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P.Sameera Sastry, Supervisor: Dr. Nalini Mallikarjuna Research scholar, Principal Scientist,
JNTU & ICRISAT Dept of Legumes Cell Biology,
ICRISAT
OBJECTIVES To test the reproducibility of the recent successful report on DH production in
chickpea via anther culture with application to chickpea varieties/cultivars available locally at ICRISAT Genetic resources unit.
Assess the effect stress pre-treatments such as cold, centrifugation, electroporation and osmotic shock on the induction of androgenesis in chickpea.
Perform preliminary experiments for DH production in pearl millet via anther culture.
In silico approach to infer protein-protein interactions involved in androgenesis in chickpea and pearl millet based on model crops via EST based interolog mapping.
Identify potential markers or triggers for androgenesis with the help of proteomics techniques.
Double Haploids??!
‘Haploid’ = gametic number of chromosomes (n)
Double haploid (DH): doubling of chromosomes either spontaneously via endomitosis or induced via chemical methods
Occur in low frequencies in nature
First observed in vivo in Datura stramonium (Blakeslee et al. 1922)
Followed by in vitro DH production in Datura (1960), tobacco, wheat.
>250 published protocols to date
Routes: Androgenesis, Parthenogenesis, Apogamy & Wide crosses
DHs- Why??
Fastest route to homozygosity
Single laboratory based generation
Represent a pure line/new variety (self-pollinated crops) or
Parental inbred line for the production of hybrid varieties (cross-pollinated crops)
Only method to develop inbred lines in self-incompatible species, dioecious species and those that suffer from inbreeding depression
Superior to RILs, retain complete homozygosity
Stages of haploids are ideal targets for transformation experiments
Convenient system for the induction of mutations and selection of mutants with desirable traits
Vital role in genomics, in the integration of genetic and physical maps & precise targetting of candidate genes
DH+MAS+ESTs = shortcut to plant breeding by improving elite lines
If a suitable protocol is available, DH technology is a rapid, cost effective and superior alternative to conventional crop improvement
DH technology, genetically improved cultivars and better management practices are among the best strategies to increase food production and meet a projected doubling of food demand in the next 40 years
Androgenesis
Development of plants from male gametes/microspores
Genetic traits of male donor plant
Deviation from microsporogenesis
Androgenesis via anther/microspore culture is the most widely used and successful method to obtain DH
Simple and efficient
Most successful species belong to Solanaceae, Cruciferae and Gramineae families
Legumes and woody plants considered recalcitrant
Major disadvantage- high genotype dependency even among species
The beginning and different modes of androgenic pathway
Factors affecting Androgenesis
Switch from microsporogenesis to androgenesis is the crucial step
Induction of microspores, commitment to microspore embryogenesis and subsequent plant regeneration- important stages DH development
Modulated by several factors:
i. Donor plant: Conditions of donor plant. Cereals exhibit high degree of success when grown in phytotron or CEF
ii. Genotype and environmental factors: High genotype dependency and combined effect of genotype-environment is a major drawback of anther/microspore culture esp. legumes
iii. Stage of microspore: Cereals- late uni- to early-binucleate stage. Dicots- mid-late uninucleate stage of microspores is the most suitable for androgenic response
iv. Media: Directs the pathway of embryo development. Osmotic pressure and pH of the medium play an important role in the maturation of microspore-derived embryos
v. Pre-treatments: Stresses such as temperature treatments, osmotic stress, and sugar starvation proved to be essential and/or enhance androgenic ability. Legumes such as chickpea and lupins require 4-13° C cold treatment of buds for few hrs- days. Heat shock and sugar starvation beneficial for cereals
Chickpea World’s second most widely grown legume after
soybean
Two distinctive- desi and kabuli
Predominantly self-pollinated crops with a very low out-crossing level of 0–1%
DH in Chickpea Legume species are considered recalcitrant to DH production
Anther/microspore culture protocols available for Medicago, Glycine, Pisum, Lupinus but have NOT been VALIDATED.
Not used as a routine tool for breeding in any grain or pasture legume including chickpea
First report on DHs in chickpea was in 2009 by Grewal et al.
