SEMINAR on RECENT STUDIES ON SYNTHETIC SEED PRODUCTION IN HORTICULTURAL CROPS

1. Presented By
Milan N. Thummar
Seed Science and Technology.
Department of Genetics and Plant Breeding.
C. P. College of Agriculture
S. D. Agricultural University
Sardarkrushinagar.
Reg. no.:- 04-AGRMA-2294-2020
Major Advisor
Dr. D. G. Patel
Associate Research Scientist,
Cotton Research Station,
S. D. Agricultural University
Talod-383215,
Sabarkantha.
RECENT STUDIES ON SYNTHETIC SEED PRODUCTION
IN HORTICULTURAL CROPS
Minor Advisor
Dr. Y. A. Viradiya
Assistant Research scientist,
Department of Seed technology.
S. D. Agricultural University
Sardarkrushinagar.

2. CONTENTS
2
Introduction
The Concept of Artificial Seed
Scope of Synthetic Seed
Land Marks on Synthetic Seed
Types of Artificial Seed
Procedure for Production of Artificial Seed
Artificial Seed Production and Plant Conversion
Advantages of Artificial Seeds
Case Studies
Limitations
Conclusions

3. • Seed serves several functions to the plant like nourishment of the embryo,
dispersal to a new location and dormancy during unfavorable environment
• The concept of synseed was first conceived by Murashige in 1978
• Encapsulated somatic embryos, which functionally mimic seed and can develop
into seedling under suitable condition
• The production of Synthetic seed is useful for plants which do not produce viable
seeds.
• Under in vitro conditions, encapsulation is the most effective approach for
protecting micro propagules and converting them to synseeds
• Artificial seeds have further advantages in storage, handling and shipping due to
small sized
• Artificial seeds have been used to grow a variety of fruits, vegetables, cereals,
orchids, ornamentals, and forest trees
INTRODUCTION
3

4. What is synthetic seed?
• Synthetic seeds are artificially
encapsulated somatic embryos or
other vegetative parts such as shoot
buds, cell aggregates, auxiliary buds,
or any other micropropagules which
can be sown as a seed and converted
into a plant under in vitro or in vivo
conditions
Rihan et al. (2017)
• Botanically- Seed is a ripened ovule
• Genetically- Connecting link between
two generations for transfer of traits
• Agriculturally- Any plant part with
regeneration capacity
What is seed?
4
Fig. 1

5. Artificial
seed coat Somatic
embryo
Artificial
endosperm
The Concept of Artificial Seeds
Rihan et al. (2017)
5
Fig. 2: concept of Artificial seed

6. Artificial seed Vs Natural seed
6
Fig. 3: Diagrammatic representation of seed A. Artificial seed B. Natural Seed
A. B.

7. Synthetic Seed
Propagation Conservation
Short to medium
term storage
Long term storage
Transportation
• Rare and endangerd plants
• Elite genotype
• Genetically engineered plants
• Seedless plants
• Commercially important
plants
• Exchange of axenic plant
material free of
undesirable contaminants
Slow-growth conservation
• Maintenance under reduced temperature and
reduced light intensity
• Use growth retardant such as ABA
• Use of minimal growth medium
• Use of osmoticum
• Reduction of oxygen concentration
Cryopreservation
• Encapsulation-dehydration
• Encapsulation-vitrification
(Synthetic seed based cryopreservation)
Scope of Synthetic seed
Suresh et al. (2021)
7

8. Year Researcher Remarks
1958
Stewart and
Coworker
Somatic embryogenesis in carrot
1977 Murashige
Somatic embryos were first time encapsulated with
encapsulating material
1978 Murashige
Gave first idea of synthetic seeds and proposed that somatic
embryos can be encapsulated, handled and used like a natural
seed for transport, storage and sowing
1982 Kitto and Janaick First report on synthetic seed in carrot
1984
Reden and
Coworker
Developed a technique for hyrdogel encapsulation ( Calcium
Alginate as Coating agent) of individual somatic embryos of
alfalfa
Land Marks on Synthetic Seed
8

