ELSEVIER Plant Science 121 (1996) 47- 61 Genetic transformation of regal pelargonium (Pelargonium Xdomesticum ‘Dubonnet’) by Agrobacterium tumefaciens M.R. Boase, S.C. Deroles, C.S. Winefield, S.M. Butcher, N.K. Borst, R.C. Butler .VCIV Zcc~luuclIus/i/ut~ ji/r Crop md Foot/ Rescwd~. Prirtrtr Bug 4005. Lrrirz, ,Yen Zethd Received 3 June 1996: revised 37 August 1996: accepted 7X August 1996 Abstract The regal pelargonium cultivar, Dubonnet, has been transformed by using three binary vectors pKiwi110, pGA643 and pLN I3 in the disarmed Apdwcterium turnqfuciem strain LBA4404. Kanamycin-resistant shoots were selected in regeneration medium and about 80’%,formed roots within 21 days in rooting medium containing 300 mg/l kanamycin monosulphate. Forty untransformed shoots failed to produce roots in the same medium, became chlorotic and died within 42 days. A total of 366 kanamycin-resistant shoots were successfully exflasked. We confirmed the stable integration of four transgenes (nptI1, gusA, als, @) by Southern analysis. Thirteen plants that were regenerated from explants inoculated with LBA4404jpKiwi110, and exflasked to a containment glasshouse, were GUS positive 34 months after explants were inoculated. Copyright (0 1996 Elsevier Science Ireland Ltd. &JXYMY/.S: A,grohcterim turm$~ien~s; /I-glucuronidase; Genetic transformation; Kanamycin resistance: Regal pelargonium 1. Introduction We are employing recombinant DNA tech- niques and tissue culture technologies to transfer a range of flavonoid and plant morphology genes to commercially important pelargonium cultivars. A prerequisite for this molecular breeding ap- proach to succeed is to have a reliable transforma- tion system for the target cultivars. Boast and Smith [l] report on transient GUS expression following Agrobncteknn-mediated gene transfer to 11 pelargonium cultivars includ- ing seven regals and four zonals (P. Xhortorum L.H. Bail). Their results suggest that a wide range of pelargonium cultivars are susceptible to Agrobacteriunl turmfrciem. Pellegrineschi and colleagues [2] report on an Agrobclcteriuln-mediated transformation for the scented species referred to as ‘lemon geranium’. This transformation system used A. rhkogenes and many plant characters were altered simulta- neously in the shoots which regenerated from the 01 6X-9452,‘96f Sl5.00 Copyright Cs 1996 Elsevier Science Ireland Ltd. All rights reserved P/I SO 168.9452(96)04508-h
Transcript
ELSEVIER Plant Science 121 (1996) 47- 61
Genetic transformation of regal pelargonium (Pelargonium Xdomesticum ‘Dubonnet’) by Agrobacterium tumefaciens
Received 3 June 1996: revised 37 August 1996: accepted 7X August 1996
Abstract
The regal pelargonium cultivar, Dubonnet, has been transformed by using three binary vectors pKiwi110, pGA643 and pLN I3 in the disarmed Apdwcterium turnqfuciem strain LBA4404. Kanamycin-resistant shoots were selected in
regeneration medium and about 80’%, formed roots within 21 days in rooting medium containing 300 mg/l kanamycin monosulphate. Forty untransformed shoots failed to produce roots in the same medium, became chlorotic and died
within 42 days. A total of 366 kanamycin-resistant shoots were successfully exflasked. We confirmed the stable
integration of four transgenes (nptI1, gusA, als, @) by Southern analysis. Thirteen plants that were regenerated from explants inoculated with LBA4404jpKiwi110, and exflasked to a containment glasshouse, were GUS positive 34 months after explants were inoculated. Copyright (0 1996 Elsevier Science Ireland Ltd.
niques and tissue culture technologies to transfer
a range of flavonoid and plant morphology genes
to commercially important pelargonium cultivars.
A prerequisite for this molecular breeding ap-
proach to succeed is to have a reliable transforma-
tion system for the target cultivars.
Boast and Smith [l] report on transient GUS
expression following Agrobncteknn-mediated
gene transfer to 11 pelargonium cultivars includ-
ing seven regals and four zonals (P. Xhortorum
L.H. Bail). Their results suggest that a wide range
of pelargonium cultivars are susceptible to
Agrobacteriunl turmfrciem.
Pellegrineschi and colleagues [2] report on an
Agrobclcteriuln-mediated transformation for the
scented species referred to as ‘lemon geranium’.
This transformation system used A. rhkogenes
and many plant characters were altered simulta-
neously in the shoots which regenerated from the
01 6X-9452,‘96f Sl5.00 Copyright Cs 1996 Elsevier Science Ireland Ltd. All rights reserved
P/I SO 168.9452(96)04508-h
48 M.R. Boase et al. I Plant Science 141 (1996) 47-61
transformed roots including stature, leaf and branch production, root system architecture, flow- ering and production of aromatic oils.
Robichon and colleagues [3] report on the ge- netic transformation of the tetraploid zonal culti- var, Peiargonium Xhortorum ‘Alain’. They used a 20 min period of co-cultivation of cotyledons and hypocotyls with the disarmed A. tumefaciens strain EHAlOl, and hygromycin as the selection agent. Southern analysis of 17 plants suggested most had a truncated copy of the T-DNA. Only one out of 57 displayed a noticeable GUS activity.
