Introduction First proposed by Murashige in 1974, with successful production from different types of explants. Artificially produced by encapsulating plant tissues in a suitable matrix. They can be used for mass propagation, germplasm conservation, and genetic transformation of various plants. Advantages include production of uniform, disease-free plants, reduced cultivation time and cost, preservation of genetic diversity, and international germplasm exchange. Types of Synthetic Seeds Desiccated Synthetic Seed Hydrated Synthetic Seed Procedure for Production of Synthetic Seed Explant Selection 1. Somatic Embryos 2. Shoot Buds 3. Axillary Buds 4. Cell Aggregates 5. Protoplasts Matrix Composition 1. The concentration of the encapsulating agent 2. The type and concentration of cations & pH 3. The osmotic potential of the solution 4. The presence of additives Components Used in the Synthetic Seed Encapsulation methods for Synthetic seed Dropping Procedure The most useful encapsulation system. Drip 2-3% sodium alginate drop from at the tip of the funnel and the somatic embryos are inserted. Keep the encapsulated embryos complex m calcium salt for 20 min. Rinsed the capsules in water and then stored in a air tight container. Automatic Encapsulation process This is the quick method of artificial seed production Alginate solution with embryo is feed from supply tank Alginate capsules were planted in speeding trays using a vacuum seeder. The capsules are planted in the field using a stanhay planter A hydrophobic coating is required for mechanical handling Scope of Synthetics Seed Advantages of Synthetic Seed Limitations of Synthetic Seed Variability in germination and conversion rates can affect efficiency and reliability. Optimization of protocols for each plant species and synthetic seed type is necessary. Risk of microbial contamination during encapsulation, storage, or germination can affect quality and performance. Use of sterile techniques and appropriate additives is required. Optimization of protocols for synthetic seed production requires significant time, effort, and resources, and may vary depending on various factors. Extensive research and development is necessary to establish standardized and validated protocols.

1. Comparative Analysis of Different Explants for
Synthetic Seed Production
Sahil Sahu
PhD (Seed Science and Technology) Regd. No.- 2381909029
SOA- FAS- Department of Seed Science and Technology

2. Contents
• Introduction
• What is Synthetic Seed
• Why Synthetic Seed
• Types of Synthetic Seeds
• Procedure for Production of
Synthetic Seed
• Explant Selection
• Matrix Composition
• Encapsulating agent
• Components Used in Synthetic
Seed
• Encapsulation method for
Synthetic Seed
• Scope of Synthetic Seed
• Advantages of Synthetic Seed
• Limitations of Synthetic Seed
• Case Studies
• Future perspective
• Conclusion
• References

3. Introduction
• First proposed by Murashige in 1974, with
successful production from different types of
explants.
• Artificially produced by encapsulating plant
tissues in a suitable matrix.
• They can be used for mass propagation,
germplasm conservation, and genetic
transformation of various plants.
• Advantages include production of uniform,
disease-free plants, reduced cultivation time
and cost, preservation of genetic diversity,
and international germplasm exchange.
(Rihan et al., 2017; Murashige, 1974)

4. Natural Seed
A natural seed is defined
as the matured and
fertilized ovule of a plant.
Vegetative Propagation
It is a form of asexual
reproduction where new
plants are grown from various
parts of the plant like roots,
stems, and leaves
Synthetic Seed
A synthetic seed is an artificially encapsulated plant
propagation material that combines aspects of natural seeds
and vegetative propagation. It includes encapsulated somatic
embryos, shoot buds, cell aggregates, or any other
meristematic tissue that has the potential to regrow after
storage conditions, much like a natural seed.
What is Synthetic Seed

5. The Concept of Synthetic Seed
(Rihan et al., 2017; Ghosh and Haque, 2019)

6. Why Synthetic Seed
Heterozygosity of seeds in
cross pollinated crops
Minute seed size Presence of
reduced endosperm
No seeds are formed
E.g.- banana, pineapple
Low germination
rate

7. Types of Synthetic Seeds
I. Desiccated Synthetic Seed
a. Desiccated synthetic seeds are produced naked or polyoxymethylene
glycol encapsulated somatic embryo.
b. This type of synthetic seeds is produced in desiccation tolerant species of
plant.
Ex: Carrot, Celery embryo
II. Hydrated Synthetic Seed
a. Hydrated synthetic seeds are produced by encapsulating the somatic
embryos in hydrogels like sodium alginate, potassium alginate,
carrageenan, sodium pectate.
Ex: Alfalfa, Barley, Sandal wood
(Redenbaugh, 1986)