9. Year Researcher Remarks
1986 Rodenbaugh Hydrogel encapsulation technology
1988 Bapat and Rao Synthetic seed in sandalwood and mulberry
1994 Onishi and Coworker Automation of synthetic seeds
2011 Asmah and Coworker
Explants such as shoot tips, auxillary buds and
somatic embryos in cryoprotectant material like
hydro gel, alginate gel, ethylene glycol, dimethyl
sulfoxide (DMSO) can be used
2011 Ma and Coworker
Given synthetic seed production technique by
forming beads using encapsulated somatic embryo
with coating materials
2017 Micheli and Coworker Encapsulation of black mulberry micro cuttings
9

10. Types of Artificial Seeds
1) Hydrated seeds
• These seeds are produced by the encapsulation of hydrogel to somatic embryos.
They are produced in recalcitrant and desiccation sensitive plant species (Ara et
al., 2000)
2) Dessicated seeds
• The seeds are naked with polyoxyethylene glycol encapsulation and later
desiccated.
• This dehydration arises either by overnight drying in an unsealed petri dishes
or by reducing relative humidity/moisture of the seeds (Ara et al., 2000).
• The higher level of osmotic potential is attained by increasing the strength of
gel and on addition with several osmoticants like mannitol, sucrose, etc
(Sundararaj, 2010) to the medium that can induce the tolerance to desiccation
and it can be induced by various stresses like low temperature and deficiency of
nutrients (Pond and Cameron, 2003) etc. They are made in the somatic embryos
in order to tolerate the desiccation process (Sharma et al., 2013)
10

11. • Non zygotic embryos with bipolar
structures
• Capable of growing into complete
plants
• Process by which somaticcells
develop into differentiated embryos
Somatic embryos
11
Fig. 4 & 5: stages of somatic embryo developement
Somatic embryogenesis

12. 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
Suresh et al. (2021)
Procedure for Production of Artificial Seed
12

13. Fig. 6: Flow diagram presenting the procedure of synthetic seed production
13

14. Encapsulation Methods for Synthetic Seed
A. Dropping procedure
2% sodium alginate
14
Fig. 7: Dropping procedure of encapsulation of synthetic seed

15. B. Molding method
• This method follows simple procedure of mixing of
embryos with temperature dependent gel (eg., gel rite
and agar).
• Cells get coated with the gel at lowering of the
temperature.
15
Fig. 8: molding tray for encapsulation

16. Complexing Agent
• Calcium chloride
• Potassium chloride
• Ammonium chloride
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
• α-naphthaleneacetic acid (NAA)
• Thidiazuron
Protective Chemical and Micro-
Organism
• Rifampicin
• Cefotaxime
• Tetracycline-HCl
• Mycorrhiza
• Activated charcoal
Coating Agents
• Polylysine
• Elvax 4260
• Gantrez ES
• Glutaraldehyde
• Maleic anhydride
• Sodium alginet
Gelling Agents
• Sodium Alginate
• Carrageenan
• Locust Bean gum
• Gelrite
• Agar
• Carboxymethylcellulose
• Sodium pectate
• Tragacanth Gum
Components Used in the Synthetic Seed
16

17. In vitro propagules for
encapsulation
Crop
Somatic embryos
Papaya, Brinjal, Mango, Carrot, Sandalwood,
Brassica, Alfalfa, Lettuce and Celery,
Auxiliary buds/
Adventitious buds
Vitis, Eucalyptus, Citrus, Pineapple
Shoot tips Banana, Cardamom, Apple, Kiwifruit and Potato
17
Table1: Artificial seed production and plant conversion

18. Suresh et al. (2021)
Advantages of Artificial seeds
18
Fig. 9

19. Case studies
19

20. Optimization of the conditions for production of synthetic seeds
by encapsulation of axillary buds derived from minitubers
sprouts
in potato( Solanum tuberosum)
Hamedan (Iran) Abdollahi et al. (2016)
Material
• Sodium alginate and CaCl2
• MS medium (pH 5.7) with
• 5 mg l benzy-ladenine (BA),
• 10 mg l −1 NAA and
• 300 mg l −1 activated Charcoal
20
1