In this paper we report on the development of a genetic transformation system mediated by a dis- armed strain of Agrobacterium tumejkiens LBA4404, for the regal cultivar Pelargonium Xdo- mesticum ‘Dubonnet’ [4].
2. Materials and methods
2.1. Plant material and preparation of explants
Plants of Pelargonium Xdomesticum ‘Dubonnet’ were grown in polythene planting bags, contain- ing a steam-sterilised bark and pumice (50:50) potting mix with added fertiliser (NPK, 3:1:2). Bags were placed in a temperature controlled green-house ( 12- 16°C) and young expanding leaves were collected from near the shoot apices of non-flowering plants during May and June (late autumn-early winter). Leaves were washed in cold, running tap water for 15 min, then surface- sterilised on an orbital shaker for 15-20 min in a 0.6% sodium hypochlorite solution. A few drops of the surfactant, polyoxyethylene-sorbitan mono- laurate (Tween 20) were added to the hypochlor- ite solution to effect wetting. Leaves were then rinsed two to three times in sterile distilled water and explants were cut with a scalpel or with a 14 mm diameter, cork borer.
2.2. Plant tissue culture media and culture conditions
Four to six explants were placed adaxial side up on 25 ml of solid shoot regeneration medium in each 90 x 15 mm disposable plastic petri-dish.
Explants were precultured for l-3 days before inoculation with Agrobacterium tumefaciens. Shoot regeneration medium comprised Murashige and Skoog [5] (MS) salts, Linsmaier and Skoog [6] (LS) vitamins, 1 mg/l naphthylacetic acid (NAA), 1 mg/l benzyl-amino-purine (BAP), 30 g/l sucrose and 7.5 g/l agar (Davis Gelatine N.Z.). The medium used for growing in vitro stock plants of Dubonnet comprised MS salts, 100 mg/l myo-inositol, 0.34 mg/l thymine-HCL, 30 g/l su- crose and 7.5 g/l Davis agar. All media were adjusted to pH 5.8 with either 0.1 M NaOH or 0.1 M HCl before addition of agar, and auto- claved for 15 min at 121°C and 103 kPa. Leaf explant cultures were maintained at 20 + 3°C with a 16/8 h light/dark photoperiod at a light intensity of 20-50 pmol/m* per s from Osram 36 W grolux fluorescent tubes.
2.3. Optimization of shoot regeneration by factorial experiments
A 4 x 4, BAP versus NAA regeneration facto- rial experiment was set up in a completely ran- domised design to test if the shoot regeneration obtained from explants in the transformation ex- periments was optimal. A combination of BAP (0 1.01, 2.03, 3.04 mg/l), (0, 4.5, 9.0, 13.5 PM) and NAA concentrations (0, 0.42, 0.84, 2.51 mg/l), (0, 2.25, 4.5, 13.5 PM) was tested using a base medium which contained MS salts, LS vitamins, 30 g/l sucrose and 7.5 g/l agar (Davis Gelatine N.Z.). Leaf discs, 14 mm diam., from young ex vitro leaves were placed with their abaxial sides in contact with the medium, four per plate with two plates per treatment. Numbers of viable shoots for each plate in each treatment were counted 56 days after initial plating. The data were examined by using a Generalised Linear Model [7] with a Poisson error structure and a log link. The inter- action between BAP and NAA was not signifi- cant, so the fitted main effects model was therefore:
log(shoot number) = x + pi + yj + t
where CI is a constant, pi varies with BAP level, I’, varies with NAA level and E is random error with a Poisson distribution. The main effects were fur-
ther examined by fitting linear contrasts on the log scale to both BAP and NAA:
log(shoot number) = u + bBAP + cNAA + E
where CI, b and c are constants. This equates to fitting the product of two exponentials of BAP and NAA levels as follows:
Shoot number = e“ehBAPecNAA
This simple linear model was not significantly different at describing the data than the main effects model from the initial part of the analysis, so these linear contrasts can be used to give a reasonably simple description of the trends in the data. All analyses were carried out with Genstat 5
PI.
2.4. Determination of shoot regeneration sensitivity% to kanamycin
Leaf explants, 14 mm in diameter were plated on to shoot regeneration medium containing one of eight concentrations of kanamycin monosul- phate (Sigma) (0, 5, 10, 15, 20, 2.5, 30, 35 mg/l). Five explants from ex vitro leaves were used per plate and seven from in vitro leaves of stock plants. Each treatment was replicated three times. The number of discs with adventitious shoots was counted after 51 days for ex vitro leaves and after 71 days for in vitro leaves. The binomial data was examined with a generalised linear model [7] with a binomial error structure and a probit link [9], testing for differences in the slope and SPDSO parameters between explant sources. SPDSO, or shoot production dose 50’%, is the kanamycin concentration at which 50% of the explants pro- duced shoots. All analyses were carried out using Genstat 5 [8].
