Plant Cell Reports (1999) 18: 554–560
© Springer-Verlag 1999
I. S. Curtis · J. B. Power · P. Hedden · D. A. Ward A. Phillips · K. C. Lowe · M. R. Davey
A stable transformation system for the ornamental plant, Datura meteloides D. C.
Received: 7 September 1998 / Revision received: 16 November 1998 / Accepted: 16 November 1998
Abstract A transformation system is described for Datura meteloides using the supervirulent Agrobacterium tumefaciens strain 1065, carrying both the β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) genes between the T-DNA border sequences of the binary vector. The importance of conditions such as the preculture period of the plant tissues, wounding, bacterial dilution and incubation time were evaluated in terms of transgenic plant production. A preculture period of 2–3 days, using a 1 : 20 or 1 : 10 (vol : vol) dilution of an overnight bacterial culture, resulted in optimum shoot regeneration, with 48% from a total of 576 explants regenerating transformed shoots. Expression of the gusA and nptII genes was confirmed by a GUS fluorometric assay and by NPTII ELISA. Southern analysis revealed the integration of both transgenes, which segregated as dominant Mendelian traits in seed progeny. Key words Agrobacterium-mediated transformation · Datura meteloides · Preculture period · Transgene expression Abbreviations DIG Digoxigenin · GUS β-Glucuronidase · IAA Indole-3-acetic acid · MS Murashige and Skoog · NPTII Neomycin phosphotransferase II · NN Nitsch and Nitsch · SR medium Shoot regeneration medium
Communicated by G. Hahne I. S. Curtis · J. B. Power · K. C. Lowe · M. R. Davey (½) Plant Science Division, School of Biological Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK e-mail:
[email protected] Fax: +44-115-9513240 P. Hedden · D. A. Ward · A. Phillips IACR-Long Ashton, Department of Agricultural Sciences, University of Bristol, Long Ashton, BS41 9AF, UK
Introduction
The genus Datura is comprised of 15 species of annuals, trees and shrubs distributed over warm and temperate regions of the world. Shrubby species are grouped in the genus Brugmansia and annual species in Datura (Chittenden 1965). Previous studies have demonstrated that Datura is amenable to both tissue culture (Sharma et al. 1993) and to Agrobacterium-mediated transformation (De Cleene and De Ley 1976; Sangwan et al. 1991, 1993; Ducrocq et al. 1994). Indeed, the initiation of hairy root cultures following inoculation of tissues by Agrobacterium rhizogenes, promoted in several Datura species, higher yields of pharmaceutical products than in cultured cells (Christen et al. 1989; Robbins et al. 1991; Dupraz et al. 1994; Hilton and Wilson 1995). Cultured hairy roots are a more important source of the tropane alkaloids scopolamine and hyoscyamine than aerial parts of the plant (Christen et al. 1989). A leaf disc transformation system has been developed for Datura innoxia (Sangwan et al. 1991), using A. tumefaciens, but its application has not yet been investigated for other Datura species grown as ornamentals. D. meteloides is one of the most attractive ornamental plants of the genus, with large, strongly scented trumpetshaped flowers and slate-blue-coloured foliage. The plant is grown primarily from seed as an annual, but can be propagated from cuttings. Although cultivated as a bedding plant or a patio pot plant, it has a tall and spreading habit (height 120 cm, spread 95 cm). Hence, the development of a transformation system would enable the manipulation of plant habit, such as the expression of the rolC gene from A. rhizogenes, which has been shown to induce dwarfism in some species (Schmülling et al. 1988). Additionally, the expression of genes which modify gibberellin biosynthesis may provide an alternative approach for dwarfing Datura and other ornamentals (Curtis et al., in press; Hedden et al., in press). In the present study, an Agrobacteriummediated transformation system has been developed for this ornamental.