Low rates of induction and plant regeneration
High genotype and seasonal dependence
Requires application of pyramiding abiotic stresses and varying media requirements at different developmental stages
Anther culture Methodology
Culture in embryo induction media
osmotic stress for 4 days in induction media (high osmotic pressure liquid media)
Stress pre-treatments- Centrifugation, Electroporation
Excision of anthers aseptically
Surface sterilization with buffered bleach for 20 min
Cold treated at 4˚C for 2-4 days
Buds 2-3mm size, msps at uninucleate stage
Genotypes: ICC 4958, WR-315, ICCV 95423 and Arearti (ICRISAT, Hyderabad, India).
Stress pre-treatments Centrifugation: Anthers centrifuged in fixed rotor
centrifuge in 1ml Liq medium at 100-1000g for 3-15min
Electroporation: Electro Cell Manipulator ECM630 with electrodes 2mm apart. Anthers in 1ml of RM-IK subjected to three exponentially decaying pulses.
125, 150, 200 or 250v/cm with 25 Ω resistance and 25 µF capacitance delivered at 10 s interval
Osmotic shock: 4 days in induction media (high osmotic pressure) with sucrose percentage of 8.89 and 17 %
Culture media Initial experiments were carried out as per DH
protocol by Grewal et al. (2009)
Modified forms of induction and culture media were employed by changing the growth regulators.
A total of 12 different media were analyzed for the most effective medium
Media
(mg/lt)
Auxin
Concentration
(mg/lt)
Cytokinin
Concentration
(mg/lt)
CHPB Picloram 0.5 BAP 0.05
CHPZ Picloram 0.5 Zeatin 0.05
CHPT Picloram 0.5 Thidiazuron 0.05
CHDB Dicamba 0.5 BAP 0.05
CHDZ Dicamba 0.5 Zeatin 0.05
CHDT Dicamba 0.5 Thidiazuron 0.05
CH2B 2,4-D 0.5 BAP 0.05
CH2Z 2,4-D 0.5 Zeatin 0.05
CH2T 2,4-D 0.5 Thidiazuron 0.05
CH1
2,4-D
Picloram
0.5
0.26
BAP 0.09
RM-D 2,4-D 2.0 - -
RM-IK IAA 4.0 Kinetin 0.4
Results
Effect of stage of microspores: (a) Cluster of unresponsive early stage microspores, (b) Mature pollen committed to gametophytic pathway, (c) Light microscope observation of responsive enlarged (arrow head) and non-developing normal microspores.
Effect of centrifugation on androgenic response in chickpea microspores
Effect of electroporation of anthers on microspores in chickpea. DAPI stained microspores observed under Florescent microscope
(a) E-125V 25Ω 25µF, Dividing nucleus, (b) E-150V 25Ω 25µF, Binucleate microspore, (c) E-200 V 25Ω 25µF, uninucleate microspore
Effect of osmotic shock on androgenesis in chickpea. Divisions observed after 4 days in high osmotic induction medium (RM-D)
Microspores in culture observed under inverted microscope and DAPI staining after 4d in culture. (a,b) ICC 4958 subjected to cold pre-treatment for 3 d and centrifugation of anthers at 150g for 10min in RM-D medium. (a)Cellularization of microspore, (b) enlarged microcpore. (c,d) ICCV 95423 subjected to cold pre-treatment for 3 d and centrifugation of anthers at 150g for 10min in RM-IK medium.
Multicellular microspores of chickpea after 30 days in culture
(a) At culture (b)-(d) ICC 4958 after 10days in culture in Ch1PB media, trinucleate (b), five nucleate with both vegetative and generative nuclei diving (c), 3 (d) and 4 celled microspores respectively (e). (f) 1month in culture in M3, 4 celled. (g) 25days in culture in Ch1a, 8 celled microspore. (h) 1month in culture in Ch1PB media, multicellular structure.
Androgenic response of the four chickpea genotypes tested against the 12 media
Summary of AC results
Very low induction rates in all the four genotypes, Arearti being the least responsive.
