Somatic embryogenesis ; 27 march 15. 3.00 pm
-
Upload
avinash-sharma -
Category
Education
-
view
166 -
download
1
Transcript of 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.
3/27/2015 Deptt of Plant Biotechnology 2
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.
3/27/2015 Deptt of Plant Biotechnology 3
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.
3/27/2015 Deptt of Plant Biotechnology 4
Stages of Organogenesis
3/27/2015 Deptt of Plant Biotechnology 5
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.
3/27/2015 Deptt of Plant Biotechnology 6
3/27/2015 Deptt of Plant Biotechnology 7
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%) 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.
3/27/2015 Deptt of Plant Biotechnology 8
Importance of Somatic Embryogenesis:
Higher propagation rate.
Suitable in Suspension culture.
Artificial seed production.
Labour savings.
3/27/2015 Deptt of Plant Biotechnology 9
Stages of Somatic Embryogenesis:-
3/27/2015 Deptt of Plant Biotechnology 10
cytokinin
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.
3/27/2015 Deptt of Plant Biotechnology 11
3/27/2015 Deptt of Plant Biotechnology 12
Crops Explants Results References
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.
3/27/2015 Deptt of Plant Biotechnology 13
Direct Somatic Embryogenesis
• ffff
3/27/2015 Deptt of Plant Biotechnology 14
Yadav et al., 2014
Indirect Somatic Embryogenesis
3/27/2015 Deptt of Plant Biotechnology 15
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.
3/27/2015 Deptt of Plant Biotechnology 16
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.
3/27/2015 Deptt of Plant Biotechnology 17
Indirect Somatic Embryogenesis in Cereals:
3/27/2015 Deptt of Plant Biotechnology 18
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)
3/27/2015 Deptt of Plant Biotechnology 19
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)
3/27/2015 Deptt of Plant Biotechnology 20
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
3/27/2015 Deptt of Plant Biotechnology 21
Table 1: Per cent of callus induction from immature embryos of five maize inbreds on different media compositions:-
3/27/2015 Deptt of Plant Biotechnology 22
Table 2: Percent somatic embryogenesis induction in immature embryos of five maize inbred on different media compositions
3/27/2015 Deptt of Plant Biotechnology 23
3/27/2015 Deptt of Plant Biotechnology 24
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.
3/27/2015 Deptt of Plant Biotechnology 25
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 .
3/27/2015 Deptt of Plant Biotechnology 26
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.
3/27/2015 Deptt of Plant Biotechnology 27
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.
3/27/2015 Deptt of Plant Biotechnology 28
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.
3/27/2015 Deptt of Plant Biotechnology 29
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(%).
3/27/2015 Deptt of Plant Biotechnology 30
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
3/27/2015 Deptt of Plant Biotechnology 31
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
3/27/2015 Deptt of Plant Biotechnology 32
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.
3/27/2015 Deptt of Plant Biotechnology 33
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.
3/27/2015 Deptt of Plant Biotechnology 35
Table 4. Effect of BAP + NAA
3/27/2015 Deptt of Plant Biotechnology 36
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
3/27/2015 Deptt of Plant Biotechnology 37
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.
3/27/2015 Deptt of Plant Biotechnology 38
3/27/2015 Deptt of Plant Biotechnology 39