somatic embryobenesis in cereals crops

1. WELCOME
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2. In vitro Regeneration System for
Indirect Somatic Embryogenesis in
Cereal Crops.
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AVINASH SHARMA
ID. No:- PALB 3235
Sr. M.Sc. (Plant Biotechnology)

3. CONTENTS
 Introduction.
 Importance of Somatic Embryogenesis.
 Types of Somatic Embryogenesis.
 Somatic Embryogenesis in Monocots and Dicots.
 Importance of Indirect Somatic Embryogenesis in
Cereals.
 Indirect Somatic Embryogenesis in Cereal crops.
 Case Study.
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4. Organogenesis and Somatic Embryogenesis:
 The development of adventitious organs or
primordia from undifferentiated cell masses in
tissue culture by the process of differentiation is
called Organogenesis.
 It produces either root organ or shoot organ.
 It contain vascular bundles.
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5. Stages of Organogenesis
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1)Shoot tip in the medium 2)Shoot callus 3)Growth of stem 4)Root
initiation5)Hardening 6)Whole plant

6. Organogenesis in Cereal Crops:
Crops Explants Results References
1) RICE cv“Tainan
11” (TN11) and
“Ai-Nan-Tsao 39”
(ANT39)
Immature
seeds
Callus induction:- MS+ 10µM 2,4-D
(75%) in ANT 39; (88%) in TN11.
Shoot Regeneration:- MS + 10µM
NAA + 20µM KIN (80%) in ANT39;
(0%) in TN11.
Lee et al., 2013.
2) Wheat cv (AS-
2002; GA-2002).
Immature
embryo
Callus induction:- MS+ 4mgl-¹
(92.75%) in AS-2002; (91.25%) in
GA-2002 .Shoot Regeneration:- MS
+ 1.0 mgl¹ (41.19%) in GA-2002.
Mahmood et al.,
2012.
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Crops Explants Results References
3) Sorghum Mature embryo
or Immature
embryos
Callus induction:- MS + pCPA
2.0mgl¯¹+ BAP 5.0mgl¯¹ (90%) in
mature embryo, (95%) in
immature embryo
Regeneration:- MS + 2.0mgl¯¹+
IAA 0.5mgl¯¹ (39% ) in mature
embryo and (48%) in immature
embryo.
Nirwan et al., 2004.
4) Maize
Genotypes
(CML 427)
Seeds Callus induction:- N6 + 5µM
DICAMBA ( 86.67%) in CML 427;
Shoot regeneration MS +
13.3µm BAP in CML 427.
Matazu et al., 2014.

8. Somatic Embryogenesis:
 Somatic embryogenesis is a process by which
somatic cells or tissues, including haploid cells
develops into differentiated embryos and to
regenerate plants.
 Stewart et al., (1958): First induced embryo
through suspension culture in carrot.
 Reinert (1959): Produce embryo from callus in
carrot through suspension culture.
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9. Importance of Somatic Embryogenesis:
Higher propagation rate.
Suitable in Suspension culture.
 Artificial seed production.
 Labour savings.
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10. Stages of Somatic Embryogenesis:-
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cytokinin

11. Somatic Embryogenesis in Cereal Crops:
Crops Explants Results References
1) Rice
var.(MR
219)
Seeds Callus induction:- 1mgl¹ 2,4-D + 10mg/l +
10mgl¹ NAA (78%) in MR 219; Embryo to
Plants :- MS + 3mgl¹+ 0.5mgl¯¹ NAA (88%) in
MR219.
Zuraida et al., 2012.
2) Wheat
loc .var.
(GA-2002;
Sahar) .
Seeds Callus induction:- MS + 2mgl¯¹ 2,4-D (71%)
in Sahar; MS + 4mgl¯¹ (82.60%) in GA-2002;
Shoot regeneration:- MS + 4mgl¯¹(88%) in
GA-2002; MS + 4mgl¯¹ (73.51%) in Sahar.
Munazir et al., 2010.
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Crops Explants Results References
3) Maize
cv. (Gaurav)
Immature
embryos
Callus induction:- MS + 5.0mgl¯
2,4-D (77.77%) in Gaurav;
Shoot regeneration:- MS +
2mgl¯¹BAP (35-48%) in Gaurav.
Joshi et al., 2010.
4) Sorghum
Genotypes
(IS 3566,
SPV 475)
Seeds Callus induction :- MS + 2mgl¯¹
2,4,5-T + 1.0 mgl¯¹ Zeatin (72%)
in IS 3566; MS + 2.0mgl¯¹ 2,4,5-
T + 1.0mgl¯¹ Zeatin (80%) in SPV
475. Regeneration :- MS + 2.5
mgl¯¹ TDZ (14 Shoots) in IS
3566; MS + 3mgl¯¹ TDZ (13
shoots) in SPV 475.
Pola et al., 2006.