Max response is 6.1 %
Combined pre-treatment of cold temperature for 4 days, centrifugation at 150g for 10 min and induction in high osmotic media (RM-D) was the most effective for chickpea genotypes tested
Most promising medium which supported multicellular microspores was CHPB medium composed of 8.8% sucrose, picloram and BAP
Rapeseed, barley and tobacco have been considered model species
Several genes which are differentially expressed at specific stages of microspore embryogenesis
Three main categories: (1) cellular response to the stress; (2) suppression of the gametophytic program; and (3) expression of the embryogenic program (Simarro and Neuz, 2008)
Genetic basis of Androgenesis
Interologs
Pictorial representation of interologs: Transferring protein interactions into different organisms
• Major consideration for building the network connection • In silico analysis often integrates multiple data types including
the gene co-expression, co-localization, functional category, and the occurrence of orthologs or interologs to derive a global network in a species
List of Query genes
SNo. GENE NCBI ID ORGANISM
FUNCTION
1 EM2 P22701.1 Triticum aestivum
Protection for the cytoplasm during the desiccation stage of embryo development, induced by osmotic stress and ABA
2 NtEPc BAA75495.1
Nicotiana tobacum
Marker protein for embryogenic dedifferentiation of immature pollen grains in vitro
3 phi-GST P30111.1 Triticum aestivum
Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles
4 ADH3 P10848 Hordeum vulgare
Alcohol dehydrogenase 3
5 SERK2 AEE31686.1
Arabidopsis thaliana
Somatic embryogenesis receptor kinase 2
6 AGL15 AAA65653.1
Arabidopsis thaliana
MADS-Box family protein expressed in developing embryos
7 ZmAE1 NP_001105113.1
Zea mays Androgenic embryo1
8 EcLTP AAF14232.1
Hordeum vulgare
Expressed in the early stages of microspore divisions
List of Query genes (Cont.) SNo. GENE NCBI ID ORGANISM FUNCTION
9 BBM AAM33803.1 Arabidopsis thaliana
Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth
10 BnmNAP P17333.1 Brassica napus Seed storage protein
11 AGP11 Q9FVE0.2 Arabidopsis thaliana
Developmental roles such as differentiation, cell-cell recognition, embryogenesis and programmed cell death
12 FtSH Q39102.2 Arabidopsis thaliana
ATP-dependent zinc metalloprotease FTSH 1, cholroplastic
13 Bl1 AAC49810.1 Arabidopsis thaliana
Involved in protein protein interactions, with SERK2
14 RIC2 Q8GYU0.1 Arabidopsis thaliana
Involved in pollen tube growth regulation through its interaction with ARAC11/ROP1
15 Cysteine protease 1 precursor
114958 Zea mays Cystatin which suppresses host immunity by inhibiting apoplastic cysteine proteases
16 Phytepsin precursor P42210.1 Hordeum vulgare Involved in the breakdown of propeptides of storage proteins in protein-storage vacuoles
List of Query genes (Cont.)
SNo. GENE NCBI ID ORGANISM FUNCTION
17 20S proteasome subunit Alpha-5
Q9LSU1.1 Oryza sativa Cleavage of peptide bonds with very broad specificity
18 26S proteasome regulatory subunit-8
Q9FHY0.2 Arabidopsis thaliana
ATP-dependent degradation of ubiquitinated
19 VAL1/MybTF Q8W4L5.1 Arabidopsis thaliana
Transcriptional repressor of gene expression involved in embryonic pathways, such as LEC1, ABI3, and FUS3
20 Maltase O04893.1 Hordeum vulgare Hydrolysis of terminal, non-reducing (1->4)-linked alpha-D-glucose residues with release of alpha-D-glucose.
21 Invertase AAA63802.1 Arabidopsis thaliana
Hydrolytic clevage of sucrose, regulates carbohydrate partitioning, developmental processes, hormone responses and biotic and abiotic interactions
22 HSP AAB28591.1 Hordeum vulgare Expressed during seedling development
23 14-3-3A P29305.1 Hordeum vulgare Cell death in non-enlarged microspores
24 14-3-3C Y14200) Hordeum vulgare Higer expression in dividing microspores
25 AGP AET04659.1 Medicago truncatula
Stimulates microspores embryogenesis from non-responsive genotypes.
Steps involved in analysis of ESTs for interolog mapping
Chickpea-Sequence similarity analysis using BLASTp and tFASTx
GENE NCBI
ID
ORGANI
SM FUNCTION BLASTp
Covera
ge Score
Identi
ty tFASTx Bits Score
Consens
us
EM2 P22701.