21. Fig. 10 A: Potato minituber 2
months after harvest and storage at
3–50C, showing axillary buds
(arrows).
Fig. 10 B: Axillary buds
encapsulated in calcium alginate
beads
Fig.10 C: Shoot regrowth and
elongation.
Fig .10 D: Shoot and root
emergence from encapsulated
buds.
Fig.10 E: Conversion of regrowing
buds into plantlets in 7*7 cm, coco
peat-containing pots from miniature
tubers.
Fig.10 F: Plantlet obtained from
encapsulated axillary buds after 4 weeks
of culture. 21

22. Table 2: Effect of three concentrations of sodium alginate and two concentrations of CaCl2
on regrowth rate and speed of encapsulated buds from ‘Sante’ potato after 2 weeks
of culture
Sodium alginate (%) CaCl2 (%)
Regrowth
Rate (%) Speed
2.5
1 56 ab
0.76 a
1.5 44 bc
0.24 c
3
1 61 a
0.46 b
1.5 56 ab
0.48 d
3.5
1 11 d
0.06 d
1.5 33 c
0.28 c
(Different letters indicate significant differences according to Duncan’s multiple test)
They measured regrowth speed using following formula
Regrowth speed =
n1
t1
+
n1
t1
+ ⋯ +
nn
tn
Where,
n1, n2, ..nn are the number of emerged buds at times t1, t2, …, tn measured in days
22

23. Table 3: Effect of explant size on regrowth rate and speed of encapsulated buds after 2 weeks
of culture of two potato cultivars
Cultivar Bud size (mm)
Regrowth
Rate (%) Speed
Sante
1-2 33b
0.25bc
2-3 78a
1.04a
Agria
1-2 19c
0.14c
2-3 39b
0.34b
Table 4: Effect of the concentration of MS salts on regrowth rate and speed of two sizes of
encapsulated buds of two potato cultivars after 2 weeks of culture.
Bud size (mm) Cultivar
MS medium
strength
Regrowth
Rate (%) Speed
1–2
Sante
Full 39 c
0.32 bc
Half 28 cd
0.18 bc
Agria
Full 22 cd
0.17 bc
Half 17 d
0.11 c
2–3
Sante
Full 89 a
1.13 a
Half 67 b
0.95 a
Agria
Full 39 c
0.3 bc
Half 39 c
0.38 b
23
(Different letters indicate significant differences according to Duncan’s multiple test)

24. Table 5: Effect of three different substrates in the conversion of growing buds into
plantlets after 4 weeks.
Cultivar Substrate
Root length
(mm)
Shoot length
(mm)
Stem
diameter
(mm)
Number of
leaves
Sante
Coco peat 117 a
77 a
2.1 a
4.7 a
Perlite 97 b
53 b
1.6 b
3.3 b
Soil mixture 67 c
43 c
1.4 cd
2.3 c
Agria
Coco peat 62 c
42 c
1.5 c
3.7 ab
Perlite 53 d
32 d
1.4 d
2.3 c
Soil mixture 32 e
26 e
1.2 e
1.0 d
24
(Different letters indicate significant differences according to Duncan’s multiple test)

25. Somatic embryogenesis, encapsulation, cold storage, and growth
of hybrid Citrus [C. paradisi Macf. (‘Duncan’) × C. reticulata
Blanco. (‘Dancy’)]
shoot tip segments
Sari, (Iran) Gholami and Kaviani (2018) 25
2
Fig. 11 a: Shoot tips encapsulated in 3%
Na-alginate and 100 mM CaCl2 Synthetic
seeds are diaphanous and asymmetrical
Fig. 11 b: Shoot tips encapsulated in 4%
Na-alginate and 100 mM CaCl2.
Synthetic seeds are firm and isometric
Fig. 11 c: Shoots emerging from
encapsulated shoot tips.
Fig. 11 d: Root induction in MS medium
supplemented with 5 mg l −1 IBA.

