Kiwi fruit) SB’s Arachis hypogaca (Ground nut) SE’s Asparagus cooper SE’s Brassica comperiris (Mustard) SB’s Daucos ceroto (Carrot) SE’s Malus Punila (Mill) (Apple root stock M. 26) SB’s Mangifera indica L. (Mango cv. Amrapali) SE’s Psodium ghajana (Guava) SE’s Solanum melangena (Egg plant) SE’s Vitis Vinifera (Grape) SE’s Zingiber Officinale Pose (Ginger) SB’s Cucumber (Cucumis sativus) SE’s Poinied gourd (Trichosruthus dauca) SB’s Cynbidium gigenieum (Orchid) PLB’s SB – Shoot bud, PLB – Protocol like bodies, SE – Somatic embryos Sources: Ara et al., 2000; Latif et al., 2007; M. A. Malru, 2009; Tabassum et al., 2010 Table.2 Effect of different concentrations of sodium alginate on germination of artificial seed of pointed gourd Sodium alginate (%) 3 4 5 Mean S.E (±) No. of seeds cultured 40 40 40 4.00 0.000 No. of seeds germinated 16 38 12 22.00 8.083 Significant at 5% and 1% level, respectively 669 % of seed germination 40 95 30 55.00 20.207 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 Table.3 Effect of different concentration and combinations of BAP and NAA on artificial seed proliferation from shoot tip explants of AM – 8 and AM – 15 genotype Treatments (mg/l) BAP 0.5 BAP 1.0 BAP 1.5 BAP 2.0 Mean S.E (±) NAA 0.5 NAA 1.0 NAA 1.5 NAA 2.0 Mean S.E (±) 1.0 + 0.5 1.0 + 1.0 1.0 + 1.5 1.0 + 2.0 Mean S.E (±) NAA + BAP 1.0 + 0.5 1.0 + 1.0 1.0 + 1.5 1.0 + 2.0 Mean S.E (±) Days to shoot proliferation % of shoot forming explants Days to shoot proliferation 77777.00 0.000 8 8 8 8 8.00 0.000 8 8 8 8 8.00 0.000 7585++ 705571.25 6.250 65+ 70++ 45 30 52.50 9.24 55 75++ 50 30 52.50 9.242 77777.00 0.000 8 8 8 8 8.00 0.000 8 8 8 8 8.00 0.000 % of shoot forming explants 80± 90+± 725875.00 6.757 70+ 75++ 47 30 55.30 10.460 60 80++ 55 35 57.50 9.242 9 9 9 9 9.00 0.000 50 75++ 50 40 53.75 7.465 9 9 9 9 9.00 0.000 60 80++ 55 40 58.75 8.260 Significant at 5% and 1% level, respectively Since these structures do not have root meristems they should be induced to regenerate roots before encapsulation, ricconi and standard have described conversion of shoot buds of apple clonal rootstock M. 26 encapsulated after an appriate root induction treatment with IBA (24.6 µm) for 3-6 days. clonal plants and for studies of genetic transformation. However, long term maintenance of embryogenic masses in culture tubes or mechanically stirred bioreactors resources frequent transfer of tissue to fresh media which is both labour intensive and costly. Embryogenic masses Protocorms or portocorm like bodies Stable and regenerative embryogenic masses make an attractive tool for the production of In orchids such as Cymbidium giganieum, Deudrobim wardianum,, Geodorum 670 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 662-674 densiflorum, Phaius tonkervillae and Spathoglotis plicata synthetic seeds have been produced by encapsulating the protocol like bodies (PLB’s) in sodium alignate gel Corrie and Tandon have reported that the encapsulated to healthy plantlets upon transferring either to nutrient medium or directly to sterile sand and soil. These techniques have made it possible to transplant to areptically grown protocols directly in the soil, cutting down the cost of raising in vitro plantlets and their subsequent acclimatization. Cell suspension Production of synthetic seed has been reported in a number of cereals, tuberous plant, vegetables and other commercially important plants like soya bean, mustards, coffee, tobacco and cotton (Datta et al., 1999). However, because of certain inherent obstacles, the production rate of the uniform and high quality embryo is lower. Consequently the preparation of efficient and quality seed has been successfully in only a few crops like carrot (Latif et al., 2007) and alfalfa (Datta and Poirythus 1989). The primary goal of artificial seed system is to regenerate whole plantlets form artificial seeds. Synchronization of embryogenesis Following callus induction, cell suspension of cv. Royal was established in its respective callus induction media. Best suspension culture was obtained from MS-media containing 3% sucrose, 30 µM adequate sulphate, 3 µM thiamine Hcl, 580 µM NaH2PO4, 0.1% CH, 5 µM NAA and 1 µM, BAP. Cultures were placed on a gyratory shaker with rotation at 30 rpm at 22±2oC for 16 h photoperiod. The procedure adopted by Givilano et al., (1983) to synchronize the cucumber cell suspension was followed. All cells were subcultured and passed through 150 µM. Steve (100 mesh) after every 10 days sub-culturing and then incubated an gyratory shaker at 80 rpm at 22±2oC. After two successive subculturing, cells at globular stage were shifted onto modified suspension media containing CH and NAA only. Cells turned into heart and torpedo shapes after 2-3 days. They were then sub-cultured on MS basal media, devoid of growth regulators which helped in uniform maturation of somatic embryo. First report of genetically uniform production of synthetic seeds was in cucumber by standardizing culture conditions to induce somatic embryogenesis synchronously and at high frequency in cell suspension culture through following steps (Tabassum et al., 2010) Synthetic seed / encapsulation Mixture of mature cell suspension and sodium alginate at a ratio of 1:4 were dropped into 100 mM calcium chloride solution to form synthetic seeds. Callus induction Regeneration efficiency of synthetic seeds Friable, nodular and embryogenic callus is considered pre-requisite to establish fast growing cell suspension culture and subsequent regeneration. The hypocotyle pieces of Royal cultivars developed into callii when cultured on MS-media continuing 2.0 µM, 24 D and 0.5µN BAP with 3% sucrose. Shoot formation was observed when synthetic seeds were placed on neutral gel formed by dissolving phytagel in autoclaved tap water. The test tube containing neutral gel plus synthetic seeds were kept for 16 h photoperiod at 22±2oC. shoots enlarged upto 10 days in neutral gel media when 671