13. Types of Somatic Embryogenesis:-
 Two types of somatic embryogenesis
 Direct somatic embryogenesis
 The embryos initiate directly from explants in the
absence of callus formation. Embryos are formed
due to PEDCs cell.
 Indirect somatic embryogenesis
 Callus from explants takes place from which
embryos are developed. Embryos are formed due to
IEDCs cells.
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14. Direct Somatic Embryogenesis
• ffff
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Yadav et al., 2014

15. Indirect Somatic Embryogenesis
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16. Importance of Indirect Somatic
Embryogenesis in Cereals:
 Indirect Somatic embryogenesis has high Plant
regeneration capacity.
 Indirect Somatic embryogenesis reduces the breeding
cycle.
 Indirect somatic embryogenesis are used in the crop
improvement.
 Frequency of somaclonal variation is also very high in
Indirect somatic embryogenesis
 Indirect somatic embryogenesis are better than the
Direct somatic embryogenesis.
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17. Somatic Embryogenesis in Monocots and Dicots:
Monocots
 Radicle protected by
coleorrhiza and plumule
coleoptiles in monocots.
 Cambial tissue are
absent in monocots.
 Secondary growth are
absent.
 Easy to culture in the
media.
Dicots
 Coleoptiles and
Coleorrhiza are absent.
 Cambial tissue are
present.
 Secondary growth are
present.
 Difficult to grow in the
media.
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18. Indirect Somatic Embryogenesis in Cereals:
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Crop: Japonica Rice cv. Kitaake seeds
Callus induction media Regeneration Media:
CHO source- maltose (40g/l) Hormones- NAA (0.2 mg/l) and BAP (3.0 mg/l),
Agar (0-8, 1 and 1.2%)
Hormones- 2,4-D and BAP (3.0 mg/l)
Proline (0.6 g/l) and Phytagel (0.3%)
Treatments: Either alone or in combination of Hormones, gelling agents, proline and
maltose supplemented with basic MS media.

19. Results:
Gelling agents Callus induction Regeneration
0.8% agar 52.47 (46.51) 28.33 (32.14)
1.0% agar 68.67 (55.94) 51.00 (45.56)
1.2% agar 70.67 (57.12) 38.00 (38.04)
0.3% Phytagel 92.00 (68.85) 60.54 (58.85)
0.8%agar + 0.2% Phytagel 87. 00 (68.85) 90.00 (71.83)
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Callus induction medium:
MS + 2,4-D (3.0mgl-1) + BAP (0.25mgl-1) + proline (0.6gl-1) + maltose
(40gl-1) + agar (0.8, 1.0, 1.2%) , Phytagel (0.3%) or agar in
combination with Phytagel.
Regeneration medium:
MS + BAP (3.0 mgl-1) + NAA (0.2mgl-1) + Phytagel (2gl-1) +
agar (8gl-1)

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Explants: Immature grains 5 Maize Inbred lines ( LM5, LM6, LM13, LM15 and LM16)
Treatments: Different concentrations and combinations of Auxins and cytokinins.
Auxins- Picloram (2.5, 5 and 10 mg/l), 2,4-D (3, 6 and 10 mg/ l) and NAA (5, 10 mg/l)
Cytokinins- BAP (0.5 mg/l) and Kinetin (0.75 mg/l)
Sucrose (60g/l), Agar (8gm/l)
Observations were recorded on percent response of explants to callus induction (%) and
Somatic embryogenesis(%)

21. Results
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Table 1: Per cent of callus induction from immature embryos of five maize inbreds on
different media compositions:-

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Table 2: Percent somatic embryogenesis induction in immature embryos of five maize
inbred on different media compositions