2.5. Agrobacterium strains and binary vectors
Transformation experiments were performed using Agrobacterium tumefaciens strain LBA4404. This carries the disarmed Ti plasmid pAL4404 and has Ach5 as a chromosomal background [lo]. Transgenic plants were generated using three bi- nary vectors: pKiwi110 [l l] (Fig. l(a)) carrying NOS/izptII. CaMV35slgusA and the uls gene;
pGA643 [12] (Fig. l(b)), carrying NOS/nptII, and pLN13 (K. Davies and M. Bradley pers, comm.) (Fig. l(c)) which is pGA643 plus CaMV35s/ cDFR from Antirrhinum majus (snapdragon). These three binary vectors were used respectively to assess gene transfer, to serve as a plasmid control and to transfer the Antirrhinum dihy- droflavonol-4-reductase cDNA in a co-suppres- sion attempt to manipulate the flavonoid pathway.
The T-DNA region of pKiwi110 contains three marker genes that can be expressed in plants. From the right to the left border, they are the neomycin phosphotransferase II gene @@II) which confers kanamycin resistance and is driven by the nopaline synthase promoter (nos), gusA reporter gene encoding /?-glucuronidase enzyme (GUS) under the cauliflower mosaic virus 35s promoter and a mutant Arabidopsis acetolactate synthase (als) gene under its native promoter which encodes resistance to sulfonylurea herbi- cides such as chlorsulfuron [13]. The latter is the active ingredient of GleanTM (Du Pont). The gusA gene is expressed upon transfer to plant cells but is not expressed in Agrobacterium due to the lack of a functional bacterial ribosome-binding site
[ill.
2.6. Cutture of agrobacteria, inoculation of explunts and cocultivation with agrobacteria
Agrobacterium strains were cultured in 10 mls of Luria-Bertani (LB)-broth (10 g/l tryptone, 10 g/l NaCl, 5 g/l yeast extract, pH 7.0-7.2) in 100 ml flasks on an orbital shaker for 16-20 h at 28°C and 300 x g. The following antibiotics were added to each 10 ml of LB-broth culture: (a) 20 mg/l of filter-sterilized streptomycin sulphate to select for Agrobacterium and against contaminating bacte- ria (b) 20 mg/l of filter-sterilized kanamycin monosulphate to select for the binary vectors. After culture overnight, 1.5 ml aliquots of broth cultures were centrifuged in Eppendorf tubes for 3 min at 12- 14 000 x g. The supernatant containing the antibiotics was discarded and the Agrobuc- terium cells were resuspended in lml of LB-broth. Leaf explants were dipped for 30 s to 1 min. in the resuspended Agrobacterium cultures, then blotted
50
a) pKIWI110 Xhol Xbal H3 RI RI H3_ [ RI , RI
M.R. Boase et al. / Plant Science 141 (1996) 47-61
medium containing 300 mg/l kanamycin monosul- phate to select for transformed cells and 500 mg/l ticarcillin disodium salt to prevent overgrowth of
the explant by Agrohcrcteriurn tumefuciens. Ex- plants were subcultured every three weeks to fresh selection medium and after 6 and 9 weeks, regen-
erated shoots were cut from the explants and
transferred to multiplication medium to produce larger shoots and clonal copies before rooting.
Multiplication medium comprised MS salts, B5 vitamins [14], 0.3 mg/l BAP, 0.1 mg/l gibberellic
acid (GA,) and 0.05 mg/l indole-3-butyric acid (IBA). Kanamycin monosulphate (300 mg/l) and
ticarcillin disodium salt (500 mg/l) were added to maintain selection pressure and control Agrohcrc- tcviuw growth respectively.
2.8. Root production USSLI_V in kumm~ycin
Shoots which regenerated adventitiously under 300 mg/l kanamycin monosulphate selection, and 40 untransformed adventitious shoots were as-
sayed over 48 days for their ability to survive and form roots in the presence of 300 mg/l kanamycin
monosulphate in a rooting medium. This com- prised MS salts, 100 mg/l myo-inositol, 0.34 mg/l thiamine-HCL. 30 g/l sucrose and 7.5 g/l Davis agar. Some shoots, from experiments with pGA643. pLN 13 and pKiwi110, that formed
kanamycin-resistant roots (putative transformed plants) were transferred to a containment
glasshouse for exflasking. Other shoots from
pKiwi 110 experiments that formed kanamycin-re- sistant roots were held in vitro for further expres- sion assays.
2.9. Root production in chlorsuljirron
Shoot condition and root production by twelve untransformed and 122 transformed shoots, in rooting medium containing 66.7 Llg/l Glean” her- bicide (DuPont), active agent 50 /(g/l chlorsul- furon, was recorded periodically over 56 days.
2. IO. HistochewGxd assay fbr /I-glucuronid~zse
Expression of the gusA gene was assayed by histochemical staining [I 51 of leaf material from both in vitro plants rooted in the presence of kanamycin and ex vitro plants. Leaf material
from 13 ex vitro plants was collected 34 months after explants were inoculated with LBA4404/
pKiwill0. Leaf discs, 10 mm in diameter were punctured over their entire surface with a scalpel
before incubation. Leaf discs from three plants
transformed with pLNl3 were included as a nega-
tive control. Material was incubated in a 0.63 mM solution of 5-bromo-4-chloro-3-indolyl-/?-D-glu-
curonic acid cyclohexylammonium salt (X-glut., Biosynth Ag, Switzerland) with 50 mM phosphate
buffer solution (pH 7.0) for 17 h at 37°C and cleared of chlorophyll in three washes of 95% ethanol, to unmask blue stains. Blue staining was
recorded as a GUS positive reaction. GUS expres- sion by colonies of LBA4404 containing
pKiwil10. grown on solid medium was also tested.
