embryogenesis 27 mar 15

WELCOME

3/27/2015 Deptt of Plant Biotechnology 1

In vitro Regeneration System for Indirect Somatic Embryogenesis of

Cereals Crops.


AVINASH SHARMA ID. No:- PALB 3235

Sr. M.Sc. (Plant Biotech)

EMBRYOGENESIS:- Plant embryogenesis refers to the process of development

of plant embryos, being either a sexual or asexualreproductive process that forms new plants.

Embryogenesis may occur naturally in the plant as a resultof sexual fertilization, and those embryo are called zygoticembryos and develop into seeds, which can germinate andgive rise to seedlings.

Plant cells can also be induced to form embryos in planttissue culture; these embryo are called somatic embryos.


Stages of Embryogenesis:-


Types of Embryogenesis:-

• Two types of embryogenesis:-

A) Zygotic embryogenesis

B) Somatic embryogenesis


Zygotic Embryogenesis:-

The zygotic embryo is formed following doublefertilization of the ovule, forming the plantembryo and the endosperm which together gointo the seed, this process is known as zygoticembryogenesis.

Seeds may also develop without fertilizationthrough pathways referred to as apomixis.


Somatic Embryogenesis:-

Somatic embryogenesis is a process by whichsomatic cells or tissues develops intodifferentiated embryos.

Embryos regenerate from somatic cells ortissues ( haploid or diploid etc) it is termed asSomatic Embryogenesis.



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 haveproduced somatic embryos.

SE occur most frequently in tissue culture as an alternativeorganogenesis for regeneration of whole plant.

In literature, somatic embryos are referred to by many names suchas embryo like structures, adventitious or vegetative embryos,Embryoids; and the process is termed as adventitious , asexual orsomatic embryogenesis.

Stages of Somatic Embryogenesis:-


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.


Induction

Auxin required for induction

Pro embryonic masses are formed.

2,4-D are mostly used.

NAA, DICAMBA are also used.


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)


Maturation

Require complete maturation 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.


Morphological Stages of Maize cv. Gaurav


Routes of Somatic Embryogenesis:-Two routes to 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.


Examples of Direct Somatic Embryogenesis:-


Figure :- Isolation of mature embryo from imbibed cereal grain. (a) A curved-tip scalpelblade is inserted beneath the Coleoptilar region of the Mature embryo; (b) With a swift andsmooth scooping motion the mature embryo is dislodged from its attachment to thescutellum; (c) Isolated mature embryo which will be inoculated with abaxial surface incontact with culture medium. Ganeshan et al., 2006.

Contd:-Mature embryos culture in the Murashige and Skoog, 1962

medium with supplements 1gm/l enzymatic caseinhydrolysate, 0.7 gm/l L-proline.

4.5 µM of TDZ and 4.4 µM of BAP are best combination ofgrowth regulators in which Durum Wheat produces 35number of shoots per explant and Mature embryos of CDCDancer oat produces 16 shoots per explant.

Explants for direct embryogenesis include microspores,ovules, scutellum, endosperm, embryos and seedlings.


Indirect Somatic Embryogenesis:-

In Indirect SE, callus is produced from explants.

Embryoids(suspensory cell to cotyledon) areproduced from callus tissue.

Explants are roots, shoots, leaf cells, anthers, seedsetc.

Steps involved in Plant regeneration of Rice varietythrough Indirect SE:-


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)

(i)3/27/2015 Deptt of Plant Biotechnology 20

Factors affecting Somatic Embryogenesis:-

1) Genotype:-

Genetically engineered / transgenic plant does not regenerate through SE because due to variation.

Methylation occurs in the DNA during mitosis then SE occurs. If Methylation occur in the cytosine bases or H3 protein then SE get stop.



2) Explant:-

Totipotent somatic cell are used.

Immature inflorescence and Scutellar tissue ofimmature seeds are used for the research. Ex:-Triticum aestivum .

Epidermis, Procambial tissue are also producedsomatic embryo.


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

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.


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.


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.

Addition of Uniconazole, Paclobutrazol inhibit somatic embryogenesis.

GA higher in suspensory embryo than the proper embryo. So GA requires early embryo development.


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 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.


8) Phytosulfokine

It modulate the culture media.

It promote somatic embryogenesis by activating cell division of embryogenic cells, in presence auxin.

Phytosulfokine increases the cell through differentiation process.

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.


Differences between Zygotic and Somatic embryo:-

Zygotic embryo

Fertilized egg or zygote.

Contain seed coat.

Produce seed.

Plantlets are healthy.

Not like to mother plant.

Propagation is low.

Somatic embryo

Sporophytic cells.

Did not contain seed coat.

Only form embryo.

Plantlets are weaker

Like to mother plant.

Propagation is high.


Advantages and Disadvantages of Somatic Embryogenesis:-

Higher propagation rate.

Suitable for Suspension culture.

Artificial seed production.

Somaclonal variation.

Germplasm conservation.

Labour savings.


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.


Introduction Rice is the staple diet for two

billion people world wide .

It is the major food for over half of those living Asia.

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.


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 .


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.


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.


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.


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(%).


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


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


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.


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 45

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.


Table 4. Effect of BAP + NAA


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


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 unclearand comphrensive work in future it by studying theinteraction of various factors thereby entire picture ofregulatory mechanism of embryogenesis would betransparent.


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.


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 MaizeInbred Lines for their response to Somatic Embryogenesis,J. Cell and Tiss. Res, 13(1): 3557-3563.

SAH, SK., KAUR, A., SANDHU, JS., 2014, High FrequencyEmbryogenic Callus Induction and Whole PlantRegeneration 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 SomaticEmbryogenesis and Regeneration of Rice (APMS – 6B),Euro. J. Mol. Biol. Biochem., 1(1):13-17.


embryogenesis 27 mar 15

Education

plant embryogenesis

plant embryos

plant cells

plant tissue

plant biotechembryogenesis

somatic embryos

somatic cells

zygotic embryos