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