555
Fig. 1 T-DNA restriction map of pVDH65. The NPTII gene, driven by the nopaline synthase promoter (Pnos), was inserted near to the left border (LB) of the T-DNA. The GUS-intron gene under the CaMV 35S promoter (P35S) with a CaMV 35S terminator sequence (T35S) was sited near to the T-DNA right border (RB). The horizontal line of 2.7 kb represents the HindIII restriction fragment released when genomic DNA of transgenic plants was digested with HindIII restriction enzyme
Materials and methods
riod, explants were transferred to SR medium containing 200 mg l–1 kanamycin sulphate and 200 mg l–1 cefotaxime. Once the explants had developed dark green organogenic calli (21–28 days post-inoculation), they were transferred to the same SR medium containing cefotaxime, but with kanamycin sulphate at 100 mg l–1. Forty-two days post-inoculation, explants were transferred individually to 40ml aliquots of SR antibiotic medium in 175-ml-capacity screwcapped glass jars (Beatson Clarke, Rotherham, UK), to allow stem elongation. Excised shoots (approx. 1 cm in height) were transferred individually to jars containing MS0 medium with 50 mg l–1 kanamycin sulphate, 30 g l–1 sucrose, 0.8% (wt/vol) agar (Sigma), at pH 5.8. Rooted plants were potted in a mixture (6 : 6 : 1 by vol) of Levington M3 compost, John Innes No. 3 compost (J. Bentley, Barrow-on-Humber, UK) and Perlite (Silvaperl, Gainsborough, UK), acclimatised to glasshouse conditions (Curtis et al. 1994) and grown to maturity. After selfing the regenerated (T0) plants, selection for transgenic seedlings (T1 generation) was performed by germinating seeds on MS0 medium, semi-solidified with 0.8% (wt/vol) agar and supplemented with 200 mg l–1 kanamycin sulphate, in 9-cm-diameter Petri dishes (20 ml medium; seven to ten seeds/dish). The seeds were surface sterilised prior to sowing (Curtis et al. 1994).
Plant material Seeds of D. meteloides cv. Evening Fragrance (supplied by Thompson and Morgan, Ipswich, UK) were sown in 9-cm-diameter pots containing Levington M3 compost (Fisons, Ipswich, UK). After germination, plants were grown in the glasshouse under natural daylight, with supplementary lighting (61 µmol m–2 s–1, Daylight fluorescent tubes) at 26 ±2 °C (day) and 20 ±2 °C (night). Six to 8 weeks from sowing, fully expanded leaves (from nodal regions 3–8 from the top of the plant downwards) were harvested and used for experimentation. Bacterial strain and plasmids A. tumefaciens strain 1065 was chosen for transformation studies due to its efficiency in transforming a range of target plants (Curtis et al. 1994; Drake et al. 1996; Knoll et al. 1997). This bacterium was strain LBA4404 (Hoekema et al. 1983), carrying pVDH65 (Fig. 1), a modified binary vector pMOG23 (Sijmons et al. 1990) with 35S.gusintron and nos.nptII.nos genes located between T-DNA left and right borders. The presence of pTOK47 (Jin et al. 1987) in the bacterium conferred supervirulence. Strain 1065 was maintained as described by Curtis et al. (1994). Production of transgenic plants Leaves from glasshouse-grown plants were surface sterilised by immersion in 7% (vol : vol) Domestos bleach solution (Lever Industrial, Runcorn, UK) for 5 min, followed by three rinses with sterile distilled water. Explants (ca 1 cm2) were excised from detached leaves; some were scored with a scapel on their abaxial surface, at right angles to the mid-rib. Explants were incubated, abaxial surface down, in Petri dishes (eight discs/dish), each containing 20 ml of shoot regeneration (SR) medium. The latter consisted of Murashige and Skoog (1962) (MS) macro and micro salts (Sigma), Nitsch and Nitsch (1969) (NN) vitamins (Sigma), 20 g l–1 sucrose, 0.5 g l–1 MES, 5 mg l–1 2-iP, 0.1 mg l–1 indole-3-acetic acid (IAA) and 0.7% wt/vol agar (Sigma), pH 5.7. IAA was filter sterilised and added to the autoclaved medium after cooling to 50 °C. Leaf explants were incubated on SR medium for 0, 1, 2, 3 and 4 days (preculture), at 26 ±2 °C (16 h photoperiod, 25 µmol m–2 s–1, Daylight fluorescent tubes). Precultured explants were floated in 20-ml aliquots of MS0 liquid medium (full strength MS macro and micro salts and vitamins, 30 g l–1 sucrose, but lacking growth regulators, pH 5.8) for 5, 10 or 20 min, containing three dilutions of an overnight culture of Agrobacterium (1 : 20, 1 : 10, 1 : 5 vol : vol). Control explants were floated on MS0 liquid medium. Explants were blotted dry on sterile filter paper and incubated on SR medium. After a 2-day cocultivation pe-