Flower material was collected when available, from five Dubonnet plants, 29 months after ex-
plants were inoculated with LBA4404ipKiwi 110. Tissue was GUS assayed for 17 h overnight at
37°C and cleared of flavonoid pigments by three washes of 95% ethanol.
-7.11. DNA isolutiotl and ctnalysi.s
DNA was isolated from non-transformed re-
generated plants and kanamycin-resistant shoots using a CTAB method based on that of Doyle
and Doyle [16]. The apex and youngest leaves
(0.5-1.0 g fresh weight) were excised and ground in liquid nitrogen to a fine powder. The tissue was suspended in extraction buffer (7% w/v) CTAB, 1.4 M NaCl. 3% (vjv) 2-mercaptoethanol, 20 mM EDTA and 100 mM Tris-HCl, (pH 8.0) preheated to 60°C. The mixture was then homogenized us- ing a Polytron PT3000 with a PT!DA 3012,QTS probe at 27 000 x g for 30 s. This homogenization treatment increased the final yield of DNA up to four times that of unhomogenized mixture, with- out visible degradation of the DNA when elec- trophoresed on a 1% agarose gel. 1 x TBE buffer
52 M.R. Boase er al. I Plant Science 141 (1996) 47-61
(90 mM Tris-borate, 2 mM EDTA). After ho- mogenization the mixture was incubated at 60°C for 30 min, extracted with an equal volume of chloroform and centrifuged at 1600 x g for 10 min. Nucleic acids were precipitated from the isolated aqueous phase by the addition of 2/3 volumes, of ice-cold isopropanol followed by 30 min on ice. The precipitate was centrifuged at 10000 x g for 10 min at 0°C and dried in air. The pellet was then washed by resuspending in 5 ml of wash buffer (76% v/v ethanol, 10 mM ammonium acetate), incubated for 20 min at O”C, centrifuged at 1600 x g for 10 min and re- suspended in 1 ml of TE buffer (10 mM Tris- HCl, pH 7.4, 1 mM EDTA). RNA was removed by incubating with 10 pug of RNAse A for 30 min at 37°C after which a further 1 ml TE buffer was added. The digested mixture was precipitated by adding 1 ml of 7.5 M ammo- nium acetate followed by 2.5 volumes of ethanol. After incubating for 30 min on ice the DNA was pelleted at 10 000 x g, 0°C for 10 min. The purified DNA was finally resuspended in 500 ~1 of TE buffer.
For Southern analysis 400 ~1 of the above samples were digested with restriction enzymes and separated electrophoretically on a 0.7% agarose gel. The DNA was then transferred onto Amersham ‘Hybond’ N+ blotting mem- brane under alkaline conditions following the manufacturer’s instructions. Control plasmid DNA (digested with the same enzymes) was in- cluded in all blots. Probe DNA was labelled via random priming with a32P dCTP. Unincorpo- rated label was removed using the method as decribed in Maniatis et al. [17].
3. Results
3.1. Shoot regeneration
Shoot regeneration occurred via adventitious organogenesis from leaf veins on the abaxial sides of the explants and explant margins (Fig. 2(a)), often via calluses. The first shoots were visible by 21 days and regeneration continued for at least nine weeks, allowing multiple har-
vests. There were up to six independent shoots per explant.
Fig. 3 shows the results from the BAP versus NAA regeneration factorial experiment. The highest average number of shoots per plate was obtained with 4.5 PM BAP and 2.25 PM NAA. Biometric analyses revealed that the statistical interaction between BAP and NAA levels over the range of concentrations examined was not significant (Fig. 4). This implies that the effect of BAP on shoot numbers can be examined in- dependently of the effect of NAA and vice versa.
3.2. Sensitivity of shoot regeneration to kanamycin
No shoots were produced on ex vitro explants in media containing more than 20 mg/l kanamycin sulphate or on in vitro explants at kanamycin concentrations above 10 mg/l (Fig. 5). SPDSO, the kanamycin concentration at which 50% of the explants produced shoots, SPD90, the kanamycin concentration at which 90% of the explants gave shoots, and the curve slope varied significantly (P < 0.05) with explant source (Table 1).
3.3. Production of transgenics
More than a 1000 kanamycin-resistant shoots of Dubonnet were produced in vitro. Kanamycin-resistant shoots (456), more than 80% of which possessed kanamycin-resistant roots were exflasked to glasshouse containment for genetically modified organisms. A total of 366 plants (80%) survived for at least 3 months after exflasking (Fig. 2(b)). One hundred had been inoculated with LBA4404/pGA643, 253 with LBA4404/pLN13 and 13 with LBA4404/ pKiwill0. A further 257 plants of Dubonnet were produced in vitro by inoculating explants with LBA4404/pKiwi 110. These plants were used in vitro for assays of marker gene expres- sion. A further 35 untransformed in vitro shoot regenerants were produced, rooted and exflasked to assess somaclonal variation.
