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 asexual
reproductive process that forms new plants.
 Embryogenesis may occur naturally in the plant as a result
of sexual fertilization, and those embryo are called zygotic
embryos and develop into seeds, which can germinate and
give rise to seedlings.
 Plant cells can also be induced to form embryos in plant
tissue 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 double
fertilization of the ovule, forming the plant
embryo and the endosperm which together go
into the seed, this process is known as zygotic
embryogenesis.
Seeds may also develop without fertilization
through pathways referred to as apomixis.

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.

 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.
 In literature, 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:-

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 scalpel
blade is inserted beneath the Coleoptilar region of the Mature embryo; (b) With a swift and
smooth scooping motion the mature embryo is dislodged from its attachment to the
scutellum; (c) 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 produces 35
number of shoots per explant and Mature embryos of CDC
Dancer 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) are
produced from callus tissue.
Explants are roots, shoots, leaf cells, anthers, seeds
etc.
Steps involved in Plant regeneration of Rice variety
through Indirect SE:-

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

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 of
immature seeds are used for the research. Ex:-
Triticum aestivum .
Epidermis, Procambial tissue are also produced
somatic 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
Addition of Uniconazole,
Paclobutrazol inhibit
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
 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±0.35

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 unclear
and comphrensive work in future it by studying the
interaction of various factors thereby entire picture of
regulatory mechanism of embryogenesis would be
transparent.

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 Maize
Inbred Lines for their response to Somatic Embryogenesis,
J. Cell and Tiss. Res, 13(1): 3557-3563.
SAH, SK., KAUR, A., SANDHU, JS., 2014, High Frequency
Embryogenic Callus Induction and Whole Plant
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 OF Protocol for Somatic
Embryogenesis and Regeneration of Rice (APMS – 6B),
Euro. J. Mol. Biol. Biochem., 1(1):13-17.

Embryogenesis 27 mar 15

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