Histochemical and fluorometric assays for β-glucuronidase (GUS) activity Seven days after inoculation of leaf explants with Agrobacterium, one or two explants/dish per treatment were selected randomly for GUS histochemical assay (Jefferson et al. 1987). GUS activity was detected in pollen from mature plants by floating dehisced anthers in reaction buffer. GUS activity in transgenic plants was quantified by fluorometric analysis of leaf protein extracts (Gartland et al. 1995). Such extracts were also used for neomycin phosphotransferase II (NPTII) ELISA (see below). Control samples were from plants regenerated from uninfected explants. NPTII ELISA of regenerated plants Detection and quantification of NPTII protein in crude leaf extracts was by NPTII ELISA (5 Prime → 3 Prime, Boulder, Colo.) (Curtis et al. 1995). Southern hybridisation of transgenic plants Genomic DNA was extracted from leaves (approx. 1 g fresh weight) of untransformed and transformed plants (Dellaporta et al. 1983). Two DNA digests were set up, separately, for each plant sample, using 10 µg of DNA for each digest. EcoRI was used to determine the number of T-DNA fragments which had integrated into the genome of transformed plants; HindIII was used to determine whether the gusA gene had integrated as a single 2.7-kb fragment (Fig. 1). Digested DNA was electrophoresed in 0.8% (wt/vol) agarose gels and blotted onto positively charged nylon membranes (Boehringer Mannheim, Lewes, UK). The latter were probed first with a gusA gene digoxigenin (DIG)-labelled probe (McCabe et al. 1997) and, after stripping, with a nptII gene DIG-labelled probe. Phenotypic characterisation of regenerated plants Plants were characterised 4 months after transfer to the glasshouse in terms of their height, internodal length, maximum leaf length and width (leaves and internodes were measured from the eight nodal regions from the top of the plant downwards) and time to anthesis. Seed weight (three random samples, each of 50 seeds/plant) was determined as an indirect measure of fertility.
556 Table 1 Percentage of explants regenerating shoots. Shoot regeneration was assessed 3 months after Agrobacterium inoculation (24 explants/treatment, eight explants/dish). Control explants were culPreculture (days)
Abaxially wounded
tured on medium lacking antibiotics (Bacterial dilution as vol : vol, for three incubation times: 5, 10 and 20 min) Bacterial dilution
0 (control)
1 : 20
5 min
10 min 20 min
5 min
1 : 10
10 min 20 min
5 min
1:5
10 min 20 min
5 min
10 min 20 min
0
+ –
88 100
92 96
75 96
0 8
8 13
13 8
0 8
4 8
4 8
0 8
0 4
4 4
1
+ –
83 96
83 96
96 100
21 42
17 50
17 58
25 38
29 58
29 50
17 29
13 17
8 17
2
+ –
88 83
83 92
83 88
13 50
29 83
25 75
17 42
29 88
29 75
13 21
8 29
8 38
3
+ –
88 79
83 96
92 83
17 38
33 88
38 79
13 46
42 75
38 83
13 33
13 42
17 42
4
+ –
92 83
83 92
92 88
29 42
33 50
33 46
25 50
29 58
33 58
8 29
21 38
21 46
Chromosome number Excised root tips (1 cm long) from 11 transgenic and 10 non-transformed plants were analysed to determine whether there was any deviation in chromosome complement from the normal diploid number (2 n = 2 x = 24). Roots were immersed in 2 mM hydroxyquinoline solution (150 min) followed by fixation in ethanol : acetic acid (3 : 1, vol : vol) and storage at –20 °C (3 days). The roots were removed from the fixative and hydrated (3 min) in 0.075 M KCl solution. Cell walls were hydrolysed in 1 M HCl (10 min, 65 °C) before staining (3 h) at room temperature in Feulgen reagent (BDH, Poole, UK). Stained root tips were removed and macerated in approximately 20 µl of ethanoic orcein, squashed beneath a cover slip and viewed by light microscopy. Statistical analysis Data are expressed as the mean ±SE where appropriate. Statistical differences between experimental treatments were assessed by ANOVA using the Minitab statistical computing system (Minitab, State College, Pa.). Phenotypic differences shown by transformed and non-transformed plants were analysed by Student’s t-test. The segregation of the gusA gene in the pollen of regenerated plants and of nptII and gusA genes in R1 seedlings were assessed by chi-square analysis.
