Somatic embryogenesis and synthetic seed productionGaurav Chandra GyanwaliM.Sc BiotechnologyTU, Nepal*

What are synthetic seeds?

Synthetic seeds are encapsulated somatic embryos The seeds may be dry or hydrated. Dry seeds are either naked or encapsulated in polyoxyethylene glycol (Polyox) followed by desiccation. Hydrated seeds are produced by encapsulation in hydrated gels.*

What are synthetic seeds?

*Alginate encapsulated synthetic seeds (left) and synthetic seed derived plantlets in orchids (Saiprasad, 2001)

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Somatic embryogenesis It is the process of production of embryos from somatic cells (not as a result of gametic fusion) The somatic embryos are commonly called embryoids or non-zygotic embryos Can be induced directly or indirectly Somatic embryogenesis proceeds in two steps: induction and maturation Induction requires high auxin and low NH4+, while maturation requires low or no auxin and high sucrose (6-40%)

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Somatic embryogenesis../2derived from single cell which divide to form meristemoid Cells of meristemoid contain dense cytoplasm, large starch grains, large nucleus and nucleolus with high dehydrogenase activity These cells later become cutinised and lose cytoplasmic connection with the surrounding- the proembryo stage The proembryo first forms globular structure and then proceeds to form heart and torpedo embryoids, respectively In the last step, the embryoids develop into cotyledonary embryoid with distinct polarity, i.e. radicle and plumule

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Production of synthetic seeds*

Production of synthetic seeds from somatic embryos In order to use somatic embryos for artificial seed production, they should be optimized. Then there should be the synchronization of somatic embryo production The somatic embryos thus produced should then be separated and subjected to mass scale production of somatic embryo They are then subjected to standardization of encapsulation and artificial endosperms

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Optimization of somatic embryo production Should be done on species specific, explants specific manner There is no sure shot mechanism, optimal conditions should be found out empirically trying out different culture conditions, hormonal regimes, media, etc Conditions optimal for one explants may not suit that for other explants even from the same species The embryos thus produced should be vigorous and of high quality such that they can produce plants in frequencies comparable to natural seeds.Synchronization of embryo maturation is major hurdle to commercial synthetic seed production

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Optimization of gelling agents and encapsulation of somatic embryos The gelling agents used should have low toxicity to somatic embryos, should be cheap, rapidly gelling and should have moderate viscosity. Several gelling agents like agar, alginate, carboxy methyl cellulose, carrageenan, sodium pectate, etc. have been tested Alginate has so far found to be the best due to its enhanced encapsulation and rigidity of the capsules against mechanical injury

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Optimization of gelling agents and encapsulation of somatic embryos../2 Somatic embryos are mixed with sodium alginate gel (0.5 5% w/v) and dropped into calcium salt solution (CaCl2 (30100 mM), Ca(NO3)2 (30100 mM)). As a result of ion-exchange reaction, sodium ions are replaced by calcium ions forming calcium alginate beads or capsules surrounding the somatic embryos. The size of the capsule is controlled by varying the inner diameter of the pipette nozzle.

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Optimization of gelling agents and encapsulation of somatic embryos../3 Hardening of the calcium alginate is modulated with the concentrations of sodium alginate and calcium chloride as well as the duration of complex formation. The use of a dual nozzle pipette in which the embryos flow through the inner pipette and the alginate solution through the outer pipette is suitable for encapsulation*

Optimization of artificial endosperm Somatic embryos lack true endosperm. This has been a major limitation in imposing dormancy and stress tolerance in artificial seeds So they need to be supplied with artificial sources of nourishment and growth regulators, but the protocols are not well established as yet.*

Advantages of using synthetic seedsEase of handling (during production/storage)Easy to transportPotential for long term storage without loss of viabilityMaintain the clonal nature (i.e., genetic uniformity) of the resulting plantsServes as a channel for new plant lines produced through biotechnological advances directly to the greenhouse or fieldAllows economical mass propagation of elite plant varieties.

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Limitations Limited production of viable micropropagules useful in synthetic seed production. Anomalous and asynchronous development of somatic embryos. Improper maturation of the somatic embryos that makes them inefficient for germination and conversion into normal plants. Lack of dormancy and stress tolerance in somatic embryos that limit the storage of synthetic seeds. Poor conversion of even apparently normally matured somatic embryos and other micropropagules into plantlets that limit the value of the synthetic seeds and ultimately the technology itself.*

Somatic embryogenesis and synthetic seed prodcutionSomatic embryogenesis and synthetic seed prodcution*