1
Triticum
aestivum
Protection for
the cytoplasm
during the
desiccation stage
of embryo
development,
induced by
osmotic stress
and ABA
XP_0045
06729.1
EMB-1
protein-
like
100 1.00E
-37 77
gi|146494
922
CAH
1-
70I6
35.6 0.075 0
NtEPc BAA75
495.1
Nicotiana
tobacum
Marker protein
for embryogenic
dedifferentiation
of immature
pollen grains in
vitro
XP_0045
08586.1
early
nodulin-
like
protein 1-
like
98 8.00E
-64 55
gi|146478
626
CAH
1-
32O2
40.7 0.0036 0
phi-
GST
P30111
.1
Triticum
aestivum
Conjugation of
reduced
glutathione to a
wide number of
exogenous and
endogenous
hydrophobic
electrophiles
XP_0044
95978.1
Glutathio
ne S-
transferas
e F13-
like
74 2.00E
-56 41 null null null 0
ADH3 P10848 Hordeum
vulgare
Alcohol
dehydrogenase 3
XP_0045
02578.1
Alcohol
dehydrog
enase 1-
like
98 2.00E
-61 55
gi|146485
017
CAH
1-
46L1
34.5 0.75 0
GENE NCBI ID ORGAN
ISM
FUNCTI
ON BLASTp
Coverag
e Score Identity tFASTx Bits Score
Consens
us
SERK2 AEE316
86.1
Arabidop
sis
thaliana
Somatic
embryog
enesis
receptor
kinase 2
XP_0044
96399.1
Somatic
embryog
enesis
receptor
kinase 2-
like
99 0 89 gi|14646
7403
CAH1-
10L1 99.1 2.20E-20 1
AGL15 AAA656
53.1
Arabidop
sis
thaliana
MADS-
Box
family
protein
expresse
d in
developi
ng
embryos
XP_0045
16227.1
Agamous
-like
MADS-
box
protein
AGL15-
like
88 2.00E-87 56 gi|14646
2182
CAH1-
13M1 47.8 3.50E-05 1
ZmAE1 NP_0011
05113.1 Zea mays
Androge
nic
embryo1
XP_0045
03421.1
LOW
QUALIT
Y
PROTEI
N:
carbon
catabolite
repressor
protein 4
homolog
3-like
[Cicer
arietinum
]
54 0.34 27 null null null 0
GENE NCBI
ID
ORGAN
ISM
FUNCT
ION
BLAST
p Coverage Score Identity tFASTx Bits Score
Consens
us
EcLTP AAF142
32.1
Hordeum
vulgare
Expresse
d in the
early
stages of
XP_0045
16173.1
Non-
specific
lipid
transfer
protein
1-like
94 6.00E-26 46 null null null 0
BBM AAM33
803.1
Arabidop
sis
thaliana
Ectopic
expressio
n of
BABY
BOOM
triggers a
conversi
on from
vegetativ
e to
embryon
ic growth
XP_0044
92865.1
AP2-like
ethylene-
responsi
ve
transcript
ion
factor
BBM-
like
[Cicer
arietinu
m]
68 7.00E-
148 57
gi|14648
3640
CAH1-
42P1 42.5 0.0034 0
BnmNA
P P17333.1
Brassica
napus
Seed
storage
protein
XP_0044
94200.1
Signal
recogniti
on
particle
54 kDa
protein,
chloropla
stic-like
28 1.6 23 gi|14647
8257
CAH1-
33M7 36 0.089 0
GENE NCBI ID ORGAN
ISM
FUNCTI
ON BLASTp
Coverag
e Score Identity tFASTx Bits Score
Consens
us
AGP11 Q9FVE0.
2
Arabidop
sis
thaliana
Develop
mental
roles
such as
differenti
ation,
cell-cell
recogniti
on,
embryog
enesis
and
program
med cell
death
XP_0045
11414.1
WEB
family
protein
At5g167
30,
chloropla
stic-like
67 0.057 29 gi|14649
8285
CAH1-
59C1 46.1 7.80E-05 0
FtSH Q39102.