Results
Regeneration of shoots from leaf explants inoculated with Agrobacterium To determine whether the gus reporter gene had been transferred and expressed in Agrobacterium-inoculated explants, randomly selected leaf explants from all treatments were analysed for GUS activity. Control (uninoculated) explants failed to exhibit GUS activity (Fig. 2 A); those inoculated with Agrobacterium stained blue (Fig. 2 B). The first signs of dark-green, kanamycin-resistant organogenic calli occurred 21–28 days post-inoculation (Fig. 2 C). Such tissues also exhibited GUS activity (Fig. 2 D). Shoot regeneration from leaf explants was assessed 3 months after
Agrobacterium inoculation (Table 1). More explants regenerated shoots (P < 0.05) when a preculture treatment was employed. Wounding of the abaxial surface of leaf explants inoculated with Agrobacterium reduced (P < 0.05) shoot regeneration, causing the explants to undergo necrosis at wounded sites (Fig. 2 E). Explants without additional wounding exhibited less browning (Fig. 2 F). Uninoculated explants cultured on medium without antibiotics exhibited a high regeneration response (640 explants regenerated shoots from a total of 720, producing a total of 2084 shoots), regardless of the wounding treatment. Uninoculated explants cultured on shoot regeneration medium supplemented with kanamycin sulphate failed to regenerate shoots. Regenerated shoots (Fig. 2 G), from all treatments, were assessed for GUS activity over a period of 6 months from Agrobacterium inoculation of leaf explants. A 2- or 3-day preculture treatment (P < 0.05) increased the production of GUS-positive transformed shoots compared to other treatments (Fig. 3 A). However, a 4-day preculture period gave (P < 0.05) more GUS-negative shoots compared to the other treatments. Explants wounded on their abaxial surface regenerated fewer (P < 0.05) GUS-positive shoots than other explants, regardless of treatment (Fig. 3 B). Treatments involving either a 1 : 20 (vol : vol) or 1 : 10 (vol : vol) bacterial dilution for inoculating explants regenerated (P < 0.05) more GUS-positive shoots than a 1 : 5 (vol : vol) dilution (Fig. 3 C). However, there was no significant difference in the effect of incubation period on the production of GUS-positive shoots (Fig. 3 D). All GUS-positive shoots rooted on semi-solidified medium containing 50 mg l–1 of kanamycin sulphate (Fig. 2 H). Shoots which failed to exhibit GUS activity died on such medium. NPTII and GUS activities of regenerated plants Forty-three randomly selected GUS-positive shoots, representing all treatments, which rooted on medium contain-
557
Fig. 2 GUS histochemical staining and regeneration of shoots from leaf explants inoculated with Agrobacterium tumefaciens strain 1065. A Uninoculated explant after 7 days of culture (bar: 2 mm). B Explant showing GUS activity 7 days after inoculation (bar: 2 mm). C Organogenic calli from an explant cultured on 200 mg l–1 kanamycin sulphate, 28 days post-inoculation (bar: 8 mm). D GUS activity in organogenic calli initiated from an explant inoculated with Agrobacterium strain 1065 (bar: 7 mm). E An abaxially wounded explant cultured on 200 mg l–1 kanamycin sulphate, showing browning, 40 days post-inoculation (bar: 8 mm). F Explant with no additional wounding, 40 days after bacterial inoculation (bar: 7 mm). G Regeneration of shoots from a leaf explant, 62 days after Agrobacterium inoculation (bar: 3 mm). H GUS-positive plant rooted on medium containing 50 mg l–1 kanamycin sulphate (bar: 6 mm). I, J GUS histochemical staining of pollen grains from a non-transformed (I) and a transgenic plant (J) (bar: 24 µm)
ing kanamycin sulphate, were assayed for NPTII and GUS activities. These plants had greater mean (±SE) NPTII activity (P < 0.05) (1.34 ±0.30 ng NPTII protein mg–1 total protein) and GUS fluorometric activity (28.35 ±3.53 µM 4-MU mg–1 protein min–1) compared to plants regenerated from uninoculated explants, which failed to show NPTII and GUS activities.