26. Fig. 12 a: Direct somatic embryogenesis on excised
immature seed in globular stage marked by arrow.
Fig. 12 c: Somatic embryos at different stages of
embryogenesis. Ge: globular embryo,
Ce:cotyledonary embryo.
Fig. 12 b: Compact and light green callus.
Fig. 12 d: Production of plantlets after 6
months on germination medium.
26

27. Table 6: Influence of different storage durations
27
Four replicates, each containing 5 synthetic seeds were used for each treatment. Values are expressed as
mean ± standard error (SE). A1: Na-alginate 4% (w/v) + liquid MS medium + 50 g l−1 sucrose + 10 mg l−1
BAP + 1 mg l−1 NAA + 100 mM CaCl2.
Encapsulated shoot tips (A1) Non-encapsulated shoot tips
Storage time 8045** 5923**
Error 120.9 61.72
CV (%) 27.8 56.56
Storage time (weeks)
0 80 ± 5.44a 65 ± 6.25a
1 72 ± 5.35a 45 ± 4.25b
2 60 ± 6.25b 15 ± 7.44c
3 55 ± 7.45b 00 ± 00d
4 34 ± 8.44c 00 ± 00d
5 28 ± 5.25c 00 ± 00d
6 15 ± 9.94d 00 ± 00d
7 12 ± 5.55d 00 ± 00d
8 00 ± 00e 00 ± 00d
LSD (α < 0.05) 9.78 6.99

28. Fig. 10 Effect of media type (MS medium with or without hormones) on the conversion of
encapsulated shoot tips of hybrid citrus after cold storage.
MS with hormones: solid
MS medium + 50 g l−1
sucrose + 10 mg l−1 BAP + 1
mg l−1 NAA . The bars
represent mean ± SE.
28

29. Table 7: Effect of three different media on Somatic embryo development stage
B1 :- MS medium supplemented with 500 mg l−1 malt extract and 30 g l−1 sucrose
B2 :- MS medium supplemented with 500 mg l−1 malt extract and 50 g l−1 sucrose
B3 :- MS medium supplemented with 500 mg l−1 malt extract, 50 g l−1 sucrose and 3 mg l−1 BAP.
29
Indirect
embryogenes
is (%)
Embryogenic
callus (%)
Globular
(%)
Heart (%)
Torpedo
(%)
Cotyledonary
(%)
Plantlet
growth
(%)
Media 2132** 2217** 2258** 2211** 2326** 2032** 1949**
Error 39.59 19.53 266.3 34.11 57.12 15.78 150
CV (%) 13.03 12.79 51.2 20.93 31.95 19.57 59.84
Media
B3
68.80 ±
4.02a
51.60 ±
3.02a
49.35 ±
3.84a
45.07 ± 6a
42.25 ±
3.27a
40.05 ± 4.15a
39.40 ±
2.30a
B2
48.70 ±
1.22b
41 ± 2.79b
38.10 ±
3.52a
34.20 ± 1.28a
28.70 ±
1.98b
24.10 ± 2.31b 22 ± 1.49b
B1
27.50 ±
4.44c
11 ± 1.78c 8.23 ± 4.60b 4.45 ± 2.70b 0 ± 0c 0 ± 0c 0 ± 0c
LSD 11.251 5.625 26.14 16.87 5.625 5.625 22.503
Data are the mean values of 20 replications. Values are expressed as the mean ± SE. Means within columns with the same letter were not
statistically different at p < 0.05 according to the Duncan's multiple range test.

30. Somatic embryogenesis, biochemical alterations and synthetic seed
development in two varieties of coriander (Coriandrum sativum L.)
Ali et al.(2018)
New Delhi (India)
30
3
Fig. 13 a: Globular embryos at induction stage Fig. 13 b: Somatic embryos at proliferation stage
Fig. 13 c and d: Somatic embryos at maturation stage

31. Fig. 14 Synthetic seed development and plantlet formation in Coriandrum sativum (RS).
Fig. 14 a: Encapsulated
somatic embryos
Fig. 14 b: Synthetic seeds
on the germination medium
Fig. 14 c:Germinating synthetic
seeds
Fig. 14 d: Rooted plantlet Fig. 14 e: Synthetic seed derived
plant, grown in outdoor condition.
31