2
Arabidop
sis
thaliana
ATP-
dependen
t zinc
metallopr
otease
FTSH 1,
cholropla
stic
XP_0045
00893.1
ATP-
dependen
t zinc
metallopr
otease
FTSH,
chloropla
stic-like
100 0 83 gi|14650
0064
CAH1-
58B2 116.3 3.00E-25 1
BI 1 AAC498
10.1
Arabidop
sis
thaliana
Involved
in protein
protein
interactio
ns, with
SERK2
XP_0045
02878.1
Brassinos
teroid
LRR
receptor
kinase-
like
96 0 68 gi|14646
7403
CAH1-
10L1 166.7 1.90E-40 1
GENE NCBI ID ORGAN
ISM
FUNCTIO
N
BLAS
Tp
Coverag
e Score
Identit
y tFASTx Bits Score
Consens
us
RIC2 Q8GYU0
.1
Arabidop
sis
thaliana
Involved in
pollen tube
growth
regulation
through its
interaction
with
ARAC11/R
OP1
XP_00
449204
5.1
Uncharac
terized
protein
LOC101
492616
51 2.00E-15 54 null null null 0
Cysteine
protease
1
precurso
r
NP_0011
4958.1 Zea mays
Cystatin
which
suppresses
host
immunity
by
inhibiting
apoplastic
cysteine
proteases
XP_00
449902
6.1
Cysteine
proteinas
e RD21a-
like
97 0 66 gi|146484
684
CAH1-
47A2 69.9 2.30E-11 1
Phytepsi
n
precurso
r
P42210.1 Hordeum
vulgare
Involved in
the
breakdown
of
propeptides
of storage
proteins in
protein-
storage
vacuoles
XP_00
450767
1.1
Aspartic
proteinas
e-like
isoform
X1
94 0 71 gi|146480
755
CAH1-
40O1 74 1.30E-12 1
GENE NCBI ID ORGAN
ISM
FUNCTIO
N
BLAS
Tp
Coverag
e Score Identity tFASTx Bits Score
Consens
us
20S
proteas
ome
subunit
Alpha-5
Q9LSU1
.1
Oryza
sativa
Cleavage
of peptide
bonds with
very broad
specificity
XP_0
0449
7162.
1
Proteaso
me
subunit
alpha
type-5-
like
isoform
X1
100 5.00E-
172 96
gi|14645
5688
CAH1-
1G23 37.2 0.047 0
26S
proteas
ome
regulato
ry
subunit-
8
Q9FHY
0.2
Arabido
psis
thaliana
ATP-
dependent
degradation
of
ubiquitinate
d
XP_0
0449
8915.
1
26S
proteaso
me non-
ATPase
regulator
y
subunit
RPN12
A-like
100 1.00E-
116 64 null null null 0
VAL1/
MybTF
Q8W4L
5.1
Arabido
psis
thaliana
Transcripti
onal
repressor of
gene
expression
involved in
embryonic
pathways,
such as
LEC1,
ABI3, and
FUS3
XP_0
0450
0370.
1
B3
domain-
containi
ng
protein
Os07g06
79700-
like
isoform
X3
93 0 48 null null null 0
GENE NCBI ID ORGAN
ISM FUNCTION
BLAST
p
Cover
age Score Identity tFASTx Bits Score
Consens
us
Maltase
O04893.
1
Hordeu
m
vulgare
Hydrolysis of
terminal,
non-reducing
(1->4)-linked
alpha-D-
glucose
residues with
release of
alpha-D-
glucose.
XP_004
486491.
1
Alpha-
glucosid
ase-like
isoform
X1
99 0 60 gi|14647
3773
CAH1-
28A1 114.9
6.40E-
25 0
Invertas
e
AAA63
802.1
Arabido
psis
thaliana
Hydrolytic
clevage of
sucrose,
regulates
carbohydrate
partitioning,
developmenta
l processes,
hormone
responses and
biotic and
abiotic
interactions
XP_004
515216.
1
Beta-
fructofur
anosidas
e,
insoluble
isoenzy
me 1-
like
96 0 64 null null null 0
HSP AAB285
91.1
Hordeu
m
vulgare
Expressed
during
seedling
development
XP_004
502738.
1
small
heat
shock
protein,
chloropla
stic-like
100 9.00E
-66 47 null null null 0
GENE NCBI ID ORGAN
ISM
FUNCTI
ON BLASTp
Coverag
e Score Identity tFASTx Bits Score
Consens
us
14-3-3A P29305.
1
Hordeu
m
vulgare
Cell
death in
non-
enlarged
microsp
ores
XP_004
489124.
1
14-3-3A
like 98
1.00E-
161 85
gi|14647
1021
CAH1-
25L1 115.7
1.70E-
25 1
14-3-3C Y14200
Hordeu
m
vulgare
Higer
expressi
on in
dividing
microsp
ores
XP_004
489124.
