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Agrobacterium mediated transformation of chickpea ( Cicer arietinum L.) embryo axes.pdf

Agrobacterium mediated transformation of chickpea ( Cicer arietinum L.) embryo axes.pdf

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Agrobacterium mediated transformation of chickpea ( Cicer arietinum L.) embryo axes
Agrobacterium mediated transformation of chickpea ( Cicer arietinum L.) embryo axes

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Published by: Muhammad Atif Raza Attari on Sep 10, 2013
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Plant Cell Reports (2000) 19:235–240
Q
Springer-Verlag 2000Communicated by H. LörzK.V. Krishnamurthy
7
K. Suhasini
7
A.P. SagarePlant Tissue Culture Division, National Chemical Laboratory,Pune 411 008, IndiaM. Meixner
7
T. Pickardt
7
O. Schieder (
Y
)Institute for Applied Genetics, Free University of Berlin,Albrecht-Thaer-Weg 6, 14195 Berlin, Germanye-mail: schieder
6
zedat.fu-berlin.deFax:
c
49–30–8384345A. de KathenDep. of Molecular Genetics, University of Hannover,Herrenhauserstr. 2, 30419 Hannover, Germany
K.V. Krishnamurthy
7
K. Suhasini
7
A.P. SagareM. Meixner
7
A. de Kathen
7
T. PickardtO. Schieder
 Agrobacterium 
mediated transformation of chickpea(
Cicer arietinum 
L.) embryo axes
Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999
Abstract
Embryo axes of four accessions of chickpea(
Cicer arietinum
L.) were treated with
 Agrobacteriumtumefaciens
strains C58C1/GV2260 carrying theplasmid p35SGUSINT and EHA101 harbouring theplasmid pIBGUS. In both vectors the GUS gene isinterrupted by an intron. After inoculation shootformation was promoted on MS medium containing0.5mg/l BAP under a selection pressure of 100mg/lkanamycin or 10mg/l phosphinothricin, depending onthe construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemicallocalization of GUS activity in regenerated shoots.Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered.T-DNA integration was confirmed by Southern anal-ysis by random selection of putative transformants. Theanalysis of 4 plantlets of the T1 progeny revealed thatnone of them was GUS-positive, whereas the presenceof the
npt 
II gene could be detected by polymerasechain reaction.
Key words
Chickpea
7
Transformation
7
 Agrobacterium
Abbreviations
BAP 
: Benzylaminopurine
7
PPT 
:Phosphinothricin
7
MS
: Murashige and Skoog (1962)
7
GUS
:
-Glucuronidase
7
NPTII 
: Neomycinphosphotransferase
Introduction
Chickpea (
Cicer arietinum
L.), an important grainlegume, suffers from heavy losses due to fungaldiseases and insect pests, mainly
 Ascochyta rabiei
andchickpea pod borer (Singh et al. 1994). Although wildspecies of Cicer have numerous desirable traits, thecross-incompatibility between the wild and cultivatedvarieties has deterred improvement of the crop byconventional plant breeding techniques (van Rheenenet al. 1993). The introduction of specific genes intochickpea could be achieved by genetic engineering. Theprerequisites for a successful gene transfer of desirabletraits is the establishment of an efficient transformationprotocol.To date, only a few reports on transformation areavailable in chickpea. Transformed callus was obtainedusing wild strains of 
 Agrobacterium
(Mohapatra andSharma 1991; Islam et al. 1994), while hairy roots wereobtained by treating chickpea plants with root-inducingstrains of 
 Agrobacterium
(Siefkes-Boer et al. 1995).Transformed plants were obtained by Fontana et al.(1993) and Kar et al. (1996) by treating seed-derivedembryo axes deprived of the apical meristem with
 Agrobacterium tumefaciens
strain LBA 4404harbouring the binary vector pBI121. The inheritanceof the transgenes was not demonstrated in thesestudies.In the present report, we describe the combinationof a simple protocol for multiple shoot formation frommature seed-derived embryos with
 Agrobacterium
mediated gene transfer. From four cultivars of chickpea, kanamycin-as well as phosphinotricin-resistant plants were recovered. The integration of theT-DNA was demonstrated by Southern analysis. More-
 