32. Table 8: Effect of different concentrations of 2,4-D on callus induction and somatic
embryogenesis from hypocotyl explants of ‘Rajendra Swathi’ and ‘Co-1’ varieties
of Coriandrum sativum
32
Rajendra swati Co-1
2,4-D (mg/l)
Callus
induction (%)
Embryogenic
callus
induction
frequency
No. of
embryos
formed/cultur
e (0.5 g)
Callus
induction
(%)
Embryogenic
callus
induction
frequency
No. of
embryos
formed/cultu
re (0.5 g)
0.5 86.0±4.0 a 55.3±2.5 b 39.3±2.1 b 89.3±3.0 b 52.0±2.64 b 36.3±2.08 c
1 89.3±4.2 a 77.6±3.2 a 63.0±4.5 a 96.0±2.3 a 72.8±3.0 a 51.0±2.64 d
1.5 74.0±4.0 b 58.0±2.6 b 44.5±2.5 b 80.7±4.1 c 54.2±2.51 b 41.5±2.0 b
2 47.3±1.1 c 45.2±2.3 c 31.0±3.0 c 64.0±2.0 d 42.0±3.0 c 27.0±3.0 d
Values are expressed as mean standard deviation, mean values within a column followed by different
letters are significantly different (at p= 0.05) according Duncan’s multiple range test.

33. Table 9: Somatic embryo differentiation and germination frequency in ‘RS’ and ‘Co-1’ on
different concentrations of NAA, BA and GA3 supplemented MS medium
Values are expressed as mean standard deviation, mean values within a column followed by different
letters are significantly different (at p = 0.05) according Duncan’s multiple range test.
33
NAA (mg/l)
BA (mg/l)
GA3 (mg/l)
Rajendra Swathi Co-1
Embryo
differentiatio
n
Conversion
rate
Embryo
differentiatio
n
Conversion
rate
0 0.5 0.25 0 54.6±3.0 c 0 51.3±3.0 c
0 1 0.25 0 68.0±3.4 b 0 64.0±3.4 b
0 1 0.5 0 83.3±4.6 a 0 76.7±4.1 a
0 1.5 0.5 0 63.6±3.0 b 0 54.0±2.0 c
0.5 0.25 0 78.7±4.1 a 0 74.0±4.0 a 0
1 0.25 0 61.3±3.0 b 0 55.3±3.0 b 0
1 0.5 0 56.0±3.4 b 0 47.2±2.3 c 0
1.5 0.5 0 39.3±3.0 c 0 34.0±2.0 d 0

34. Table 10:Effect of different concentrations of sodium alginate and calcium chloride on the
conversion rate of encapsulated somatic embryos on 1.0 mg l-1 BA and 0.5 mg l-1
GA3 MS medium
Alginate
(%)
Calcium chloride
(mM)
Conversion rate (%)
Rajendra Swathi Co-1
2 weeks 4 weeks 2 weeks 4 weeks
2
75 36.7±2.3 d 45.3±3.0 d 29.3±2.3 d 38.0±3.5 d
100 42.6±3.0 c 48.6±2.3 cd 36.6±3.0 c 43.3±3.0 c
125 30.0±2.0 e 37.3±3.0 e 24.0±2.0 e 32.6±2.3 e
3
75 46.0±3.4 c 52.0±3.4 c 41.3±3.0 e 51.3±3.0 b
100 63.3±4.2 a 74.0±4.0 a 64.0±3.4 a 70.6±4.1 a
125 52.0±3.4 b 57.3±2.3 b 45.3±2.3 b 54.7±3.0 b
4
75 21.3±2.3 f 24.6±3.0 g 18.0±2.0 f 24.0±2.0 f
100 25.3±3.0 ef 30.0±2.0 f 21.3±2.3 e 26.6±2.5 f
125 12.0±2.0 g 20.6±2.3 g 10.6±1.15 f 19.3±1.1 f
Values are expressed as mean standard deviation, mean values within a column followed by different
letters are significantly different (at p= 0.05) according Duncan’s multiple range test.
34