8. Procedure for Production of Synthetic Seed
Greenhouse and field planting
Mass production of synthetic seed
Standardization of artificial endosperm
Standardization of encapsulation
Mass production of somatic embryos
Synchronize and singulate somatic embryos
Mature somatic embryos
Establish somatic embryogenesis
(Rihan et al., 2017)

9. Procedure for Production of Synthetic Seed
(Gantait and Kundu, 2017)

10. Explant Selection
• The choice of explants is
crucial for the success of
synthetic seed production.
• Explants are plant tissues used
as the source of cells for
encapsulation.
• Different types of explants can
be used, each with its own
advantages and disadvantages.
(Nondgam, 2016)

11. 1. Somatic Embryos
• Most commonly used explants.
• Resemble zygotic embryos in morphology and development.
• Advantages: Ability to germinate and convert into plants without
intermediate steps, ease of handling and encapsulation.
• Limitations: Low viability and germination rates, loss of desiccation
tolerance, susceptibility to microbial contamination.
(Lyngved, 2009)

12. 2. Shoot Buds
• Meristematic tissues that can develop into shoots and leaves.
• Advantages: higher viability and germination rates, better desiccation
tolerance, lower risk of abnormalities.
• Disadvantages: need for an intermediate step of rooting before
conversion into plants, lower genetic stability and uniformity,
difficulty of handling and encapsulation due to size and shape.
Shoot bud
(Nondgam, 2016)

13. 3. Axillary Buds
• Special type of shoot buds located in the axils of leaves or bracts.
• They can be used for synthetic seed production of plants with a
sympodial growth habit.
• Advantages: ability to produce multiple shoots and plants,
preservation of the original genotype and phenotype, compatibility
with cryopreservation.
Axillary bud Axillary bud (Nondgam, 2016)

14. 4. Cell Aggregates
• Clusters of cells derived from callus or
suspension cultures.
• They can be used for synthetic seed
production of plants with a
recalcitrant or non-embryogenic
response to tissue culture.
• Advantages: high multiplication rate,
ease of handling and encapsulation,
possibility of genetic modification.
• Disadvantages: low viability and
germination rates, loss of desiccation
tolerance, susceptibility to microbial
contamination, occurrence of
somaclonal variation.
Callus, from which cell aggregates are derived
(Nondgam, 2016)

15. 5. Protoplasts
• Cells stripped of their cell walls.
• They can be used for synthetic
seed production of plants with a
high potential for somatic
embryogenesis.
• Advantages: ability to fuse with
other protoplasts or cells,
possibility of genetic
modification, ease of handling and
encapsulation.
• Disadvantages: low viability and
germination rates.
Protoplasts
(Nondgam, 2016)

16. Matrix Composition
• Mix of the encapsulating agent and additives.
• Forms a protective gel around the explants.
• Influences the synthetic seeds’ physical properties like gel strength,
viscosity, porosity, and water content.
• Affects chemical properties such as pH and osmotic potential.
• Influences biological properties like nutrient supply.
• Impacts the viability, germination, conversion, and characteristics of
synthetic seeds.
• It is essential to optimize the matrix composition for each plant
species to achieve the best results.
(Chandra et al., 2016)

17. 1. The concentration of the encapsulating agent
• The encapsulating agent’s
concentration determines the
gel’s strength and viscosity.
• Higher concentration = stronger
gel, but less permeability.
• The optimal concentration varies
from 1% to 5%, depending on
the agent and plant species.
(Sharma and Shahzad, 2012; Chandra et al., 2016)

18. 2. The type and concentration of cations & pH
• Cations affect the gelation of the agent.
• Different cations like calcium, magnesium, barium, etc., have different
effects on the gel properties and explant development.
• Calcium is commonly used but can inhibit cell division, and interfere
with germination and conversion.
• The optimal concentration varies from 50 mM to 200 mM (millimolar).
• The pH affects the gelation, matrix stability, and explant viability.
• A low pH can result in a weak and unstable gel, while a high pH can
result in a strong and stable gel but can affect the viability and vigour.
• The optimal pH varies from 5.5 to 7.5.
(Chandra et al., 2016)

