2. MUBASHIR HUSSAIN PHD SCHOLAR 13-arid-3282

3. Synthetic Seed Technology: The magical concept in seed Biotechnology

4. Contents:  Why there is a need to produce synthetic seeds?  Procedure for the production of artificial seeds.  Somatic embryogenesis and its types.  Explants for initiation of SE.  Distinct stages in development of Somatic Embryogenesis.  Separation of somatic embryos:  Importance of Somatic embryogenesis.  Artificial seeds and its methods for production.  Types of artificial seeds.  Pretreatment of synthetic seeds before germination.  Importance of Artificial seeds.  Limitations of Artificial seeds.

5. Why there is a need to produce synthetic seeds?  If plants are unable to produce seeds.  Seeds which are produced by plants are not viable.  Seeds which are produced by plants are not in large number.  Conditions are not suitable for plants to produce seeds.

8. Somatic embryogenesis  Somatic/Asexual embryogenesis is the regeneration of embryo like structure from the somatic cells without gametic fusion (Tabassum et al., 2010). 1. Direct Somatic embryogenesis 2. Indirect somatic embryogenesis

9. Type of Somatic embryogenesis 1. Direct Somatic Embryogenesis: If the somatic embryos differentiate directly from the explants without an intervening callus phase it is called direct somatic embryogenesis. 2. Indirect Somatic Embryogenesis: If the somatic embryos differentiate indirectly from the explants after an intervening callus phase it is called indirect somatic embryogenesis.

10. Somatic embryos Somatic embryos are defined as bipolar structure containing both shoots and roots apices that are anatomically and physiologically comparable to zygotic embryos. Characteristics of somatic embryos: 1. Origin is single cell. 2. Polarity is bipolar. 3. Vascular connection with callus/explant is absent. 4. Somatic embryos can be easily separated from explants/callus.

11. Explants for initiation of SE  1. Embryonic or young seedling tissues  2. Excised small tissues from young inflorescence  3. Scutellum  4.Young roots  5. Petioles  6. Immature leaf  7. Immature hypocotyls  8. Nucellus tissues Somatic embryos

12. Distinct stages in development of Somatic Embryogenesis  1. Single cells  2. Group of cells  3. Globular stage  4. Heart shaped embryo  5. Torpedo stage embryo

13. Conti.........  Somatic embryogenesis may develop from single cells or from a small group of cells.  Repeated cell divisions lead to the production of a group of cells that develop into an organized structure known as “globular” stage embryo.  Further development results in heart and torpedo stage embryos from which plants can be regenerated.  Signs of tissue differentiation become apparent at the globular stage and apical meristem are apparent in heart shaped embryo.

14. Conti.........  Somatic embryogenesis usually proceeds in two distinct stages. a) Embryo initiation b) Embryo production In the initial stage (embryo initiation), a high concentration of 2,4-D is used. In the second stage (embryo production) embryos are produced in medium with no or very low level of 2,4-D.

15. Conti.........  Most of times, we see different stages in embryogenesis  Embryos take about 7-10 days to reach its torpedo stage.  As soon as embryo reach to torpedo stage it will quickly germinate so we have to provide different types of stress to prevent the germination.  To cope up with this problem we increased the sucrose concentration upto 6%. Further to prevent the germination we will transfer to MS medium containing ABA.

16. Separation of somatic embryos: Cultures having somatic embryos are shifted to MS liquid medium in Jars inside the laminar air flow cabinet and then these jars are placed on orbital shaker for shaking cultures. The cultures shake for almost 6 hours to completely separate somatic embryos from each other.

18. Importance of Somatic embryogenesis 1. Clonal propagation. The mass production of adventitious embryos in cell culture is one of the way of clonal propagation. 2. For genetic transformation 3. Raising somaclonal variations in tree species with the help of mutation. 4. Synthesis of artificial seeds. 5. Synthesis of metabolites.