35. Table 11:Conversion rate at temperature conditions of somatic embryos encapsulated in 3%
sodium alginate and 100 mM CaCl2, after storage. MS was added with 1.0 mg l-1 BA
and 0.5 mg l-1 GA3
35
Storage
temperature
Storage duration
(weeks)
Regeneration (%)
Rajendra Swathi Co-1
2 weeks 4 weeks 2 weeks 4 weeks
-20°C
1 12.6±2.3 f 13.3±1.1 f 6.6±1.1 f 9.3±1.15 e
3 0 0 0 0
4oC
1 57.3±4.1 a 62.0±4.0 a 54.0±3.5 a 58.6±3.0 a
3 38.0±2.0 b 44.0±3.4 b 31.3±3.0 b 37.3±3.0 b
5 24.0±2.0 d 32.0±2.0 c 19.3±2.3 d 27.3±3.0 c
7 10.6±1.1 f 18.6±1.1 e 09.3±1.1 f 16.0±2.0 d
25°C
1 33.3±3.0 c 42.6±3.0 b 27.3±2.3 c 34.6±2.3 b
3 18.0±2.0 e 27.3±2.3 d 13.3±1.15 e 19.3±1.15 d
Values are expressed as mean standard deviation, mean values within a column followed by different
letters are significantly different (at p= 0.05) according Duncan’s multiple range test.

36. The Optimized Protocols for Production, Adaptation and
Keeping of the Produced Artificial Seeds from Encapsulated
Lateral Buds in Stevia Rebaudiana
Shaafi et. al. (2021)
Hamedan (Iran)
36
4
Fig. 15 a: The branches of
original plant including
lateral buds.
Fig. 15 b: Encapsulated lateral
buds with 2.5% sodium alginate
and 1% calcium chloride.
Fig. 15 c: Emerging and
elongation of the shoot of the
artificial seed.
Fig. 15 d: Shoot and root
emergence from produced
artificial seeds.
Fig. 15 e: Three different types of the used seedbed of
artificial seed for adaptation of produced planets
including; potting soil (e1), perlite (e2) and cocopeat (e3).
Fig. 15 Steps of production of artificial seeds with lateral buds of Stevia rebaudiana.

37. Table 12: Effect of three concentrations of sodium alginate and three concentrations of
CaCl2 on the germination percentage and placement of the explants in the center of
the capsules after 3 weeks of culture
Sodium alginate
(%)
CaCl2 (%) Germination (%)
Placement of the
explants in the capsule
(%)
2
0.75 25 c
28.75 d
1 25 c
25.00 c
1.25 25 c
28.75 d
2.5
0.75 25 c
42.50 c
1 46.25 a
83.75 a
1.25 28.75 b
77.50 b
3
0.75 21.25 d
17.50 g
1 21.25 d
21.25 f
1.25 17.5 e
17.50 g
Different letters indicate significant differences according to Duncan’s multiple test range with
p ≤ 0.05.
Materials: MS liquid medium, BAP, NAA ,Sodium alginate and Calcium chloride.
37

38. Explants size
(mm)
Culture medium
type
Regrowth
Rate (%) Speed
1.00-1.99 MS 31.00 c
0.09 bc
B5 31.00 c
0.07 c
2.00-3.00 MS 68.00 a
0.24 a
B5 50.00 b
0.13 b
Table 13: Effect of explants size and culture medium on regrowth in encapsulated buds after 3
weeks of culture
38
Different letters indicate significant differences according to Duncan’s multiple test range with p ≤ 0.05.

39. Explants type Bud size (mm) Regrowth speed
Lateral bud 1.00-1.99 0.10 bc
Lateral bud 2.00-3.00 0.23 a
Apical bud 1.00-1.99 0.06 c
Apical bud 2.00-3.00 0.14 b
Table14: Effect of explants type and explants size on regrowth speed of capsulated buds after 3
weeks of culture
Seed application
method
Substrate
Number of
leaflets
Stem diameter
(mm)
Direct seed
application
cocopeat 5.75 c
1.27 bc
perlite 4.75 cd
1.22 c
garden soil 4.25 d
1.12 c
Seed germinated on
MS
cocopeat 10.20 a
1.88 a
perlite 9.33 ab
1.50 b
garden soil 7.25 b
1.30 bc
Table 15: Effect of seed application method and seedbed type on the number of leaflets and
stem diameter of encapsulated lateral buds after 3 weeks of culture
39
Different letters indicate significant differences according to Duncan’s multiple test range with p ≤ 0.05.

