1. Synthetic Seeds :Prospects and Limitations
By: Tinku kumar sharma

2. Definition:
Synthetic seeds are encapsulated somatic embryos, shoot buds, cell aggregates or any other tissue that can be used for sowing as a seed or that possesses the ability to convert into a plant under in vitro or ex vitro conditions and that can retain this potential also after storage.(Capuano et al.,1998)

3. Synthetic seeds were first introduced in the 1970s as a novel analogue to the plant seeds. The production of Synthetic seeds is especially useful for plants which do not produce viable seeds. It represents a method to propagate these plants. Synthetic seeds are small sized and this provides further advantages in storage, handling, shipping and planting
Synthetic seeds

4. What makes a synthetic seed?
Synthetic seeds are produced by encapsulating a plant propagule in a matrix which will allow it to grow into a plant. Plant propagules consist of shoot buds or somatic embryos that have been grown aseptically in tissue culture. In culture, these plant propagules easily grow into individual plants as we have the capacity to control its growth using chemicals provided in the culture media.
In the production of Synthetic seeds , an artificial endosperm is created within the encapsulation matrix. The encapsulation matrix is a hydrogel made of natural extracts from seaweed (agar, carageenan or alginate), plants (arabic or tragacanth), seed gums (guar, locust bean gum or tamarind) or microrganisms (dextran, gellan or xanthan gum.). These compounds will gel when mixed with or dropped into an appropriate electrolyte (copper sulphate, calcium chloride or ammonium chloride). Ionic bonds are formed to produce stable complexes. Useful adjuvants such as nutrients, plant growth regulators, pesticide and fungicide can be supplied to the plant propagule within the encapsulation matrix. In most cases, a second coat covering the artificial endosperm is required to simulate the seed coat.

5. Synthetic seed
Somatic embryo
Artificial endosperm

6. Need for Synthetic seed production technology
Characteristics of Clonal Propagation Systems
Micropropagation
Low volume, small scale propagation method
Maintains genetic uniformity of plants
Acclimatisation of plantlets required prior to field planting
High cost per plantlet
Relatively low multiplication rate
Greenhouse cuttings
Rooting of plantlets required prior to field planting
Multiplication rate limited by mother plant size
Artificial seeds
High volume, large scale propagation method
Direct delivery of propagules to the field, thus eliminating transplants
Lower cost per plantlet
Rapid multiplication of plants.

7. Ease of handling while in storage
Easy to transport
Has potential for long term storage without losing viability
Maintains the clonal nature of the resulting plants
Serves as a channel for new plant lines produced through biotechnological advances to be delivered
directly to the greenhouse or field
Allows economical mass propagation of elite plant varieties
Synthetic seeds have the potential for providing an inexpensive plant delivery system.
It also provides rapid bulking up for the production of individual genetically engineered plants.
Advantages of Artificial or Synthetic Seeds over Somatic Embryos for Propagation

8. Basic Requirements for Production of Synthetic Seeds
Production of high quality, vigorous somatic embryos that can produce plants with frequencies comparable to natural seeds.
Inexpensive production of large number of high quality somatic embryo with synchronous maturation and high conversion frequency.
Synchronized embryoid development.

9. Steps of Synthetic Seeds Production
Establishment of somatic embryogenesis
Maturation of somatic embryos
Synchronization and singulation of somatic embryos
Mass production of somatic embryos
Standardization of encapsulation
Standardization of artificial endosperm
Mass production of synthetic seeds
Green house and field planting

10. Procedure of Synthetic Seed Production

11. How are synthetic seeds made?
Shoot buds cut from shoot cultures can be used for artificial seed production. They are cut to 2-3mm in size and placed in the encapsulation matrix.

12. What are somatic embryos?
Somatic embryos are bipolar structures with both apical and meristematic regions, which are capable of forming shoot and root respectively. A plant derived from a somatic embryo is sometimes referred to as an ‘embling’.

13. Somatic Embryos vs Zygotic Embryos and Their Advantages
Develop from somatic cells
Can be used to produce duplicates of a single genotype.
It does not involve sexual recombination, thus allow specific and directed changes to be introduced into desirable elite individuals by inserting isolated gene sequences into somatic cells.
Develop from zygote (i.e. fusion product of male and female gametes)
Cannot be used.
It involves sexual recombination.