19. 3. The osmotic potential of the solution
• Determined by the concentration of solutes added to the matrix.
• Affects the water content and water loss of the matrix, and the
viability and development of the explants.
• High osmotic potential can protect explants from microbial
contamination and enhance desiccation tolerance but can also cause
plasmolysis, osmotic stress, and metabolic inhibition.
• Optimal osmotic potential varies from -0.5 MPa to -1.5 MPa.
(Chandra et al., 2016)

20. 4. The presence of additives
• Enhances the performance of synthetic seeds by improving the
properties of the matrix and the development of the explants.
• Different types of additives can be added to the matrix, such as
activated charcoal, polyethylene glycol, sucrose, amino acids,
vitamins, hormones, antibiotics, fungicides, etc.
• Each additive has specific benefits, like improving optical properties,
stability, mechanical strength, permeability, osmotic potential,
nutrient supply, and preventing microbial infection.
(Chandra et al., 2016)

21. Encapsulating Agent
1.Role: Forms a protective layer around explants, crucial for synthetic seed
production.
2.Criteria for Selection:
1. Biocompatible, biodegradable, non-toxic.
2. Good gelation properties.
3. Good permeability.
4. Good mechanical strength.
5. Good optical properties.
6. Low cost and easy availability.
3.Alginate: Most widely used, extracted from brown algae, meets all criteria.
4.Other Agents: Agar, gelatin, carrageenan, pectin, chitosan, cellulose,
starch. Often used with additives to improve performance.
5.Choice of Agent: Depends on plant species, type of explants, matrix
composition. Optimization is essential for best results.
(Chandra et al., 2016)

22. Components Used in the Synthetic Seed
Gelling Agents
• Sodium Alginate
• Carrageenan
• Locust Bean gum
• Gelrite
• Agar
• Carboxymethylcellulose
• Sodium pectate
• Tragacanth Gum
Protective Chemical and Microorganism
• Rifampicin
• Cefotaxime
• Tetracycline-HCl
• Mycorrhiza
• Activated charcoal
Complexing Agent
• Calcium chloride
• Potassium chloride
• Ammonium chloride
Coating Agents
• Polylysine
• Elvax 4260
• Gantrez ES
• Glutaraldehyde
• Maleic anhydride
• Sodium alginate
Growth Regulator
• Gibberellic acid (GA3)
• Abscisic acid (ABA)
• Zeatin
• kinetin
• meta-topolinriboside (m-TR)
• 6-benzylaminopurine (BA)
• 2,4 - Dichlorophenoxy acetic acid (2, 4-D)
• Indole -3-acetic acid (IAA)
• 6-benzyaminopurine
• a-naphthaleneacetic acid (NAA)
• Thidiazuron

23. Encapsulation methods for Synthetic seed
A. Dropping Procedure
i. The most useful encapsulation
system. Drip 2-3% sodium
alginate drop from at the tip of
the funnel and the somatic
embryos are inserted.
ii. Keep the encapsulated embryos
complex m calcium salt for 20
min.
iii. Rinsed the capsules in water and
then stored in a air tight
container.
(Roy and Tulsiram, 2013)

24. B. Automatic Encapsulation
process
This is the quick method of
artificial seed production
i. Alginate solution with embryo is
feed from supply tank
ii. Alginate capsules were planted
in speeding trays using a
vacuum seeder.
iii. The capsules are planted in the
field using a stanhay planter
iv. A hydrophobic coating is
required for mechanical
handling
The planet gear
outer periphery
A Punched
(perforated) plate
Orifices
of 5 mm
stand
(Shelar et al., 2021)

25. Scope of Synthetics Seed

26. Advantages of Synthetic Seed
(Ghosh and Haque, 2019)

27. Limitations of Synthetic Seed
1. Variability in germination and conversion rates can affect
efficiency and reliability. Optimization of protocols for each plant
species and synthetic seed type is necessary.
2. Risk of microbial contamination during encapsulation, storage, or
germination can affect quality and performance. Use of sterile
techniques and appropriate additives is required.
3. Optimization of protocols for synthetic seed production requires
significant time, effort, and resources, and may vary depending on
various factors. Extensive research and development is necessary to
establish standardized and validated protocols.
(Ghosh and Haque, 2019)