40. BAP
concentration
(mg/l)
NAA
concentration
(mg/l)
Regrowth
Shoot length (cm)
Rate (%) Speed
0
0 71 b
0.3 e
1.2 k
0.5 71 b
0.3 e
1.3 jk
1 73 b
0.3 de
1.4 ij
1.5 75 b
0.3 bcde
1.4 hi
0.5
0 73 b
0.3 cde
1.5 h
0.5 73 b
0.3 bcde
1.5 gh
1 75 b
0.3 bcde
1.6 fg
1.5 75 b
0.3 bcde
1.6 ef
1
0 73 b
0.3 bcde
1.7 de
0.5 75 b
0.3 bcde
1.7 cd
1 75 b
0.3 bcde
1.8 bcd
1.5 75 b
0.3 bcd
1.8 bc
1.5
0 75 b
0.3 ab
1.8 ab
0.5 83 a
0.4 a
1.9 a
1 75 b
0.3 abc
1.8 ab
1.5 75 b
0.3 bcd
1.8 abc
Table 16: Effect of the different concentrations hormones BAP and NAA on regrowth rate,
speed and shoot length encapsulated lateral buds after 3 weeks of culture.
40
Different letters indicate significant differences according to Duncan’s multiple range test.

41. Maintenance methods
Time of storage
(day)
Regrowth
Rate (%) Speed
Without MS medium at
4°C
0 71.2 a
0.3 a
30 71.2 a
0.3 a
60 53.7 b
0.2 abc
90 50.0 b
0.1 d
Inside MS medium at 4°C
0 71.2 a
0.3 a
30 71.2 a
0.3 a
60 71.2 a
0.3 ab
90 53.7 b
0.1 cd
Inside liquid
paraffin at 4°C
0 71.2 a
0.3 a
30 71.2 a
0.3 a
60 71.2 a
0.2 bc
90 67.5 ab
0.1 cd
Table 17: Effect of three different maintenance methods and four times of storage at 4°C on
regrowth rate and speed of encapsulated lateral buds after 3 weeks of culture
41
Different letters indicate significant differences according to Duncan’s multiple range test.

42. Effective use of synthetic seed technology in the regeneration of
Cymbidium aloifolium using protocorm-like bodies.
Verma and Pathak (2020)
Chandigarh (India) 42
5
Fig. 16 a: Spherical, non-leaky and
firm seeds with 3% sodium alginate
and 100 mM calcium chloride.
Fig. 16 b: Multiple shoot
formation (M + BAP (1 mg l −1 )).
Fig. 16 c: Formation of protocorm-like
bodies (PLBs) (M + IBA (1 mg l −1 ) +
BAP (1 mg l −1 )).
Fig. 16 d & e: Formation of long roots and complete plantlet
formation (M + IAA (1 mg l −1 )).

43. Fig. 16 f & g:Multiplication of PLBs
(M + IAA (1 mg l −1 ) + KN (1 mg l −1 )).
Fig. 16 h & i: Formation of leaf
primordia(M + 2, 4- D (1 mg l −1 )).
Fig. 16 j: Multiplication of PLBs and
complete plantlet formation (M).
Fig. 16 k: Complete plantlet
formation with well-developed
roots (M + BAP (1 mg l −1 )).
Materials: Calcium chloride, Sodium alginate, MS medium, IAA, BAP, KN and
IBA
43

44. Fig. 15: Effect of temperature and storage on the conversion frequency of synthetic seeds in
Cymbidium aloifolium.
44

45. Table 18: Effect of different growth additives on time taken for initiation response and plantlet
formation (days) in synthetic seeds immediately after their preparation in
Cymbidium aloifolium .
Additives (1 mg l−1
)
Time taken for
initiation response
(days)
Time taken for
plantlet formation
(days)
Remarks
MS 25 45
Protocorm-like bodies
(PLBs) multiplication
MS + IAA 38 58
Formation of plantlets with
long roots
MS + 2,4-D 34 54 –
MS + BAP 30 51 PLBs multiplication
MS + IAA + KN 40 60 PLBs multiplication
MS+ IBA + BAP 42 62 PLBs multiplication
45

46. Synthetic Seed Preparation, Germination and Plantlet
Regeneration of Litchi (Litchi chinensis Sonn.)
Das et al. (2016)
Bhagalpur (India)
a b c
d e f
g h
f
i
46
6
Fig. 16 a: Embryogenic calli
originated from zygotic embryos of
cultivar Bedana
Fig. 16 b: Globular somatic
embryos differentiated from
embryogenic calli.
Fig. 16 c: Cotyledonary stage somatic
embryos
Fig. 16 d: Encapsulation of somatic
embryos
Fig. 16 e: Germinating ESEs
showing emergence of root
Fig. 16 f: Emergence of shoot meristem
at the tip of elongated somatic embryo

