somatic embtyogenesis in cereals crops

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

3. CONTENTS
 Definition of Somatic Embryogenesis.
 Stages of Somatic Embryogenesis.
 Process involved in formation of Somatic Embryos.
 Types of Somatic Embryogenesis.
 Factors affecting Somatic Embryogenesis.
 Case Study.
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4. Stages of ZYGOTIC Embryogenesis:-
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5. Somatic Embryogenesis:-
 Somatic embryogenesis is a process by which
somatic cells or tissues develops into differentiated
embryos.
 Embryos regenerate from somatic cells or tissues (
haploid or diploid etc) it is termed as Somatic
Embryogenesis.
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 Somatic embryogenesis was first induced in
suspension culture (Stewart et al, 1958) and in callus
culture (Reinert, 1959) of carrot, Umbelliferae and
Solanaceae dicotyledonous families have produced
somatic embryos.
 SE occur most frequently in tissue culture as an
alternative organogenesis for regeneration of whole
plant.
 Somatic embryos are referred to by many names such
as embryo like structures, adventitious or vegetative
embryos, Embryoids; and the process is termed as
adventitious , asexual or somatic embryogenesis.

7. Stages of Somatic Embryogenesis:-
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8. Contd:-
 INDUCTION
 Development and Maturation
 Globular
 Heart stage
 Torpedo
 Germination and Conversion
• Globular stage: Embryo
is small and round
(multicellular).
• Heart stage (Bilateral symmetry):
Shape changes to heart
shape with more
cotyledon development.
• Torpedo shaped stage:
Consists of initial cells
for the shoot/root
meristem.
• Mature stage: Embryo
becomes cylindrical.
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9. Induction
Auxin required for induction
 Pro embryonic masses are formed.
 2,4-D are mostly used.
 NAA, DICAMBA are also used.
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10. Development
• Auxin must be removed for embryo development.
• Continuous use of Auxin inhibits embryogenesis.
• Stages are similar to those of Somatic embryogenesis:-
 Globular
 Heart
 Torpedo
 Cotyledonary
 Germination (Conversion)
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11. Maturation
 Embryo are mature with apical meristem, radicle and
cotyledons.
 Often obtained repetitive embryony.
 Storage protein production necessary.
 Often require ABA for complete maturation.
 ABA often required for normal morphology.
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12. Morphological Stages of Maize cv. Gaurav
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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. Examples of Direct Somatic Embryogenesis:-
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Figure :- Isolation of mature embryo from imbibed durum grain.
Isolated mature embryo which will be inoculated with abaxial
surface in contact with culture medium.
Ganeshan et al., 2006.

15. Contd:-
 Mature embryos culture in the Murashige and Skoog,
1962 medium with supplements 1gm/l enzymatic
casein hydrolysate, 0.7 gm/l L-proline.
 4.5 µM of TDZ and 4.4 µM of BAP are best combination
of growth regulators in which Durum Wheat produced
35 number of shoots per explant and Mature embryos
of CDC Dancer oat produced 16 shoots per explant.
 Explants for direct embryogenesis include microspores,
ovules, scutellum, endosperm, embryos and seedlings.
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16. Indirect Somatic Embryogenesis:-
 In Indirect SE, callus is produced from explants.
 Embryoids are produced from callus tissue.
 Explants are roots, shoots, leaf cells, anthers, seeds
etc.
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17. a) Formation of callus b) Greening of callus c) Embryo at globular stage
d) Torpedo stage of embryo e) Cotyledonary stage and regeneration
of embryo f-g) Multiple shoot regeneration h) Complete plantlets i)
Hardening of plantlets. (Rice Variety:- Swarna)
Mondal et al., 2011
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)
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Steps involved in Plant regeneration of Rice variety through Indirect SE:-

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20. Factors affecting Somatic Embryogenesis:-
1) Genotype:-
 Genetically engineered / transgenic plant does
not regenerate through SE.
 Methylation occurs in the DNA during mitosis
then SE occurs. If Methylation occur in the
cytosine bases or H3 protein then SE get stop.
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2) Explant:-
 Totipotent somatic cell are used.
 Immature inflorescence and Scutellar tissue of
immature seeds are used for the research. Ex:-
Triticum aestivum .
 Epidermis, Procambial tissue are also produced
somatic embryo.

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3) Auxin:-
 Polar transport of auxin produces somatic embryo.
 Auxin concentration will be more then somatic
embryogenesis get stop. Ex:- Maize.
 Auxin induces indirect somatic embryogenesis in monocots.
 During Proembryonic phase, 2,4-D generates DNA Hyper
methylation so that cells in a highly active mitotic stage.
 High concentration of auxin produces root in somatic
embryo.
 2,4-D is one of the growth regulator that produces callus
from cereals and conc. of 2,4-D 0.1-10µM

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4) Cytokinins:-
 Cytokinin promote axial growth.
 Cytokinin produces globular embryo from initial
embryo.
 Cytokinin combination with auxin, induces somatic
embryogenesis and produce callus in cereals.
 Cytokinin ratio more than auxin then it produces
Shoots.