14. Somatic Embryogenesis
A Young Plant
Isolated single cells from leaves
Doublet formation after first mitosis in culture
Torpedo stage embryo
Cell colony following many mitosis
Heart shape embryo
Globular embryo

15. Encapsulation
Encapsulation is necessary to produce and to protect synthetic seeds. the encapsulation is done by various types of hydrogels which are water soluble. the gel has a complexing agent which is used in varied concentrations.
Gelling agent (% w/v)
Complexing agent (μM)
Sodium alginate (0.5 – 5.0)*
Sodium alginate (2.0) with Gelatin (5.0)*
Carragenan (0.2 – 0.8)
Locust beam gum ( )
Gelrite (0.25)
Calcium salts (30 –100)
Calcium chloride (30 –100)
Potassium chloride
Ammonium chloride (500)
temperature lowered
*Alginate hydrogel is frequently selected as a matrix for synthetic seed because of its moderate viscosity and low spinnability of solution, low toxicity for somatic embryos and quick gellation, low cost and biocompatibility characteristics.

16. Principle and Conditions for Encapsulation with Alginate Matrix
The major principle involved in the alginate encapsulation process is that the sodium alginate droplets containing the somatic embryos when dropped into the CaCl2.2H2O solution form round and firm beads due to ion exchange between the Na+ in sodium alginate with Ca2+ in the CaCl2 . 2H2O solution. The hardness or rigidity of the capsule mainly depends upon the number of sodium ions exchanged with calcium ions. Hence the concentration of the two gelling agents i.e., sodium alginate and calcium CaCl2.2H3O and the complexing time should be optimized for the formation of the capsule with optimum bead hardness and rigidity.
Sodium alginate (3%) upon complexation with CaCl2. 2H3O (1%) for half an hour gives optimum bead hardness and rigidity for the production of viable synthetic seeds (Vakeswaran, 2001).

17. Artificial Endosperm and storage
Somatic embryos lack seed coat (testa) and endosperm that provide protection and nutrition for zygotic embryos in developing seeds.
To augment these deficiencies, addition of nutrient and growth regulators to the encapsulation matrix is desired, which serves as an artificial endosperm.
Addition of nutrients and growth regulators to the encapsulation matrix results in increase in efficiency of germination and viability of synthetic seeds.
These synthetic seeds can be stored for longer period of time, even up to six months without losing viability when stored at 20C.
In addition, a number of useful materials i.e. nutrients, fungicides, pesticides, antibiotics and microorganisms (Rhizobia) can be incorporated into encapsulation matrix
Incorporation of activated charcoal improves the conversion and vigour of encapsulated somatic embryos.
To induce desiccation tolerance, a wide variety of chemical or physical stresses can applied during the development of somatic embryos (ABA, Proline, Triazoles, High sucrose concentration, nutrient deprivation, chilling, high density inoculum and thermal shock).

18. Somatic embryos formed from cultured plant parts are ideal for artificial seed production.

19. Using a sterile 10ml pipette, the shoot bud / somatic embryo is drawn up with some encapsulation matrix

20. The shoot bud or somatic embryo is dropped into the complexation solution and a capsule is formed and allowed to harden. Capsule hardness can be controlled by the concentration of the complexation solution and the complexation time. Size of the capsule is determined by the size of the shoot bud or somatic embryo and the inner diameter of the pipette used.

21. The capsules or artificial seeds are collected by decanting off the complexation solution and rinsed in water. The artificial seeds should be pliable enough to cushion and protect the embryo, yet allow germination and growth of the shoot bud or somatic embryo. It should be rigid enough to withstand rough handling during manufacture, transportation and planting. For the artificial seeds to remain dormant until planting, a thin layer of water-soluble resin is used to coat the encapsulation matrix.

22. b) Artificial or synthetic seed derived plantlets in orchid.
a) Artificial or synthetic seed produced in orchids by alginate encapsulation.
b) Artificial or synthetic seed derived plantlets in orchid. a b

23. Utilization of Synthetic Seeds
Multiplication of non-seed producing plants, hybrids or propagation of polyploid plants with elite traits.
Propagation of male or female sterile plants for hybrid seed production.
Germplasm conservation of recalcitrant species.
Conservation of transgenic plants.
Production of disease free plants

24. Crops: Synthetic seed production and plant conversion Reported
In vitro propagules
Crops
Somatic embryos
Alfaalfa, celery, brinjal, carrot, brassica, lettuce, potato, sweet potato, cucumber, asparagus, rice, horse raddish, wheat, triticale, maize, sorghum, soyabean, sugarcane, coffee, tobacco and cotton
Axillary or adventitious buds
mulburry, eucalyptus and grape
Shoot tips
Banana, cardamom and Carum carvi

25. Potential Uses of Artificial Seeds
Delivery Systems:
Reduced cost of transplants
Direct greenhouse and field delivery of:
Elite, select genotypes
hand-pollinated hybrids
Genetically engineered plants
Sterile and unstable genotypes
Large-scale mono cultures
Mixed-genotypes plantations
Carrier for adjuvants such as microorganisms, plant growth regulators, pesticides, fungicides, nutrients and antibiotics
Protection of meiotically unstable, elite genotypes.
Can be conceivably handled as seed using conventional planting equipment.
Germplasm conservation and cryopreservation
Propagation with a low cost, high volume capabilities of seed propagation.
Can be produced with in short time ( one month) at anytime and in any season of a year
Dormancy period can be reduced to a great extent thereby shortening the life cycle of plant.
Production and transport of pathogen free propagules across the international borders avoiding bulk transportation of plants, quarantine and spread of diseases.
Storage of genetically heterozygous plants or plants with unique gene combination.
Analytical Tools:
Comparative aid for zygotic embryogeny.
Production of large numbers of identical embryos.
Determination of role of endosperm in embryo development and germination
Study of seed coat formation.
Study of somaclonal variation.