Southern analysis of transformed plants A random sample of 11 putative transformed plants (plants 1 and 2 from 1-day preculture treatment, plants 3–5 from
558 B 1000
GUS positive
400
800
GUS negative
Number of shoots
A 500
Number of shoots
Fig. 3 Effect of culture conditions on the regeneration of transformed shoots. Total number of GUS-positive and GUSnegative shoots, 6 months after Agrobacterium inoculation, relative to preculture of explants (A), wounding of explants (B), Agrobacterium dilution (C) and incubation period (D)
300 200
600 400
100
200
0
0 0
1
2
3
4
Normal
Wounded
Pre-culture time (days) C
D
400
500
Number of shoots
Number of shoots
400 300
200 100
300 200 100
0
0 1:20
1:10
1:5
Bacterial dilution (v:v)
2 days preculture, plants 6–8 from 3 days preculture, plants 9–11 from 4 days preculture) from the 43 GUS-positive plants were characterised at the molecular level. Southern hybridisation confirmed the presence of both the gus and nptII genes in transformed plants. Southern blots of genomic DNA from transgenic plants 1–11, digested with EcoRI, revealed one to six copies of each of the gusA (Fig. 4 A) and nptII genes (Fig. 4 B). DNA extracts digested with HindIII and probed with the gusA gene demonstrated that the gusA gene had integrated into the plant genome as a single 2.7-kb fragment (Fig. 4 C). Phenotypes of regenerants and segregation of transgenes in the seed progeny of transgenic plants The 11 transgenic plants characterised by molecular analyses were compared phenotypically with 10 non-transformed regenerants. There was no significant difference between transformed and control plants in terms of plant height (transgenic 161.7 ±3.3 cm, non-transformed 158.9 ±3.4 cm), internodal lengths (5.5 ±0.2 cm, 5.5 ± 0.2 cm), maximum leaf lengths (9.8 ±0.3 cm, 10.2 ± 0.3 cm), maximum leaf widths (8.0 ±0.4 cm, 8.1 ±0.3 cm) and time to anthesis (111.2 ±1.1 days, 111.6 ±1.3 days). In addition, there was no significant difference between the seed weights of transformed (840 ±20 mg) and control (840 ± 30 mg) plant populations. The chromosome numbers for both transformed and control plants was 2 n = 2 x
5
10
20
Incubation time (min)
= 24. Pollen grains from freshly dehisced anthers of nontransformed plants failed to exhibit GUS activity (Fig. 2 I). In transgenic plants, the number of GUS-stained (Fig. 2 J) to non-stained pollen grains approximated to a 1 : 1 ratio. T1 seeds (17–44) from each of the 11 selfed (T0) transgenic plants were sown on MS0 agar medium containing 200 mg l–1 of kanamycin sulphate to study the segregation of antibiotic resistance. There was a high probability (50–100%) that the segregation of kanamycin-resistant to kanamycin-sensitive seedlings agreed with a ratio of 3 : 1. In addition, the concentration of kanamycin sulphate used in germinating seeds of transgenic Datura was efficient in preventing the growth of R1 seedlings from selfed (R0) non-transformed plants. All T1 seedlings which grew on medium containing 200 mg l–1 of kanamycin sulphate also exhibited GUS activity.