47. Fig. 16 (g) in somatic embryos
roots are elongated in liquid
medium
Fig. 16 (h)-(i) development of study root and shoot
systems in semi-solid medium.
Fig. 16 (j) In vitro grown litchi
plantlets in semi solid medium
Fig. 16 (k) acclimatized in vitro litchi
plantlets were transferred into field soil.
Material: B5 and NN media,non-encapsulated somatic embryos (NSEs) and
encapsulated somatic embryos (ESEs).
47

48. Table 19: Response of Non-encapsulated somatic embryos (NSEs) and Encapsulated somatic
embryos (ESEs) on agar medium
Exp No.
Concentration of
macro salts
Germination (G)
(%)
Conversion (C)
of plants (%)
G/C
1 Full strength 10 6.64 1.49
2 Half strength 21.7 13 1.55
3 Quarter strength 69.1 36 1.91
4 One eighth strength 35.7 24.3 1.46
Table 20:Influence of B5 (Gamborg medium) macrosalt on germination and plantlet
development
48
Exp. No.
Conditions of
somatic embryos
Germination (%) Dead (%)
Arrested
germination (%)
Plantlets (%)
1 NSEs 23.7 ± 3.7 30.0 ± 6.5 44.1 ± 3.1 8.0 ± 1.6
2 ESEs 44.3 ± 4.5 18.0 ± 3.8 17.2 ± 4.3 16.0 ± 1.6
Mean value of three independent experiments ± SE and petri plates are in triplicate in each experiment.
Mean value of three independent experiments ± SE and petri plates are in triplicate in each experiment.

49. Table 21: Responses of ABA-treated and non-treated ESEs on agar medium
Exp. No.
Sucrose
concentration
(%)
Survival of somatic embryos (%)
Without ABA With ABA
1 1 5 7
2 3 15 56.7
3 6 30 46.7
4 9 61 92.3
Table 22: Effect of sucrose with or without ABA in NN basal medium (Nitsch and Nitsch
medium) on the survival of somatic embryos after 4 - 6 weeks of culture
49
Exp. No.
Concentration
s of ABA (µM)
Germination
(%)
Dead (%)
Arrested
germination
(%)
Plantlet (%)
1 0 78.0 ± 3.1 17.5 ± 1.7 4.0 ± 1.6 42.1 ± 1.9
2 0.004 72.0 ± 2.1 20.5 ± 1.1 5.5 ± 1.1 39.1 ± 1.9
3 0.02 69.8 ± 3.0 20.5 ± 2.9 6.1 ± 1.5 36.1 ± 3.4
4 0.04 23.3 ± 3.2 31.3 ± 2.3 39.5 ± 2.1 12.2 ± 1.1
5 0.2 8.0 ± 1.6 41.5 ± 2.3 38.5 ± 1.1 3.0 ± 1.6
6 0.4 0 64.7 ± 1.5 40.0 ± 1.6 0
Mean values of three independent experiments ±SE. P value is 0.0337.

50. Limitation
• The major impediment is the high cost involved in the large-scale
production of good quality viable micro propagules
• In many cases, loss in tissues viability and occurrence of
somaclonal variations frequently limits the use of synthetic seed
technology
• Low production rate of viable micropapules, anomalous and
asynchronous development of somatic embryos are other major
problems involved
• Inefficient germination and poor survival which may be due to the
lack of nutrient and oxygen supply is also a major limitation
• Conventional encapsulation process is labor intensive
50

51. Conclusions
• Encapsulation in 3 % sodium alginate with 1 % CaCl2 and MS
basal medium as matrix were found best among all combinations
of encapsulation
• Coco peat is the best commercial substrate for regrowth and
conversion of synseed
• Artificial seeds offer an ideal delivery system enabling easy
flexibility in handling and transport as compared to large parcels
of seedlings or plants
• Storage at 4°C is effective for long term preservation of artificial
seeds
51

52. Thank You