26. Milestones in Synthetic Seed Production
Steward et al. (1958)
Report on somatic embryogenesis in carrot
Murashige (1978)
First to moot the idea of embryogenesis
Kitto and Janick (1982)
First report on synthetic seed in carrot using embryos, roots and callus with polyoxyethylene encapsulation
Redenbaugh et al. (1986)
Calcium alginate as coating agent to produce synthetic seeds in celery and alfaalfa
Gray et al. (1987)
Desiccation of somatic embryos
Mckersie et al. (1989)
Synthetic seed in hybrid alfalfa
Kamada et al. (1989)
Stress induced somatic embryogenesis in carrot and its application to synthetic seed production.
Datta and Potrykus (1989)
Storage of barley synthetic seeds for six months
Bapat and Rao (1989)
Synthetic seeds in sandalwood and mulberry
Berjak et al.(1990)
Similarity in storage behavior of somatic embryos and recalcitrant seeds
Sakamoto et al. (1992)
Development of encapsulation technology for synthetic seeds in carrot
Chee and Canliffe (1992 )
Production procedure for somatic embryos of sweet potato for synthetic seed production in cv. White Star
Onishi et al. (1994)
Automation
Attree et al. (1994)
Bio-reacter production of synthetic seeds in white spruce
Tetteroo et al. (1995)
Development of desiccation tolerant carrot embryoids
Falcinelli et al. (1995)
Self breaking of synthetic seeds in coats
Sujata singh(1997)
Synthetic seed production in hybrid rice
Rout et al. (1997)
Somatic embryogenesis in Rosa hybrida
Bhatti et al. (1997)
Cryopreservation of sweet potato embryos through encapsulation
Arun kumar (1998)
Synthetic seed production in hybrid rice
Jayanthi (1999)
Synthetic seed production in sweet potato
Neelamathi (2000)
Synthetic seed production in sugarcane
Vakeswaran (2001)
Synthetic seed production in Ashwagandha
Lakshmi (2001)
Synthetic seed production in cassava
Nyende et al. (2003)
Production, storability and regeneration of shoot tips of potato encapsulated in calcium alginate hollow beads in cv. Desiree, Genet, Tigone and Tomensa
Kantha rajah (2004 )
Somatic embryogenesis in eggplant.
Nyende et al. (2005)
Yield and canopy development of field grown potato plants derived from synthetic seeds.

27. Limitations:
Although results of intensive researches in the field of synthetic seed technology seen promising for propagating a number of plant species. Practical implementation of the technology is constrained due to the following reasons:
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 conservation into normal plants.
Lack of dormancy and stress tolerance in somatic embryos that limit the storage of synthetic seeds.
Poor conservation of even apparently normally matured somatic embryos and other micropropagules into plantlets that limit the value of the synthetic seeds and ultimately the technology it self .
Somaclonal variation is the genetic variation arising through tissue and cell culture. If it happens, the basis of uniformity of synthetic seed will be defeated.

28. Applicability and Feasibility of Artificial Seed Production Technology:
In order to be useful, synthetic seed must either reduce production costs or increase crop value. The relative benefits gained, when weighed against development costs, will determine whether its use is justified for a given crop species. Considering a combination of factors, including improvement of the existing embryogenic systems, relative cost of seed as well as specific application for synthetic seed of seedless water melon would actually cost less than conventional seed, providing a benefit at the outset of crop production. Although embryogenic systems for this crop do not exist, the benefit that could be conferred by use of synthetic seed would be very great. Value added aspects that would increase crop worth are numerous and include cloning of elite genotypes, such as genetically engineered varieties, that cannot produce true seed.

29. Conclusions:
Plant propagation using artificial or synthetic seeds developed from somatic embryos opens up new vistas in agriculture. Artificial seeds make a promising technique for propagation of transgenic plants, polyploid with elite traits and plant lines with problems in seed propagation. Being clonal in nature the technique cut short laborious selection procedure of the conventional recombination breeding and can being the advancements of biotechnology to the doorsteps of the farmers in a cost effective manner. It also offers tremendous potential in germplasm conservation. However further research is needed to perfect the technology so that it can be used on a commercial scale.

30. Thanks