19. Differences between organogenesis and somatic embryogenesis Organogenesis Somatic embryogenesis 1. OriginMany cellsSingle cell 2. PolarityUnipolarBipolar 3. Vascular connection with callus or explant Presentabsent 4. Separation from Callus or explant Not easily separatedEasily separated

21. Artificial or synthetic seeds  When the somatic embryos derived from plant tissue culture (Somatic embryogenesis) are encapsulated by hydrogel and such encapsulated somatic embryos behave like true seeds if grown in soil and can be used as substitute of natural seeds. Methods for encapsulation of Somatic embryos: 1. Gel Complexation 2. Molding Method

22. A) Gel Complexation  In this method isolated somatic embryos are mixed with 0.5- 5% (w/v) sodium alginate and dropped into 30-100 mM calcium nitrate solution. Surface complexation begins immediately and drops are gelled completely within 30 seconds.

23. B) Molding Method  In this method isolated somatic embryos are mixed in a temperature dependent gel such as gelrite are placed in the well of microtiter plate and it forms gel when the temperature is cooled down.

24. Four types of synthetic seeds have been proposed on the basis of embryos and its encapsulation:  Uncoated desiccated somatic embryos e.g orchard grass  Coated desiccated somatic embryos e.g Carrot  Coated hydrated somatic embryos e.g Alfalfa  Uncoated hydrated embryos ( in a fluid drilling gel) e.g Carrot

25. Plants produced from synthetic seeds sown in vitro and in soil :  In Vitro 1. Apium graveiolens 2. Brassica sp. 3. Carrot 4. Cotton 5. Alfalfa 6. Rice 7. Maize  In Soil 1. Apium graveolens 2. Carrot 3. Alfalfa

26. Common hydrogel used for encapsulation: Sr. No GelComplexing Agent 1Sodium alginateCalcium Salt 2Sodium alginate with gelatinCalcium Chloride 3 Carnagenan with locust beam gun Potassium or ammonium chloride 4GelriteTemperature lowered

27. Pretreatment of synthetic seeds before germination:  Before germination, synthetic seeds were given self breaking treatment by dipping the synthetic seeds in 200mM KNO 3 solution for 5 minutes and then rinsing in sterile water for 2 minutes.  Seeds treated with KNO 3 solution germinated earlier than seeds without pretreatment with KNO 3.  It took almost 30 days for sprouting when synthetic seeds were not subjected to pretreatment and germinate within one week when treated with 200mM KNO 3 solution.  Seeds swell up after pretreatment with KNO 3 solution which increase the ease in sprouting of seeds.

28. Plantlets regeneration from the somatic embryos: Plantlets regeneration from the somatic embryos:

29. Plantlets regeneration from the synthetic seeds:

31. Importance of Artificial seeds:  Stored upto a year without loss in viability.  Easy to handle and useful as units of delivery.  Directly sown in the field without hardening.  Artificial seeds are available within a short time.  Artificial seeds technology is season independent.

32. Conti……..  Artificial seeds show no dormancy.  Help to study the role of endosperms and seed coat formation.  Artificial seed coating also has the potential to hold and deliver beneficial adjuviants such as growth promoting thioazobacteria, plant nutrients and control agents.

33. Limitations of Artificial seeds  Cost of production is high.  When the artificial seeds are stored at low temperature, the embryos show a characteristic drop in conversion.  Artificial seeds are less vigorous as compared to natural seeds. The simple reason is that due to lack of storage protein, starch or any chemical that is present in natural seeds in sufficient quantity.  Although we provide coating to the synthetic seeds but that coating is not as protective as natural seeds coating.

34. Conti………  Artificial seeds show abnormal development of seedling particularly abnormal plumule development.  Germination of synthetic seeds is comparable to natural seeds but further conversion into seedling is quite less as compared to natural seeds.  Artificial seeds are more sensitive than natural seeds so we can not directly sown in the field for germination.  It is a time taking procedure.  Special skills are required to do so but still there are chances of attack of pathogens.  Germination of natural and artificial seeds is almost equal where as further conversion into seedling is 80-100% in natural seeds and 50% in artificial seeds.

35. References:  Gantait, S., S. Kundu, N. Ali and N.C. Sahu. 2015. Synthetic seed production of medicinal plants: a review on influence of explants, encapsulation agent and matrix. Acta Physiol Plant., 37: 98.  Nadeem, A., Siddique, M. Mujeeb, M. Rashid and K. Hussain. 2013. Synthetic Seed Production; its Relevance and Future Panorama. American J. PharmTech Research., 3: 3.

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37. Suggestions and recommendations ? 37