over, the presence of the
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 (
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).
strains and plasmidsFor inoculation of embryo axes we used
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
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-
l aliquot of an
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
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
3% sucrose at a final density of 1
cells/ml.CocultivationThirty injured embryo axes at a time were incubated in 2ml of
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
C under a light intensity of 140
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
) and decapitated (
) embryo axis used forcocultivation.
Response of embryo axes toincreasing concentrations of kanamycin (mg/l).
GUS activityin leaf and shoot bud, respectively, of putative transformedshoots.
(Unstained) histological section of the shoot bud shown in
Transformed shootgrafted on the epicotyl of an in vitro-grown seedling.
Graftedshoots transferred to soil at the beginning of the hardening phase.
Southern analysis of putative primary transformants (
):genomic DNA digested with
III probed with a [32P]-labelled0.85-kb
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
1/4) or multiple (2/1
7/1) copies have been integrated.
PCR analysis of 4 T1 progeny plants deriving from lines 15/1 (
), 14/4 (
) and 13/1 (
4 and 5
). Position 1860–2222 of Tn5, whichis part of the coding region of
II (363bp), was amplified. The
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
C in the presence of 1m
X-gluc in 0.1
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 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
III (Boehringer/Germany), separated on 0.8%agarose gels, transferred to Hybond N
nylon membrane(Amersham) and hybridised with a 0.85-kb NPTII-
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
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