236
over, the presence of the
npt 
II-gene in the T1 progenycould be detected by the polymerase chain reaction.Although the current protocol is still labour-consumingdue to its relatively low transformation efficiency, itmay have general application in the transformation of chickpea.
Materials and methods
Plant materialSeeds of chickpea (
Cicer arietinum
L.) cultivars ‘PG1’, ‘PG12’ and‘Chafa’ were obtained from Mahatma Phule Agricultural Univer-sity, Rahuri, and cv ‘Turkey’ was obtained from the local marketin Berlin, Germany. Seeds were surface-sterilized according tothe protocol described by Suhasini et al. (1994) and soaked over-night (16h) in sterile distilled water. The embryo axes weresubsequently separated from the cotyledons and injured by theremoval of the shoot and root meristems (Fig.1A).
 Agrobacterium
strains and plasmidsFor inoculation of embryo axes we used
 A. tumefaciens
strainscarrying binary vectors: (1) C58C1/GV2260 containingp35SGUSINT and (2) EHA101 (Hood et al. 1986) containingpIBGUS. Plasmid p35SGUSINT (see Fig.1H) is a pBin19 deriva-tive, which carries the GUS gene with a ST-LS1-derived intron(Vancanneyt et al. 1990). Plasmid pIBGUS was constructed byintroducing the
Hind
III fragment of p35SGUSINT (the GUSgene containing the intron; de Kathen and Jacobsen 1995) intopIB16.41 (Strauch et al. 1988) carrying a procaryotic gentamycinresistance and 35S-PAT and Nos-NPTII as plant selectablemarkers. A 500-
m
l aliquot of an
 Agrobacterium
culture takenfrom glycerol stocks was suspended in 5ml of YEB medium (VanLarebeke et al. 1977) with 50mg/l kanamycin (GV2260/p35SGUSINT) or 40mg/l geneticin (EHA101/pIBGUS) andgrown at 200rpm on a rotary shaker at 28
7
C for 16h. The culturewas subsequently spun at 5000rpm at RT for 10min in an Eppen-dorf centrifuge and the pellet resuspended in 2ml liquid MSmedium with 0.5mg/l BAP
c
3% sucrose at a final density of 1
!
10
5
cells/ml.CocultivationThirty injured embryo axes at a time were incubated in 2ml of 
 Agrobacterium
culture for 20min, blotted dry on a sterile filterpaper and cocultivated in 55-mm petri dishes on gelrite-solidified(0.3%) MS medium (Murashige and Skoog 1962) supplementedwith 0.5mg/l BAP and 3% sucrose. Cocultivation was performedfor 72h at 25
7
C under a light intensity of 140
m
mol m
–2
s
–1
provided by cool-white fluorescent tubes and a 16-h photoperiod.After cocultivation the explants were rinsed three times withsterile distilled water containing 500mg/l cefotaxime (Claforan,Hoechst, Frankfurt/Main, Germany), blotted dry on sterile filterpaper and cultured in 250-ml glass jars containing shoot regener-ation medium (MS salts, 0.5mg/l BAP, 3% sucrose, 0.3% gelrite)supplemented with 500mg/l cefotaxime and either 100mg/l kana-mycin (Sigma Chemical, USA) or 10mg/l PPT (DL-PPT,obtained from Hoechst, Germany) depending on the agrobac-terial construct used for treating the embryo axes.Selection and regeneration of plantsThe responses of plant tissue to kanamycin and PPT and theoptimal selection pressure were determined by culturing injuredembryo axes (50 per treatment) on shoot regeneration medium
P
Fig.1 A
Entire (
a
) and decapitated (
b
) embryo axis used forcocultivation.
Bar 
: 1.25mm.
B
Response of embryo axes toincreasing concentrations of kanamycin (mg/l).
C, D
GUS activityin leaf and shoot bud, respectively, of putative transformedshoots.
 sm
Shoot meristem.
E
(Unstained) histological section of the shoot bud shown in
D
.
Bar 
:125
m
m.
F
Transformed shootgrafted on the epicotyl of an in vitro-grown seedling.
G
Graftedshoots transferred to soil at the beginning of the hardening phase.
H
Southern analysis of putative primary transformants (
T0
):genomic DNA digested with
Hind
III probed with a [32P]-labelled0.85-kb
npt 
II fragment. The different sizes of the hybridising frag-ments indicate the integration of the T-DNA into the plantgenome: the number of hybridising fragments shows that 1 (lines6/1
c
1/4) or multiple (2/1
c
7/1) copies have been integrated.
I
PCR analysis of 4 T1 progeny plants deriving from lines 15/1 (
lane 2
), 14/4 (
 3
) and 13/1 (
4 and 5
). Position 1860–2222 of Tn5, whichis part of the coding region of 
npt 
II (363bp), was amplified. The
npt 
II-containing vector pHP23 served as a positive control(see above) containing 25, 50, 75, 100, 200, 300 or 400mg/l kana-mycin or 2, 5, 10 or 20mg/l PPT under the conditions statedabove for cocultivation. The cultures were scored after 4 weeks.Inhibition of shoot proliferation from mature embryo axes wasobserved on medium containing 100mg/l kanamycin (Fig.1B) or10mg/l PPT.Approximately 160–170 explants per experiment were coculti-vated, and the experiments were repeated three times. Thecultures were subcultured at 3-week intervals on shoot regenera-tion media to retain the selection pressure under the same condi-tions as stated above. The concentration of cefotaxime was halfedat each subculture. Green shoots growing in the presence of 100mg/l kanamycin or 10mg/l PPT were scored for GUS activityat different intervals of time. Mature plants were generated bygrafting shoots of an appropriate length onto 5-day-old etiolatedseedlings of chickpea. Grafting was carried out as described byPickardt et al. (1995).Tissue staining for gus activityTissues were incubated for 12h at 37
7
C in the presence of 1m
X-gluc in 0.1
NaPO
4,
pH7.4, 0.1% Triton X-100, 0.5m
potas-sium ferricyanide and potassium ferrocyanide and 10m
EDTA(Stomp 1992), and subsequently bleached in 95% ethanol beforeobservation. The tissue was further processed by passing itthrough toluene and embedding in wax. The embedded tissue wasthen cut into 10-
m
m sections, mounted, dewaxed and viewedunder the microscope. Shoots derived from untreated embryoaxes served as controls.Southern hybridisation and polymerase chain reaction (PCR)The presence and integration of the NPTII gene was analysed bySouthern blot (T0) and polymerase chain reaction (T1). GenomicDNA was isolated from non-transformed plants and putativetransformants according to the protocol of Rogers and Bendich(1985) with modifications. For Southern blotting the DNA wasdigested with
Hind
III (Boehringer/Germany), separated on 0.8%agarose gels, transferred to Hybond N
c
nylon membrane(Amersham) and hybridised with a 0.85-kb NPTII-
Hind
III-frag-ment excised from pHP23 (Paszkowski et al. 1988). The probeswere radiolabelled according to Feinberg and Vogelstein (1983).After hybridization, the filter was exposed to X-ray film at –70
7
Cfor 5 days.For the detection of the NPTII coding sequence in the progenygenomic DNA was subjected to PCR using the following primersand conditions: forward 5
b
-TCATCTCACCTTGCTCCTG-3’
 
237

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