1
14-3-3A
like 98
3.00E-
163 86
gi|14647
1021
CAH1-
25L1 79.8
1.00E-
14 1
AGP AET046
59.1
Medicag
o
truncatul
a
Stimulat
es
microsp
ores
embryog
enesis
from
non-
responsi
ve
genotyp
es.
XP_004
504045.
1
uncharac
terized
protein
LOC101
497764
100 0 87 gi|14648
2239
CAH1-
39L7 298.8
2.20E-
80 1
Pearson correlation for co-expression of query genes
Chickpea-Consensus reached after 6-point classification scoring scheme (TRS)
Query Orthology
(a)
Pfam (b)
Functional linkages
(c)
Sub-cellular location
(d)
Interactants (e)
Interologs (f)
Total reliability
Score (a+b+c+d+e+f)
EM2
0 1 0 1 0 0 2
NtEPc
0 1 0 1 0 0 2
phi-GST
0 1 1 1 1 1 5
ADH3 0 1 0 0 1 1 3
SERK2 1 1 1 1 1 1 6
AGL15 1 1 1 0 1 1 5
ZmAE1 0 0 0 1 0 0 0
EcLTP 0 1 1 1 1 1 5
BBM 0 1 1 0 1 1 4
BnmNAP
0 1 1 1 1 1 5
AGP11 0 0 1 0 1 1 3
Query Orthology
(a)
Pfam (b)
Functional linkages
(c)
Sub-cellular location
(d)
Interactants
(e)
Interologs (f)
Total reliability
Score (a+b+c+d+e
+f)
FtSH 1 1 0 0 0 0 2
BI1 1 1 0 0 0 0 2
RIC2 0 1 0 1 1 1 4
Cysteine protease 1 precursor
1 1 0 1 0 0 3
Phytepsin precursor
1 1 0 1 0 0 3
2 20S proteasome
subunit Alpha-5
0 1 0 1 0 0 2
26S proteasome regulatory subunit-8
0 1 0 1 0 0 2
VAL1/MybTF
0 1 0 0 1 1 3
Query
Orthology
(a)
Pfam (b)
Functional linkages
(c)
Sub-cellular location
(d)
Interactants (e)
Interologs
(f)
Total reliability
Score (a+b+c+d+
e+f)
Maltase 0 1 0 1 1 1 4
Invertase 0 1 0 1 1 1 4
HSP 0 1 0 0 0 0 1
14-3-3A 1 1 0 1 0 0 3
14-3-3C 1 0 0 1 0 0 2
AGP 1 1 0 1 0 0 3
Protein interaction network built with SERK2 as potential candidate for androgenesis in chickpea
Proteomic studies
Simultaneously examine changes and classify temporal patterns of protein accumulation in different developmental stages and/or growth conditions
Complicated than gene expression and a compliment
2-DE method of choice to identify the total proteins expressed in any given tissue/cell
2-DE coupled with mass spectroscopy (MALDI-TOF/TOF MS) reveal complete proteome profile
Methodology
2-DE using Bio-Rad protocol; 17 cm IpG strip of pI 3-10, mw 14- 100 kDa
Gels were stained with colloidal CBB G250 for 48hrs, scanned and images obtained using GE scanner III
Overnight storage at 4°C, centrifuged at 10,000 rpm for 20 min at 4 0C.
Supernatant stored at -20 0C . Protein concentration determined by Bradfords assay.
Extraction of proteins from anthers at pre-culture and 4d in culture
Dry chilled power of anthers; acetone followed by hexane washes; solution of 0.05 mM Tris-HCl and 1% PVP 40 at pH 8.0 was added to the dry powder in 1:6 ratio (powder: buffer)
2-Dimensional electrophoresis gels of proteins extracted from anthers of chickpea
Virtual 2-DE • Produced using a software
JVirGel v2.0 (http://www.jvirgel.de/)
• ESTs of chickpea used as input
• Five EST sequences from the input were selected as landmarks on the experimental gel
• Protein spots in the range of pH 4-6 and 3-70 kDa
• 596 spots with their predicted pI and MW
• Presence of AGL16, WUS13 isoform X2, 14-3-3 GF14, 14-3-3C isoform X1, Glucan endo 1-3 β glucosidase14 , EMB 506 isoform X2, AGP 14, MYB4, ADH7 isoform X3, 14-3-3B isoform X1, ERF53 and ERF-LEP; indicate that chickpea is a possible candidate for androgenesis.