Discussion
This is the first report of the transformation of the commercially important ornamental, D. meteloides. Using a single regeneration medium, previously designated BM2 (Sangwan et al. 1991), it has been possible to produce transgenic plants of this species. In previous studies using D. innoxia (Sangwan et al. 1991), a two-step procedure was necessary for transgenic plant production. However, in agreement with Sangwan et al. (1991), the present study
559
Fig. 4 Southern hybridisation of genomic DNA of regenerated plants digested with EcoRI (A, B) and HindIII (C). A DIG-labelled gusA gene fragment was used as a probe (A, C) and a DIG-labelled nptII gene fragment (B). A, B Lane 1 pVDH65, lane 2 uncut DNA from plant 1, lanes 3–13 DNA from plants 1–11 regenerated from leaf explants infected with A. tumefaciens, lane 14 plant regenerated from an uninoculated leaf explant. C Lane 1 pVDH65, lanes 2–12 plants 1–11, lane 13 plant regenerated from an uninoculated leaf explant
showed that for shoot development, it was essential to reduce the concentration of kanamycin sulphate from 200 to 100 mg l–1 at the time of initiation of organogenic calli. Preculture of explants prior to Agrobacterium inoculation has improved transformation efficiency in some plants. For example, cells of precultured leaf explants of apple show an increased porosity in their walls, which may aid in the transfer of T-DNA into the cells, resulting in a significant increase in the number of transgenic plants (Sriskandarajah and Goodman 1998). A 1-day preculture period in Datura innoxia increased significantly the number of explants producing transformed calli (Sangwan et al. 1991). A 2- to 3-day preculture period yielded significantly more GUS-positive shoots compared to a 1-day treatment. Wounding of explants prior to Agrobacterium inoculation is beneficial for transformation in some species, since wounded cells release polyphenolic compounds which activate Agrobacterium vir genes (Zambryski 1992). However, in the context of the present study, leaf explants which were wounded on their abaxial surface regenerated fewer
transformed shoots than unwounded explants. Interestingly, in uninoculated explants of Datura, there was no significant difference in the number of shoots regenerated with respect to wounding. The number of agrobacteria in the inoculum is a critical factor in the transformation of some species, such as lettuce (Michelmore et al. 1987), since excessive numbers of bacteria can stress plant cells. Alternatively, if the number of agrobacteria is low, the number of transformed cells is reduced. In Datura, a 1 : 10 or 1 : 20 (vol : vol) dilution of an overnight culture of bacteria, resulted in more transformed shoots compared to a 1 : 5 (vol : vol) dilution. The length of time explants are immersed in the Agrobacterium suspension during inoculation can influence transformation. In lettuce, a 10-min incubation period increased the number of transgenic shoots compared to a treatment of 2–3 s (Curtis et al. 1994). In the present study, the number of transformed shoots was similar for incubation periods of 5, 10 and 20 min. Expression of both the gusA and nptII genes was confirmed in GUS-positive kanamycin-resistant plants by fluorometry and by NPTII ELISA, with Southern analysis demonstrating transgene integration into the Datura genome. Phenotypically, transgenic plants were identical to their non-transformed explant-derived counterparts. These results demonstrate that it is possible to introduce and to stably transmit genes into the seed progeny of D. meteloides. This Agrobacterium-mediated transformation system can be employed to transfer genes, such as those for dwarfism, to widen the genetic diversity of this species and, hence, to improve the novelty of this attractive ornamental plant. Acknowledgements This work was supported by MAFF under OC 9407. IACR-Long Ashton receives grant-aided support from BBSRC. The authors thank B. V. Case for photographic assistance.