M.R. Boase el al. II Plant Science 121 (IY96) 47-61 53
Fig. 2. Dubonnet regeneration and transformation process. A. Abaxial side of leaf disc showing sites of adventitious shoot
regeneration. B. Transgenic plants of Dubonnet transformed with pGA643 or pLNl.3. flowering in a containment glasshouse. C.
Untransformed shoot (left) failed to growth, became chlorotic and died without producing roots whereas a transgenic plant (right)
grew from a small shoot and produced multiple roots during 6 weeks in rooting medium containing kanamycin. D. GUS staining
pattern in leaf from an in vitro plant transformed with pKIWIll0 showing the strongest staining in the vascular tissue. E, Left: Gus
staining in leaf discs collected from 16 plants of Dubonnet, 34 months after inoculation with Agobacteria. Leaf discs in wells
Al --A4. Bl -B4, Cl -C4 and Dl are from each of the plants containing pKiwiI IO T-DNA. Leaf discs in wells D2-D4 arc from
control plants. Right: Close-up of punctured 10 mm disc, well C3, plant T90 showing GUS staining at 34 months.
54 M.R. Boast et al. i Plant Science 141 (1996) 47-61
Average 30’
, number of 25
shoots 20:
per plate 15:
1oj
BAP WV 13.5
Fig. 3. Shoot regeneration on Dubonnet leaf discs in media
with 16 combinations of BAP and NAA.
3.4. Root production assay in kanamycin
Adventitious shoots (444), which formed roots in rooting medium containing 300 mg/l kanamycin monosulphate, were produced from 666 explants of Dubonnet. Explants had been inoculated with Agrobacterium tumefaciens strain LBA4404, containing one of the binary vectors
3.6
3 -I
4.5 9 BAP level
13.5
r *.x vitro
in vitro
0 5 10 15 20 25 30 35 Kanamycin (m&l)
Fig. 5. Sensitivity to kanamycin monosulphate of shoot regen-
eration from in vitro and ex vitro leaves of Dubonnet.
pKiwill0, pGA643 or pLN13. Root production by kanamycin-resistant shoots
often occurred within 8 days and mostly within 21 days, after transfer to new rooting medium, in the absence of exogenous auxin. Forty untransformed adventitious shoots of Dubonnet failed to pro- duce roots in rooting medium with 300 mg/l kanamycin monosulphate, became chlorotic and
2.25 4.5 WA level
Fig. 4. Shoot regeneration from Dubonnet leaf discs. The triangles are mean log(shoot counts) for (a) each BAP concentration
averaged over the three NAA concentrations and (b) each NAA concentration averaged over the three BAP concentrations. The
lines with crosses arc the predicted response from fitting linear contrasts for (a) BAP. at NAA = 6.75 PM, i.e., mean of the 3 NAA
concentrations used and (b) NAA, at BAP = 9 ,O M, i.e.. mean of the 3 BAP concentrations used. The bars are the minimum and
maximum confidence intervals for the triangles, estimated from fitting the full model i.e., the model with both main effects and the interaction fitted.
Table 1
Probit parameters estimates from fitted curves (Fig. 5) with
standard errors in brackets (df = 26)
In vitro Ex vitro
Slope -0.2936 (0.0535) -0.1773 (0.0361)
SPDSO 6.295 (0.669) 15.39 (1.10)
SPD9O I .930 (0.955) 8.17 (1.76)
Slope in this table is the relative slope at the LD50 concentra-
tion.
died within 42 days (Fig. 2(c)). Some shoots con-
taining pGA643 and pLN13 remained green, grew
in rooting medium containing 300 mg/l
kanamycin monosulphate and produced adventi-
tious shoots within the medium. However, these
shoots would not form roots. They probably ex-
pressed lower degrees of kanamycin resistance
than the other plants. Over 60 of these plants
were exflasked and over one third produced roots
ex vitro and survived.
Two hundred and seventy shoots of Dubonnet
that were regenerated under 300 mg/l kanamycin
selection from explants inoculated with LBA4404/
pkiwil 10, were analysed for resistance to
kanamycin (root production in the presence of
300 mg/l kanamycin monosulphate), for expres-
sion of gusA and chlorsulfuron resistance (root
production in 50 Llg/l chlorsulfuron, i.e., 66.7 /(g/l
Glean herbicide). None of the 12 untransformed
shoots produced roots in 50 pg/l chlorsulfuron.
Of 270 shoots that arose under 300 mg/l
kanamycin selection, 128/258 (50%) formed roots
in the presence of 300 mg/l kanamycin, only
881’270 or 33% were GUS positive (Fig. 2(d)) and
15: 122 ( 12%) produced roots in rooting medium
containing chlorsulfuron. Only 13/122 plants
transformed with pKiwi110 (11%) expressed all
three genes. These 13 plants were chosen for
exflasking into glasshouse containment.
