WELCOME

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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)

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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Stages of ZYGOTIC Embryogenesis:-

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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.

Stages of Somatic Embryogenesis:-

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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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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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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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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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Morphological Stages of Maize cv. Gaurav

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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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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.

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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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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a) Formation of callus b) Greening of callus c) Embryo at globular staged) Torpedo stage of embryo e) Cotyledonary stage and regenerationof 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)

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Steps involved in Plant regeneration of Rice variety through Indirect SE:-

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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 developingsomatic embryo.

It improve photosynthetic activity, Extra storage reserves invitro germination.

Hypo cotyledon are used as explant then GA inhibit somaticembryogenesis.

GA higher in suspensory embryo than the proper embryo.

Addition of Uniconazole, Paclobutrazol inhibit somaticembryogenesis.

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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.

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 protectingsenescing membranes against lipid per oxidation.

In Maize, Putrescine are most effective with varyingconcentration 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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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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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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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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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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Introduction Rice is the staple diet for two billion people world wide .

It is feared that world population would be around 10billion by 2050.

Diminishing of cultivated land.

Attack of pests and insects are responsible for decrease inproduction.

There is a constant need to improve crops to overcome allthese hazards.

Somatic embryogenesis in rice has been reported culture ofleaf tissue, root tissue, inflorescence and protoplast.

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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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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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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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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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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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Results:- After 3 days of culture callus started to grow from

Scutellar embryo.

Embryo derived callus subsequently started to enlargeand some yellowish to greenish nodules grew aroundexplants after ten days.

After 2 months of culture calli almost covered theexplants surface.

For callus induction MS medium supplemented withdifferent concentration of 2,4-D(0, 1.0, 1.5, 2.5, 3.5and 5 mg/l) was used in which 3.5 , 5 mg/l 2,4-Dshowed high callus induction percentage. It can beobserved from Table 1

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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.

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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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±0.353/27/2015 Deptt of Plant Biotechnology 42

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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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

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

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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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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References:-

Joshi, R., KUMAR, P., 2013, Regulation of SomaticEmbryogenesis in Crops: A Review, Agri. Reviews, 34(1): 1-21, 2013.

DHLLION, N. K., GOSAL, S. S., 2013, Analysis ofMaize Inbred Lines for their response to SomaticEmbryogenesis, J. Cell and Tiss. Res, 13(1): 3557-3563.

SAH, SK., KAUR, A., SANDHU, J,S., 2014, HighFrequency Embryogenic Callus Induction and WholePlant 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 OFProtocol for Somatic Embryogenesis and Regenerationof Rice (APMS – 6B), Euro. J. Mol. Biol. Biochem.,1(1):13-17.

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