Somatic embryogenesis ; 27 march 15. 3.00 pm

WELCOME

3/27/2015 Deptt of Plant Biotechnology 1

In vitro Regeneration System for

Indirect Somatic Embryogenesis in

Cereal Crops.


AVINASH SHARMA

ID. No:- PALB 3235

Sr. M.Sc. (Plant Biotechnology)

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.


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.


Stages of Organogenesis


1)Shoot tip in the medium 2)Shoot callus 3)Growth of stem 4)Root

initiation5)Hardening 6)Whole plant

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.




3) Sorghum Mature embryo or Immature embryos

Callus induction:- MS + pCPA 2.0mgl¯¹+ BAP 5.0mgl¯¹ (90%) in mature embryo, (95%) inimmature 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.

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.


Importance of Somatic Embryogenesis:

Higher propagation rate.

Suitable in Suspension culture.

Artificial seed production.

Labour savings.


Stages of Somatic Embryogenesis:-


cytokinin

Somatic Embryogenesis in Cereal Crops:


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.




3) Maize cv. (Gaurav)

Immatureembryos

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.

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.


Direct Somatic Embryogenesis

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Yadav et al., 2014

Indirect Somatic Embryogenesis


Importance of Indirect Somatic

Embryogenesis in Cereals:

Indirect Somatic embryogenesis has high Plantregeneration capacity.

Indirect Somatic embryogenesis reduces the breedingcycle.

Indirect somatic embryogenesis are used in the cropimprovement.

Frequency of somaclonal variation is also very high inIndirect somatic embryogenesis

Indirect somatic embryogenesis are better than theDirect somatic embryogenesis.


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.


Indirect Somatic Embryogenesis in Cereals:


Crop: Japonica Rice cv. Kitaake seedsCallus 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.

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)


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)


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(%)

Results


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


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



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.


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 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: 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(%).


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


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


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 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±0.353/27/2015 Deptt of Plant Biotechnology 34

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.


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

Immature embryos of APMS -6B 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 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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Somatic embryogenesis ; 27 march 15. 3.00 pm