Pearl millet 6th most important cereal in the world
Equal or even superior to rice and maize in protein and oil content
Cultivated on about 26 million hectares in semi-arid tropics of the Africa and the Indian sub-continent
India is the largest` producer of pearl millet in Asia
DHs in Pearl millet Being exploited in breeding programs to develop DH but
merely as the pollinator which will be further eliminated, resulting in haploids of the recipient species, eg. wheat, oat etc
Limited work is carried out on in vitro production of haploids in pearl millet (Dang Ha and Pernes, 1982; Nitsch, 1982; Choi et al, 1989).
Most successful protocols for DH through anther and/or microspore culture belong to cereal species i.e. rice, barley, wheat and maize
DH are quicker to generate than NILs and RILs which are costly, making genetic studies difficult in out-crossing species
Anther culture
Culture in embryo induction media
Excision of florets aseptically
Surface sterilization of whole inflorescence
Cold treatment for 7days and untreated inflorescence
Inflorescence in flag leaf, msps at uninucleate stage
Genotypes: ICMB 89111, XL-51, 4201, ICMB 841-P3, ICMB 93333 (ICRISAT, Hyderabad, India).
Culture media
A B C D E F G H I J K L M
Basal
media(Ch)+
+ + + + + + + + + + + + +
Sucrose 1.7% 1.7% 1.7% 1.7% 1.7% 1.7% 1.7% 1.7% 1.7% 1.7% 8% 8% 8%
Maltose - - - - - - - - - - - - 4%
Agar(1.6%) + + + + + + + + + - + + +
PH 5.8 + + + + + + + + + + + + +
2-4D(mg/lt) - - -- - - 2 2 2 2 2 2 - 2
NAA(mg/lt) 2 2 2 2 2 - - - - - - 2 -
Kn(mg/lt) 1 - - - 1 - - - - - -- -
Zn(mg/lt) - 1 - - - - 1 - - - - - -
TDZ(mg/lt) -- - 1 - - - - - 1 - - - -
BAP(mg/lt) -- - - 1 - - - - - - - - 1
2ip(mg/lt) - - - - 1 - - 1 - 1 1 1 -
Light phase + + + + + + + + - + - - +
Dark phase - - + + + + + + + + + + -
Results
• Maximum response w.r.t. multi cellular microspores was observed in 841-P3 (13.7 %) followed by ICMB 93333 (9.51 %) and XL-51 (7.8 %) with the least being 4201 (0.21 %)
• Cold pre-treatment adversely affected viability of microspores • 8% sucrose more favorable • Addition of 4% maltose induced pro-embryoids and globular embryos • Most effective CH+8% sucrose+4% maltose+2-4D+BAP
Effect of pearl millet genotypes on microspore viability and androgenesis
Effect of light condition and growth regulators on androgenic response in pearl millet
Effect of sucrose concentration on the viability and androgenic response of microspores in pearl millet
Pro-embryoids of pearl millet, developed after 26 days in culture
2-Dimensional electrophoresis gels of proteins extracted from anthers of pearl millet
15 days in culture Pre-culture
Overlay of virtual pearl millet protein gels on the experimental 2-DE gels using JvirGel software
Fruitful MADS-box TF, Myc like regulatory gene product and putative RAB
ABC Transporter and α Amylase
Pre-culture 15 days in culture
11 proteins known to be involved in androgenesis such as ADH, AGL6, Heat shock TF, DREB, DREB2A, MADS5, Rab7, Leafy hull sterile, Terminal flower 1, No apical meristem (NAM) and Late Embryogenic abundant like have been identified in pearl millet. The presence of these genes during the culture phase is an indication of the androgenic and embryogenic potential of pearl millet.
Proteomic studies
With virtually no data is available regarding key regulators of androgenesis in legumes; a virgin attempt was made to build a protein-protein interaction network in chickpea based on interolog mapping in silico.
Can be extended to other legume crops not only to study androgenesis but also other specialized pathways like apomixes and embryogenesis.
Prediction of the transcriptional activators or triggers for these candidates would help to build a network of genes and signaling molecules.
Influence the choice of media components and alter the design of the experiment making it a more process and target specific approach
Summary