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25. Introduction
 Rice is the staple diet for two billion people world wide .
 It is feared that world population would be around 10
billion by 2050.
 Diminishing of cultivated land.
 Attack of pests and insects are responsible for decrease in
production.
 There is a constant need to improve crops to overcome all
these hazards.
 Somatic embryogenesis in rice has been reported culture of
leaf tissue, root tissue, inflorescence and protoplast.
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26. Materials and method:-
 Explant collection:-
 Explant material for this research were rice seeds.
 Variety APMS-6B obtained from DRR (Hyderabad).
 Rice caryopses containing Scutellar region of
embryo, were isolated by removing lemma and
palea from the seeds .
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27. Surface sterilization of Seeds:-
Sterilization of rice caryopses using 70% alcohol for
3min.
Followed by shaking in 30% Chlorox containing 2-3
drops of Tween-20 on an orbital shaker at 120 rpm
for 20min.
Explants were rinsed with sterile double sterilization
water for 6 times.
Cultured onto the medium with different treatment.
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28. Preparation of Media:-
 Two basic media used in this study:-
 First one was:- half MS (Murashige & Skoog, 1962)
supplements with 500mg/l glutamine, 100 mg/l
proline.
 Second one was:- N6 media supplemented with
500mg/l L-Glutamine.
 Both media were solidified with 0.2% agar.
 pH adjusted with 5.8.
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29. Callus Induction Media:-
 Different concentrations of 2, 4-D [0.1, 1.5, 2.5,3.5
and 5 mgL-1 (w/v)] were used as the treatments for
embryogenic callus induction.
 Media were kept in dark condition for 1 week, 25±2°C
at room temperature.
 After 1 week transferred the cultures under 16 hrs
lighting , provided by fluorescent bulbs with 15.75
µmolm-²s-¹ for eight weeks.
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30. Somatic Embryo Germination Media:-
 MS medium: BAP (0, 1, 2, 3, 4and 5 mg/l),
NAA (0, 0.5, 1.0, 1.5, 2.5 and 4.0 mgL-1)
 Media were kept in the incubation room 25±2°C with
16 hrs of light provided by fluorescent bulbs and a
light intensity of 16.75 µmolm-²s-¹ for eight weeks.
 Calculation: Callus induction frequency(%)
Regeneration frequency(%).
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31. Results:-
 After 3 days of culture callus started to grow from
Scutellar embryo.
 Embryo derived callus subsequently started to enlarge
and some yellowish to greenish nodules grew around
explants after ten days.
 After 2 months of culture calli almost covered the
explants surface.
 For callus induction MS medium supplemented with
different concentration of 2,4-D(0, 1.0, 1.5, 2.5, 3.5
and 5 mg/l) was used in which 3.5 , 5 mg/l 2,4-D
showed high callus induction percentage. It can be
observed from Table 1
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32. Table 1. Callus induction percent of rice:
S. No Conc. Of 2,4-D (mgL-¹) Callus Induction Frequency % from rice
1. 0 No callus
2. 1.0 76±35
3. 1.5 80±40
4. 2.5 88±45
5. 3.5 95±30
6. 5.0 86±45
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The result showed that the increased concentration of
2,4 –D more than 3.5 mgL-¹ decreased the callus
formation percentage.

33. Contd:-
 MS media supplemented with 0.8% agar, 70gm/l
sucrose, 4gm/l Casein, 3mg/l BAP and 4 mg/l NAA
was used for derived calli.
 3 mg/l BAP concentration showed good results in
Shoot induction, it can be observed from Table 3.
 4 mg/l NAA concentration showed good results in
Shoot induction, it can be observed from Table 2.
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34. S.No. Conc. Of NAA
(mg/l)
Shoot Induction % No. of Shoots
1. 0 31.33 2.6±0.48
2. 0.5 25.65 2.5± 0.64
3. 1 33.45 3.0± 0.54
4. 1.5 41.60 3.5± 0.64
5. 2.5 45.60 4.0± 0.59
6. 4.0 48.55 4.5± 0.60
Table 2. Effect of NAA PGRs in rice
Table 3. Effect of BAP PGRs in rice
S.No. Conc. Of BAP (mg/l) Shoot Induction % No. of Shoots
1. 0 30.33 2.0±0.87
2. 1 23.45 1.8±0.48
3. 2 31.85 2.2±0.16
4. 3 40.68 3.0±0.18
5. 4 38.67 2.5±0.64
6. 5 35.45 2.4±0.35
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35. Contd:-
 MS medium + different concentrations of NAA (0, 0.5,
1.0, 1.5, 2.0 mg/l) in combination with different
concentrations of BAP (0, 1, 2, 3, 4, and 5 mg/l).
 Result showed that combination of 3mg/l BAP + 1.5
mg/l NAA showed highest result.
 Further combination increases cause the decrement of
percent of Shoot induction. It can be observed from
Table 4.
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36. Table 4. Effect of BAP + NAA
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S.No. BAP + NAA
(mg/l)
Shoot Induction
%
No. Of Shoots
1. 1 + 0.5 26.85 2.1± 0.63
2. 2 + 1.0 29.65 2.5 ±0.83
3. 3 + 1.5 39.60 3.5± 0.54
4. 4 + 2.0 35.45 3.2± 0.45
5. 5 + 4.0 30.40 3.0± 0.54

37. Immature embryos of APMS -6B seeds regenerate
through Indirect Somatic Embryogenesis
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Fig 1. Seed inoculation in MS medium Fig 2. Callus formation by 2, 4-D
Fig 3. Shoot induction by differ. Conc. Of
BAP and NAA
Fig -4 Transplantation

38. Conclusion
 Somatic embryogenesis is an efficient plant
regeneration system under in vitro.
 It is potential useful tool for genetic transformation.
 Cross linking between hormone and transcription
factors is likely to play an important part in SE.
 But mechanism of plant embryogenesis is unclear and
comphrensive work in future is necessary to be studied
with the interaction of various factors for entire picture
of regulatory mechanism of embryogenesis to be
transparent.
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