28. Case Studies

29. Performance of encapsulated explants (clump’s base and node) of
Thornfree variety of Blackberry.
Source: Regni et al., 2024. The Influence of the Explant’s Type on the Performance of Synthetic Seeds of Blackberry (Rubus
spp.) , Plants; 13(32): 1-12
CASE
STUDY-
I

30. Performance of encapsulated explants (clump’s base and node) of Chester
variety of Blackberry.
Source: Regni et al., 2024. The Influence of the Explant’s Type on the Performance of Synthetic Seeds of Blackberry (Rubus
spp.) , Plants; 13(32): 1-12
CASE
STUDY-
I

31. Effect of different concentrations of auxins (in MS medium) on callus
induction using various types of explants.
Source: Salma et al., 2019. Somatic embryogenesis-mediated plant regeneration of Eclipta alba (L.) Hassk. and its conservation
through synthetic seed technology , Acta Physiologiae Plantarum; 41(103): 1-10
CASE
STUDY-
II

32. Effect of different concentrations of auxins on callus induction of D.
rotundata using various types of explants.
Source: Manoharan et al., 2016. Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata) , Plant
Cell Tiss Organ Cult; 126: 481-497
CASE
STUDY-
III
Auxins (mg/l)
Explant type
Immature
leaf lobes
Petiole Node
Stem
internode
Axillary
bud
Root
segments
Callus obtained after 8 weeks of culture
Control - - - - - -
2,4-D
0.5 ++ ++ ++ + - +
1.5 ++ ++ ++ + - +
NAA
0.5 CWR CWR CWSR CWR CWSR CWHR
1.5 CWR CWR CWSR CWR CWSR CWHR
Picloram
0.5 ++ +++ +++ + +++ ++
1.5 ++ ++ +++ + +++ ++
Control, MS medium without auxins; +++, proliferative callus; ++, moderately proliferative; +, less
proliferative; -, no response; CWR, callus with roots; CWSR, callus with shoot and root;
CWHR, callus with hairy roots

33. Effect of origin explants on SEs induction from embryogenic calluses
(ECs).
Source: Moon et al., 2013. Improvement of somatic embryogenesis and plantlet conversion in Oplopanax elatus, an endangered
medicinal woody plant , SpringerPlus; 2(1): 1-8
CASE
STUDY-
IV
A. Number of SEs induced from different explant-derived ECs, B. Histological differences of different
explant-derived ECs, a. leaf-derived EC, b. petiole-derived EC, c. root-derived EC.

34. Effect of nutrient composition of encapsulation matrix on regrowth of
different Explants
Source: Murthy et al., 2013. Synthetic seeds - A novel approach for the conservation of endangered C. spiralis wt. and C. pusilla
, Bangladesh Journal of Scientific and Industrial Research; 48(1): 39-42
CASE
STUDY-
V

35. Effect of different concentrations and combinations of plant growth
regulators on artificial seed proliferation in shoot tip explants and nodal
explants of M. arvensis.
Source: Islam and Bari., 2012. In vitro regeneration protocol for artificial seed production in an important medicinal plant
Mentha arvensis l , J. bio-aci; 20: 99-108
CASE
STUDY-
VI
Plant growth
regulators (mg/l)
Explant type
Shoot tip explants Nodal explants
Percentage of
germination
Average
number of
shoot per
culture (mean
± SE)
Average length
(cm) of shoot
per culture
(mean ± SE)
Percentage of
germination
Average
number of
shoot per
culture (mean
± SE)
Average length
(cm) of shoot
per culture
(mean ± SE)
BAP 1.0 50 2.20 ± 0.20 4.27 ± 0.17 64 5.67 ± 0.06 4.40 ± 0.11
2.0 40 4.20 ± 0.30 4.93 ± 0.29 56 4.20 ± 0.11 5.07 ± 0.17
BAP + Kn 1.0 + 0.2 45 2.60 ± 0.23 5.40 ± 0.11 50 4.00 ± 0.11 5.40 ± 0.11
1.0 + 0.5 60 2.93 ± 0.06 5.80 ± 0.23 65 5.87 ± 0.17 5.67 ± 0.06
2.0 + 0.2 44 2.80 ± 0.11 5.13 ± 0.06 55 4.47 ± 0.17 4.93 ± 0.17
2.0 + 0.5 65 4.33 ± 0.17 6.13 ± 0.29 72 6.47 ± 0.13 6.07 ± 0.17
BAP + NAA 1.0 + 0.2 50 2.60 ± 0.11 5.33 ± 0.06 50 6.00 ± 0.11 5.40 ± 0.11
1.0 + 0.5 65 4.27 ± 0.29 6.43 ± 0.08 72 7.87 ± 0.33 5.93 ± 0.06
2.0 + 0.2 52 2.87 ± 0.17 4.00 ± 0.11 54 4.33 ± 0.29 4.27 ± 0.17
2.0 + 0.5 60 4.20 ± 0.11 5.33 ± 0.13 80 9.87 ± 0.58 5.27 ± 0.24