References Chittenden FJ (ed) (1965) The Royal Horticultural Society dictionary of gardening: a practical and scientific encyclopaedia of horticulture, vol. II, 2nd edn. Clarendon, Oxford, pp 640–641 Christen P, Roberts MF, Phillipson JD, Evans WC (1989) High yield production of tropane alkaloids by hairy root cultures of a Datura candida hybrid. Plant Cell Rep 8: 75–77 Curtis IS, Power JB, Blackhall NW, de Laat AMM, Davey MR (1994) Genotype-independent transformation of lettuce using Agrobacterium tumefaciens. J Exp Bot 45: 1441–1449 Curtis IS, Power JB, Davey MR (1995) NPTII assays for measuring gene expression and enzyme activity in transgenic plants. In: Jones H (ed) Methods in molecular biology, vol. 49. Plant gene transfer and expression protocols. Humana, Totowa, NJ, pp 149–159 Curtis IS, Davey MR, Hedden P, Phillips A, Ward DA, Thomas SG, Lowe KC, Power JB (in press) Evaluation of gibberellin 20-oxidase and rolC genes for dwarfing ornamental plants. In: Altman A, Izhar S, Ziv M (eds). IX international congress of plant tissue and cell culture. Kluwer, Dordrecht, The Netherlands De Cleene M, De Ley J (1976) The host range of crown gall. Bot Rev 42: 389–466 Dellaporta SL, Wood J, Hicks IB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–21
560 Drake PMW, John A, Power JB, Davey MR (1996) Expression of the gusA gene in embryogenic cell lines of Sitka spruce following Agrobacterium-mediated transformation. J Exp Bot 48: 151– 155 Ducrocq C, Sangwan RS, Sangwan-Norreel BS (1994) Production of Agrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos of Datura innoxia. Plant Mol Biol 25: 995–1009 Dupraz J-M, Christen P, Kapetanidis I (1994) Tropane alkaloids in transformed roots of Datura quercifolia. Planta Med 60: 158–162 Gartland KMA, Phillips JP, Vitha S, Benes K (1995) Fluorometric GUS analysis for transformed plant material. In: Gartland KMA, Davey MR (eds) Methods in molecular biology, vol 44. Agrobacterium protocols. Humana, Totowa, NJ, pp 195–199 Hedden P, Coles JP, Phillips AL, Thomas SG, Ward DA, Curtis IS, Power JB, Lowe KC, Davey MR (in press) Alteration of plant morphology by genetic manipulation of gibberellin biosynthesis. In: Genetic and environmental manipulation of horticultural crops. Proc First HRI Conf, October 1997 Hilton MG, Wilson PDG (1995) Growth and the uptake of sucrose and mineral ions by transformed root cultures of Datura stramonium, Datura candida × aurea, Datura wrightii, Hyoscyamus muticus and Atropa belladonna. Planta Med 61: 345–350 Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-regions of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: βglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 Jin S, Komari T, Gordon MP, Nester E (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J Bacteriol 169: 4417–4425 Knoll KA, Short KC, Curtis IS, Power JB, Davey MR (1997) Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): a system for Agrobacterium transformation. Plant Cell Rep 17: 96–101
McCabe MM, Power JB, de Laat AMM, Davey MR (1997) Detection of single copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis. Mol Biotechnol 7: 79–84 Michelmore RW, Marsh E, Seely S, Landry B (1987) Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens. Plant Cell Rep 6: 439–442 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163: 85–87 Robbins RJ, Parr AJ, Bent EG, Rhodes MJC (1991) Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures. Planta 183: 185–195 Sangwan RS, Ducrocq C, Sangwan-Norreel BS (1991) Effect of culture conditions on Agrobacterium-mediated transformation in Datura. Plant Cell Rep 10: 90–93 Sangwan RS, Ducrocq C, Sangwan-Norreel B (1993) Agrobacterium-mediated transformation of pollen embryos in Datura innoxia and Nicotiana tabacum: production of transgenic haploid and fertile homozygous dihaploid plants. Plant Sci 95: 99–115 Schmülling T, Schell J, Spena A (1988) Single genes from Agrobacterium rhizogenes influence plant development. EMBO J 7: 2621–2629 Sharma VK, Jethwani V, Kothari SL (1993) Embryogenesis in suspension cultures of Datura innoxia Mill. Plant Cell Rep 12: 581–584 Sijmons PC, Dekker BMM, Schrammeijer B, Verwoerd TC, Van den Elzen PJM, Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8: 217–221 Sriskandarajah S, Goodwin P (1998) Conditioning promotes regeneration and transformation in apple leaf explants. Plant Cell Tissue Organ Cult 53: 1–11 Zambryski PC (1992) Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol 43: 465–490