Of the 13 Dubonnet plants transformed with
pKiwill0 and exflasked to glasshouse contain-
ment. all were GUS positive (Fig. 2(e)), 34
months after the explants were inoculated. Blue
staining was evident around the vascular bundles
in some petioles cut transversely, within two hours of emersion in the X-glut. phosphate buffer solution. By 4.5 h, the epidermis and trichomes were also blue in this material. All material from
the three pLN13 transgenic Dubonnets, used as controls, assayed GUS negative. Colonies of LBA4404 containing pKiwi110, grown on solid
medium also assayed GUS negative. Of the flower material collected from five
Dubonnet plants, 29 months after explants were
inoculated with LBA4404,‘pKiwil IO, all five showed GUS activity in the veins of some petals and/or in small areas on the edges of some petals. Some blue staining was also seen at the base of some sepals and at the base of some carpels.
The isolated plant genomic DNA was cut with EcoRI. blotted and probed with the 3.8 kb J&I! X1101 fragment from pKiwi110 to detect the pres- ence of the 4.6 kb ALS-GUS and 4.2 kb
chloramphenicol resistance/OCS terminator re- gions (Fig. l(a) and 6(a)). Lane 1 shows the lack of these two bands in the untransformed control, and their presence in the transformed plants (open triangles--lane 2-A45, lane 5-T87. lane 6-T94 and
lane 7-T103). Lane 3 shows two bands (closed triangles) in the transformed plant K57 but there has been some internal rearrangement in the T-
DNA, indicated by the unexpected positions of these bands. This is also the case for lane 4 (T58) but the rearrangement has scattered the bands more widely.
To test for the presence of the als gene, the blot was reprobed with a 1.3 kb EcoRI fragment isolated from pKiwill0 which is designed to de- tect the 1.3 kb fragment of the ~1l.s gene adjacent to the left border (Fig. l(a) and 6(b)). Lanes 2- 7 show the expected band at 1.3 kb in the trans- formed plants. This band is absent in the untrans- formed plant----lane 1 which is the expected result under the high stringency wash conditions. Al- though this fragment of the LI/.S gene is present in
both K57 (lane 3) and T58 (lane 4), neither plant expressed chlorsulfuron resistance in vitro. By
and T103 (lane 7) did produce roots in rooting medium supplemented with chlorsulfuron.
To test for the presence of the 1.5 kb EcoRI NPTII-NOS fragment and for gene copy numbers (EcoRI right border fragments), the blot was
reprobed with a 8kb Hind111 fragment isolated from pLN3 (pKiwi110 minus the ~11s gene). This
probe also picks up the 4.2 kb chloramphenicol/ OCS EcoRI fragment (Fig. l(a)). Fig. 6(c) shows the absence of all bands in the untransformed
plant (lane 1) as expected. In the transformed plants (lanes 2-7), both the 1.5 kb NPTII-NOS fragment and the 4.2 kb chloramphenicol/OCS Eu~RI fragment are present as expected. The
minimum gene copy numbers indicated by the other bands, which are border fragments, in lanes 2-~7, vary from one to three.
3.6.2. Plunts transjkwzed with pGA643 (Fig.
7((l)). Plant and vector DNA (pGA643) was cut with
B~1rnHI/HindI11, blotted and probed with the 2.5 kb BanlHI/HindIII fragment from pGA643 (Fig. l(b) and 7(a)) to test for the presence of the kanamycin resistance gene. Lane 1 (pGA643 vec-
tor DNA) shows the expected band at 2.5 kb. The corresponding band is also visible in the three transformed plants (lanes 3,4 and 5) and absent in
the untransformed control (lane 2). This result shows that the kanamycin resistance gene is
present in an intact form in each of the transgenics.
3.6.3. Plmts tmmjbmed lvith pLN13 (Fig. 7(b)). Plant and vector DNA (pLN13) was cut with
MIjXb~I, blotted and probed with the 1.4 kb Sa/I!XbL~I fragment from pLN 13 to test for the presence of the DFR gene (Fig. 7(b)). Lane 1
(pLN 13 vector DNA) shows the expected band at
1.4 kb. The corresponding band is also visible in the four transformed plants (lanes 3, 4, 5 and 6) and absent in the untransformed control (lane 2). This result shows that the DFR gene is present in
an intact form in each of the transgenics.
3.6.4. Intt~grution of’ T-DNA into the plmt
genorw (Fig. 7(c)). To test for integration of the T-DNA into the
plant genome, both blots were reprobed with the
1.35 kb SulI/XbccI fragment from pGA643 (Fig. l(b,c) and Fig. 7(c)) containing the left border region. All the transgenics show a unique banding
pattern with none containing the expected frag- ment from either pGA643 (9.1 kb) or pLN13
( 1.35 kb) should the kanamycin-resistant pheno-
type be derived from Agrobucterium contamina- tion (Fig. 7(c,d)). Because each band corresponds to at least a single insertion event, T-DNA inser-
tions range from at least one to six copies per genome (open triangles).
In all plants tested the target genes have been transferred in an intact form and there is no evidence of Agrobacterz’urn contamination, thus leading to the conclusion that the plants are gen- uine transformants.