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5) Gibberellic acid:-
 GA promote elongations of embryo axis, cell division.
 It synthesized of photosynthetic pigments in developing
somatic embryo.
 It improve photosynthetic activity, Extra storage reserves in
vitro germination.
 Hypo cotyledon are used as explant then GA inhibit somatic
embryogenesis.
 GA higher in suspensory embryo than the proper embryo.
 Addition of Uniconazole, Paclobutrazol inhibit somatic
embryogenesis.

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6) Abscisic acid (ABA):-
 ABA control tolerance and seed dormancy during later
stage of embryogenesis.
 ABA induced somatic embryogenesis in high osmotic
stress and high temperature in auxin free medium.
 Primary embryo contain more conc. of ABA than
secondary embryo.
 Treatment of Fluridone inhibit ABA synthesis and
primary embryo does not produce secondary embryo.

26. 7) Polyamines:-
 Spermidine, Spermine and Putrescine are added as
growth regulators and secondary messenger.
 Polyamines serve as nitrogen source for plants.
 It act as a free radical scavengers by protecting
senescing membranes against lipid per oxidation.
 In Maize, Putrescine are most effective with varying
concentration of GA3.
 Spermine act as a antioxidant in a medium.
 It help in vegetative growth, pollen development,
regulation of DNA duplication, transcription of genes,
cell division, development of organs.
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27. 8) Phytosulfokine
 It modulate the culture media.
 It promote cell division of embryogenic cells, in
presence auxin.
 Phytosulfokine increases the cell through
differentiation process.
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28. 9) Phenolic compounds:-
 Phenolic compounds are inhibit somatic
embryogenesis.
 4hydroxy benzyl alcohol inhibits the globular stages.
 Vanillyl benzyl ether are inhibit the suspensor
development.
 Recently identification of 4 [(phenyl methoxy) methyl]
phenol involves in seed development stills
unknown.
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29. Advantages and Disadvantages of Somatic
Embryogenesis:-
Advantages:
 Higher propagation rate.
 Suitable in Suspension culture.
 Artificial seed production.
 Germplasm conservation.
 Labour savings.
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30. Disadvantages
 Response tissue specific (explants).
 Low frequency embryo production.
 Incomplete embryo production.
 May create unwanted genetic variation (Somaclonal
variation).
 Inability to generate large numbers of normal, free
living plantlets.
 Plantlets are weaker.
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33. 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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34. 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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35. 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 with sterile double
sterilization water for 6 times.
Cultured onto the medium with different treatment.
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36. 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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37. 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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38. Somatic Embryo Germination Media:-
 MS medium containing different concentrations of
BAP (0, 1, 2, 3, 4and 5 mg/l), in combination with
different concentrations of NAA (0, 0.5, 1.0, 1.5, 2.5
and 4.0 mgL-1) were used as treatments for the
germination of somatic embryos.
 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.
 Calculate the Callus induction frequency(%) and
Regeneration frequency(%).
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39. 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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40. Table 1. Callus induction percent of rice in Somatic
Embryogenesis
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.

41. 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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42. 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 Transplantation PGRs in rice
Table 3. Effect of Transplantation 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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43. Contd:-
 MS medium supplements with 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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44. 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

45. APMS -6B Variety 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

46. Conclusion
 Somatic embryogenesis is an efficient plant
regeneration system.
 It is potentially 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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47. Conclusion:-
 Indirect Somatic embryogenesis reduces the breeding
cycle.
 Indirect somatic embryogenesis are used in the crop
improvement.
 Indirect somatic embryogenesis are produce virus free
plants.
 Indirect somatic embryogenesis are better than the
Direct somatic embryogenesis.
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48. References:-
 Joshi, R., KUMAR, P., 2013, Regulation of Somatic
Embryogenesis in Crops: A Review, Agri. Reviews, 34
(1): 1-21, 2013.
 DHLLION, N. K., GOSAL, S. S., 2013, Analysis of
Maize Inbred Lines for their response to Somatic
Embryogenesis, J. Cell and Tiss. Res, 13(1): 3557-
3563.
 SAH, SK., KAUR, A., SANDHU, J,S., 2014, High
Frequency Embryogenic Callus Induction and Whole
Plant Regeneration in Japonica Rice Cv. Kitaake, J.
Rice Res., 2: 125.
 ANAND, P., TIWARI, A., SAXENA, A., ARNOLD, R.,
TIWARI, S., 2014, Studies on Optimization OF
Protocol for Somatic Embryogenesis and Regeneration
of Rice (APMS – 6B), Euro. J. Mol. Biol. Biochem.,
1(1):13-17.
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