36. Development of Rhodiola kirilowii axillary buds and differentiating callus
encapsulated in calcium alginate hydro gel
Source: Zych et al, 2005. Micropropagation of Rhodiola kirilowii plants using encapsulated axillary buds and callus , ACTA
BIOLOGICA CRACOVIENSIA Series Botanica; 47(2): 83-87
CASE
STUDY-
VII
Plant material used for
encapsulation
Method of
encapsulation Time of
preservation at
4°C (weeks)
% of developed
axillary buds
Time of
development in
24°C (days)
5% SA (Sodium
Alginate)
Axillary shoot buds of
plantlets developed in
vitro
+ 6 100 10-12
Differentiating callus of
hypocotyl origin
+ 6 95 8-10

37. Future Perspectives
1.Improve germination and conversion rates
1.Enhance genetic stability
1.Incorporate secondary metabolites
1.Integrate cryopreservation
1.Evaluate field performance

38. Conclusion
1.Synthetic seeds are artificial seeds produced by encapsulating plant
tissues in a suitable matrix.
2.Used for mass propagation, germplasm conservation, and genetic
transformation of medicinal plants.
3.Offers advantages over conventional methods but also faces
challenges such as variability in germination and conversion rates, risk
of microbial contamination, and need for protocol optimization.
4.Requires further research and development to overcome challenges
and improve efficiency and reliability. Opens new horizons for
conservation and utilization of medicinal plants.

39. References
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encapsulation agent and matrix. Acta Physiologiae Plantarum, 37, 1-12.
• Rihan, H. Z., Kareem, F., El-Mahrouk, M. E., & Fuller, M. P. (2017). Artificial seeds (principle, aspects and applications). Agronomy, 7(4),
71.
• Jain, S. M., & Saxena, P. K. (Eds.). (2009). Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal
plants (Vol. 547). New YorkNew York: Humana Press.
• Redenbaugh, K., Paasch, B. D., Nichol, J. W., Kossler, M. E., Viss, P. R., & Walker, K. A. (1986). Somatic seeds: encapsulation of asexual
plant embryos. Bio/technology, 4(9), 797-801.
• Zych, M., Furmanowa, M., Krajewska-Patan, A., Łowicka, A., Dreger, M., & Mendlewska, S. (2005). Micropropagation of Rhodiola
kirilowii plants using encapsulated axillary buds and callus. Acta Biologica Cracoviensia Series Botanica, 47(2), 83-87.
• Islam, M. S., & Bari, M. A. (2012). In vitro regeneration protocol for artificial seed production in an important medicinal plant Mentha
arvensis L. Journal of Bio-Science, 20, 99-108.
• Murthy, K. S. R., Reddy, M. C., & Kondamudi, R. (2013). Synthetic seeds-A novel approach for the conservation of endangered C. spiralis
wt. and C. pusilla. Bangladesh Journal of Scientific and Industrial Research, 48(1), 39-42.
• Moon, H. K., Kim, Y. W., Hong, Y. P., & Park, S. Y. (2013). Improvement of somatic embryogenesis and plantlet conversion in Oplopanax
elatus, an endangered medicinal woody plant. SpringerPlus, 2, 1-8.
• Manoharan, R., Tripathi, J. N., & Tripathi, L. (2016). Plant regeneration from axillary bud derived callus in white yam (Dioscorea
rotundata). Plant Cell, Tissue and Organ Culture (PCTOC), 126, 481-497.
• Salma, U., Kundu, S., Ali, M. N., & Mandal, N. (2019). Somatic embryogenesis-mediated plant regeneration of Eclipta alba (L.) Hassk. and
its conservation through synthetic seed technology. Acta Physiologiae Plantarum, 41, 1-10.

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41. The future of agriculture lies not just in the soil, but
in the seeds we sow and the science that guides their
growth.
“
”

42. THANK YOU