4. Discussion
We transformed the regal pelargonium cultivar, Dubonnet, by using three binary vectors, pKiwi110, pGA643 and pLN 13 in the disarmed Agrobucteriunl tumejkiens strain LBA4404. Kanamycin-resistant shoots were selected and
more than 80% formed roots, usually by day 2 1, in the presence of 300 mg/l kanamycin monosul-
Fig. 6. Southern analysis of DNA samples from plants transformed with pKiwi1 IO. (a) lane I-untransformed, lane 2-A45. lane
3-K57. lane 4-T%. lane 5-T87, lane 6-T94 and lane 7-Tl03. This blot was probed with a XbaI/XhoI fragment which is designed to
pick up two fragments-the 4.6 kb ALS-GUS and 4.2 kb OCS-chloramphenicol resistance regions. (b) lanes I -7 as for (a). This
blot was probed with a I.3 kb EcoRI fragment isolated from pKiwiI10. This fragment is designed to pick up the I.3 kb fragment
of the u/s gene adjacent to the left border.The lane loadings are the same as for (a). (c) lanes l-7 as for (a). This blot was probed
with a 8 kb Hi~~dIIl fragment isolated from pLN3. This probe picked up the right border EcoRI border fragment. the 1.5 kb E~wRI
Kan fragment and the 4.2 chloramphenicol/OCS EcoRI fragment. The lane loading order is the same as for (a). On this blot there
are two constant bands across the blot one being the 4.2 kB fragment the other being the 1.5 kB fragment. All other bands represent
border fragments.
58 M.R. Boase et al.
2SKb
9.1Kb
1 2 3 4 5
( Plant Science 141 (1996) 47-61
6
1.4Kb
2 3 4 5 6
1.3
1 2 3 4 5 6
Fig. 7. Southern analysis of DNA samples from plants transformed with pGA643 and pLNl3. (a) lane I-pGA643, lane
2-untransformed Pelatpkm, lane 3-TP8914. lane 4-TP901, lane 5-TP904. All DNA was cut with BarnHI/HindIlI and probed with
the 2.5 kb BarnHl/Hindlll fragment from pGA643. (b) lane I-pLNl3, lane 2-untransformed pelargonium, lane 3-H2, lane 4-H44,
lane 5-C23, lane 6-G24. All DNA was cut with SalI/XhaI and probed with the 1.4 kb Xbcrl fragment from pLNl3. (c and d) The
same blots as described in (a) and (b) probed with the 1.35 kb SalI,‘,Yhul fragment from pGA643.
phate. Forty untransformed shoots failed to pro- vitro leaf discs was determined, after the transfor-
duce roots in the same medium, became chlorotic mation experiments, to be 4.5 ,uM BAP and 2.25
and died within 42 days. We confirmed the stable ,LIM NAA. This result suggests the system could
integration of four transgenes (nptll, gus A, uls. be improved in future applications by reducing
@) by Southern analysis. To the best knowledge the NAA levels. There was a high random vari-
of the authors, this is the first published scientific ability in the data overall as can be seen by the
paper of an Agrobacterirlrn-mediated transforma- minimum and maximum 95% confidence limits
tion system for a regal pelargonium. for the means, Fig. 4. One possible source of this
Transgenic plants were regenerated from leaf variability could be that endogenous hormonal
pieces via organogenesis in medium containing 1 levels vary from explant to explant [18]. The
mg/l (4.44 PM) BAP and 1 mg/l (5.37 ,uM) NAA. analysis shown in Fig. 4 suggests that the exoge-
The optimum levels of BAP and NAA to max- nous phytohormones were acting independently
imise the number of shoots regenerated from ex over this concentration range in a multiplicative
59
way to affect shoot numbers, with shoot numbers non-selected genes were being expressed (see re-
decreasing significantly (P < 0.05) with increasing sults Section 3.5). The pattern of expression of
BAP levels and increasing NAA levels. There was marker genes (50% nptI1 + 33% gusA -+ 12% rrls)
a slightly stronger decrease in shoot numbers with suggests partial transfer to the plant cells of the
increasing BAP levels compared with increasing large T-DNA (25.7 kb) of the pKiwil10 binary
NAA levels. The decrease in shoot numbers sug- vector, with more efficient transfer of the genes
gests that the concentrations of exogenous phyto- adjacent to the right T-DNA border (1rptI1 gene)
hormones supplied range from near optimal to than adjacent to the left T-DNA border (u/s
supraoptimal for shoot regeneration. This range, gene). Transfer of T-DNA to plant cells is
together with the high random variability in the thought to be initiated from the right border; data overall is probably why the expected interac- therefore, some truncated T-DNA insertions are
tion between BAP and NAA in accordance with expected [27]. Further Southern analyses would be the classical Skoog and Miller model [19] was not needed to determine the frequency of truncated detected. T-DNA’s,
In contrast, Dunbar and Stephens [20] report shoot primordia regenerating on leaf explants of
seven regal cultivars by using a medium contain- ing 9.3 PM BAP and 11 ,uM NAA. Callus with
shoot primordia was transferred to a medium without NAA and one-tenth the BAP to induce shoot development. However, only 5O/o of the shoots formed were rooted.
