This document provides information about indirect somatic embryogenesis in cereal crops. It begins with an introduction to somatic embryogenesis and its importance. It then discusses the types of somatic embryogenesis, including direct and indirect somatic embryogenesis. Indirect somatic embryogenesis is described as occurring through callus formation from explants, from which embryos later develop. The document presents information on indirect somatic embryogenesis systems developed for several cereal crops like rice, wheat, maize and sorghum. It also provides a case study on the indirect somatic embryogenesis of rice variety APMS-6B, including the methods used for callus induction and embryo germination, as well as the results obtained.
Gene mapping means the mapping of genes to specific locations on chromosomes.
Such maps indicates the positions of genes in the genome and also distance between them.
Gene mapping means the mapping of genes to specific locations on chromosomes.
Such maps indicates the positions of genes in the genome and also distance between them.
OVARY CULTURE:-
"the in-vitro culturing of ovaries in an aseptic condition from the pollinated or un-pollinated flowers, in an appropriate nutrient medium and under optimal conditions." And
OVULE CULTURE:-
"Ovule culture is an experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions."
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
https://www.youtube.com/watch?v=IZwrkgADM3I
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
The presentation gives overview of production of secondary metabolites using callus culture as well as tissue culture techniques. Various batch and continuous culturing process are described on the basis of secondary metabolite to be synthesised.
A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Embryo culture is the culturing of embryos excised from the ovaries at earlier stages of their development. This technique helps to overcome problems associated with embryo development. Embryos are prevented from development by different factors like incompatibility with the female tissue, absence of endosperm etc. Hybrids produced by wide crosses usually fail to develop inside the ovaries of the mother plants. In such cases, the embryos can be rescued (the technique is called embryo rescue) and grown in culture media so as to produce viable progeny.
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
OVARY CULTURE:-
"the in-vitro culturing of ovaries in an aseptic condition from the pollinated or un-pollinated flowers, in an appropriate nutrient medium and under optimal conditions." And
OVULE CULTURE:-
"Ovule culture is an experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions."
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
https://www.youtube.com/watch?v=IZwrkgADM3I
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
The presentation gives overview of production of secondary metabolites using callus culture as well as tissue culture techniques. Various batch and continuous culturing process are described on the basis of secondary metabolite to be synthesised.
A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Embryo culture is the culturing of embryos excised from the ovaries at earlier stages of their development. This technique helps to overcome problems associated with embryo development. Embryos are prevented from development by different factors like incompatibility with the female tissue, absence of endosperm etc. Hybrids produced by wide crosses usually fail to develop inside the ovaries of the mother plants. In such cases, the embryos can be rescued (the technique is called embryo rescue) and grown in culture media so as to produce viable progeny.
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
The production of haploid plants exploiting the totipotency of microspore.
Androgenesis is the in vitro development of haploid plants originating from totipotent pollen grains through a series of cell division and differentiation.
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2. 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)
3. 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
4. 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
5. Stages of Organogenesis
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1)Shoot tip in the medium 2)Shoot callus 3)Growth of stem 4)Root
initiation5)Hardening 6)Whole plant
6. 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
7. 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%) in
immature 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.
8. 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
9. Importance of Somatic Embryogenesis:
Higher propagation rate.
Suitable in Suspension culture.
Artificial seed production.
Labour savings.
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10. Stages of Somatic Embryogenesis:-
3/27/2015 Deptt of Plant Biotechnology 10
cytokinin
11. 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
12. 3/27/2015 Deptt of Plant Biotechnology 12
Crops Explants Results References
3) Maize
cv. (Gaurav)
Immature
embryos
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.
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.
3/27/2015 Deptt of Plant Biotechnology 13
16. Importance of Indirect Somatic
Embryogenesis in Cereals:
Indirect Somatic embryogenesis has high Plant
regeneration capacity.
Indirect Somatic embryogenesis reduces the breeding
cycle.
Indirect somatic embryogenesis are used in the crop
improvement.
Frequency of somaclonal variation is also very high in
Indirect somatic embryogenesis
Indirect somatic embryogenesis are better than the
Direct somatic embryogenesis.
3/27/2015 Deptt of Plant Biotechnology 16
17. 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
18. Indirect Somatic Embryogenesis in Cereals:
3/27/2015 Deptt of Plant Biotechnology 18
Crop: Japonica Rice cv. Kitaake seeds
Callus 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.
19. 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)
20. 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(%)
21. 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:-
22. 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
25. 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.
3/27/2015 Deptt of Plant Biotechnology 25
26. 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
27. 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
28. 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
29. 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
30. 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
31. 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
3/27/2015 Deptt of Plant Biotechnology 31
32. 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.
33. 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
35. 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
37. 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
38. 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