Shoot regeneration from in vitro leaves was found to be more sensitive to kanamycin than shoot regeneration from ex vitro leaves (see re-
sults Section 3.2). Sensitivity to the selection agent affects the recovery of transgenic plants and is known to vary widely among tissues, with root
formation generally more sensitive than shoot for- mation and callus growth [21,22]. Also explant- specific kanamycin sensitivities have been
reported for mustard (Brussicu jmwu L. Czern and Coss cv. Rai5) and Vignu rudiutu cv. ML-5 (Mung bean) by Mathews [23]. Our results show that for Dubonnet, source of explant needs to be taken into account, as well as explant type, when choosing selection agent levels. In this study, kanamycin monosulphate at 300 mg/l, an order of magnitude greater than the SPDO for ex vitro leaf discs, was chosen as the selection level to reduce
the number of untransformed shoots that escaped selection and to select for high copy numbers of T-DNA’s [24,25]. The latter could lead to co-sup- pression [26].
High levels of non-expression of both selected and unselected genes have been reported in other
studies. For example, Deroles and Gardner [28]
found that 40/99 transgenic petunia plants exhib- ited reduced or no expression when leaf discs were rechallenged in a medium containing 300 mg/l
kanamycin. Atkinson and Gardener [29] found that in 55 transgenic pepino plants which had been transformed with A. turne~uc.iens strain LBA4404 containing pKiwi110. 32 (58%) did not express gltsA and 27 (49%) did not express the ~11s gene.
Southern analysis shows internal rearrange- ments and/or deletions have occurred in two
plants, K57 and T58 (Fig. 6(a,b)). This, togethei with the lack of chlorsulfuron resistance expressed in these two plants, suggests another explanation
for some of the non-expression of marker genes. Other explanations, not investigated in this
study, which could account for some of the gene silencing observed include, DNA methylation [30&32] and/or mutual transgene inactivation due
to multiple copies per nucleus (Fig. 6(c) and Fig. 7(c,d)) [33]. The structure of the integrated T- DNA’s, single copy, direct or inverted tandem repeats, and their genomic environment may es-
tablish transgene expression properties [34,35].
Plants that were regenerated from explants in- oculated with LBA4404/pKiwi 110, were examined in vitro for nptI1, gtrsA and uls expression, to determine the frequency at which selected and
The pattern of expression of marker genes (50% npf 11 + 33% ~u.FA + 12% uIS) suggests selectable markers are better placed near the left border to facilitate the identification of transgenic plants containing complete T-DNA inserts. Such binary vectors as the pGPTV series [36] the PART series [37] and pMOG410 [38] enable those plant cells
60 M.R. Boase et al. I Plant Science 141 (1996) 47-61
containing truncated T-DNA inserts to be coun- ter-selected. We are employing the transformation system reported here in combination with PART binary vectors to transfer a range of flavonoid and plant morphology genes to Dubonnet.
Thirteen plants transformed with pKiwi110, ex- hibited stable GUS expression in leaf material collected from plants in a containment glasshouse 34 months after explants were inoculated with Agrobacterium. This result indicates stable expres- sion of a nonselected gene over the long term and gives encouragement to those seeking stable inte- gration and expression of horticulturally useful trangenes in regal pelargoniums.
[2] A. Pellegrineschi, J. Damon, N. Valtorta. N.Paillard and
D. Tepfer, Improvement of ornamental characters and
fragrance production in lemon-scented geranium through
genetic transformation by Agrobacterium rhkgenes. Bio-
Technology 12 (1994) 64468.
[3] M.P. Robichon, J.P. Renou and R. Jalouzot. Genetic
transformation of Pelargonium Xhortorum. Plant Cell
Rep., 15 (1995) 63-67.
[4] J.D. Llewellyn, A check list and register of pelargonium
cultivar names: Part two, CF. International Registration
Authority for Pelargonium, Australian Geranium Society,
1985.
[5] T. Murashige and F. Skoog, A revised medium for rapid
growth and bioassays of tobacco tissue cultures. Physiol.
Plant.. 15 (1962) 473-497.
Although many self-pollinations were per- formed on transgenic plants of Dubonnet in this study, transgene inheritance studies were not pos- sible because no viable seed was produced. Chanon and Hanniford [39] report on a study of infertility in nine genotypes of regal pelargoniums and found low pollen viability, low pollen germi- nation and low seed set.
[6] E.M. Linsmaier and F. Skoog, Organic growth factor
requirements of tobacco tissue cultures. Physiol. Plant. 18
(1965) 100-127.
[7] P. McCullagh and J.A. Nelder, Generalized Linear Mod-
els, 2nd edn., Chapman and Hall, London, 1989.
The co-suppression attempt to manipulate the flavonoid pathway in Dubonnet, by using the Antirrhinum dihydroflavonol-4-reductase cDNA did result in some plants exhibiting altered pheno- types [40]. These results will be reported more fully elsewhere, once molecular analyses are com- pleted.
@I
[91
PO1
t111
[‘21
Acknowledgements [I31
We thank Dr Cathie Martin for supplying the DFR cDNA clone from Antirrhinum. Dr B. Jor- dan, Dr T. Conner and Dr J. Grant provided critical review of the manuscript. Dr P. Umaha- ran and Jenny S. Smith are thanked for providing technical assistance and Jane Riley for photo- graphic work. The Foundation for Research, Sci- ence and Technology (CO2405) is acknowledged for funding this research.
[‘41
1151
U61
u71
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[I] M.R. Boase and J.L. Smith, Expression of the GUS gene in 11 pelargonium cultivars transformed by Agrohac-
