Analysis of stable events of transformation in wheat via PEG-mediated DNA uptake into protoplasts

plan cience ~l~,l\lll~ u,I I I N I I I I ( I'l I~1 I k t l F R k I R I I ~,"~1)

Plant Science 93 (1993) 85-94

Analysis of stable events of transformation in wheat via PEG-mediated DNA uptake into protoplasts P. Ajmone Marsan a, E. Lupotto *a, F. Locatelli a, Yao-Min Qiaobt, M. Cattaneo b "lstituto Sp¢rimentale per la Cerealicoltura, Sezione di Bergamo, Via Stezzano 24, 24100 Bergamo, Italy bSezione di S. Angelo L., Via Mulino 3, 20079 S. Angelo Lodigiano (MI), Italy (Received 27 January 1993; revision received 26 April 1993; accepted 7 June 1993)

Abstract

Protoplasts derived from an embryogenic suspension culture of hexaploid wheat (Triticum aestivum L.) cv. Oderzo have been transformed via PEG-mediated DNA uptake. The chimaeric gene utilized for transformation was the neomycin phosphotransferase-ll gene under the 35S CaMV promoter. The frequency of transformation was between 1 and 2.25 x 10 -6 treated protoplasts. Calliclones were selected on 100 mg/1 kanamycin, and their resistance analysed at the cellular and molecular level. Cultures originated from transformed protoclones were highly resistant to kanamycin, neomycin and geneticin (G418); their resistance was maintained at a stable condition in the absence of selective pressure. PCR analyses showed that the selected protoclones growing on kanamycin contained the NPT-II gene and that the gene was active, as confirmed by NPT-II enzymatic assay. Several kanamycin-resistant protoclones were analyzed by Southern hybridization for the modality of gene integration. Various patterns of integration of the NPT-II gene were observed; in several cases multiple insertions and rearrangements of the integrated gene were observed. In most cases 35S CaMV promoter and the NPT-II coding region were linked on the same restriction fragment. Either complete or partial chimaeric genes were inserted into the genomic DNAs, in a variable number of copies and in different locations. The NPT-II activity detected in the calli analyzed was always high, independently from the copy number of the gene inserted. Key words." Transformation; Kanamycin; Triticum aestivum; Molecular analysis

I. Introduction

A m o n g the various methods proposed for cereal transformation, to date, two direct gene transfer into protoplasts [1] and the biolistic a p p r o a c h [2] * Corresponding author. ?On leave from Academia Sinica, Peking, China.

have reproducibly been used to produce stablytransformed plants [3-5]. More recently, a novel method o f transformation based on electroporation o f immature embryos and calli was proposed in maize [6]. While the biolistic approach, a particle bombardment, appears as a more universally adoptable method, the major constraint on the use o f direct D N A uptake into protoplasts is repre-

0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03667-K

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sented by the relative difficulty of cereal species in establishing efficient protoplast systems [7,8], until plant regeneration. In the last few years, considerable progress has been achieved in regenerating plants from protoplasts of the major cereal species: rice [9], maize [10], hexaploid wheat [11-14], barley [15], although genotype dependence still restricts the system to few responsive genotypes for each species. In hexaploid wheat (Triticum aestivum L.), the establishment of protoplast systems is still a rare event, and only recently decisive progress has been obtained in regenerating plants from protoplasts isolated from embryogenic suspension cultures [11-15]. However, fertile transgenic wheat plants have been obtained by microprojectile bombardment of regenerable embryogenic callus [5]. As a progressive step towards the regeneration of transformed wheat plants via direct gene transfer, we have developed a transformation protocol based on PEG-mediated direct DNA uptake into protoplasts. The present work describes the whole procedure deve,loped for transformation, selection and characterization of stably-transformed calliclones, and studies performed on some events of transformation for better understanding the modality of integration of a foreign gene into the wheat genome. 2. Materials and methods

2.1. Plant material Cell suspension cultures of hexaploid wheat cv. Oderzo were derived from fast-growing friable calli obtained from immature embryos as previously described [13]. At the time of protoplast isolation for the transformation experiments, suspension cultures were 5 months old (9 months after initiation of callus cultures). 2.2. Plasmid The plasmid pCGN778 was a kind gift from Calgene, Davis, California (USA). The plasmid contained the neomycin phosphotransferase-II gene (NPT-II) driven by the 35S CaMV promoter and followed by a terminal polyA 3'-end from transcript 7 of Agrobacterium tumefaciens. The complete chimaeric gene had a size of 2200 bp.

P, A/mone Marsan et al, / Pl ant Sci. 93 (1993) 85-94

Plasmid DNA used for transformation was prepared by alkaline lysis and purified on CsCI according to standard procedures. 2.3. Protoplast transJormation, culture and selection Protoplasts were isolated from 7-8-day-old suspension cultures: 10 ml suspension containing approximately 800-900 mg fresh weight cells were used for each preparation. Protoplasts were isolated as previously described [13]. Aliquots of 400-/A protoplasts suspension were dispensed into disposable screw-capped 12-ml polypropilene tubes. After 5 min heat shock at 45°C, 50 t~g sheared calf thymus carrier DNA and 10 #g circular plasmid pCGN778 DNA were added in sequence. After approximately 60 s, 400 p,1 of a 40%-PEG 6000 (Merk) solution in 0.6 M mannitol, 0.1 M CaC12.2H20 (pH 8.2) kept at 42°C were added. Incubation lasted 30 min at 22-24°C. Protoplasts were then stepwise diluted in 7 mM CaC12.2H20, 0.7 mM NaH2PO4, 3 mM MES, 0.65 M mannitol, pH 5.6, sedimented 5 min at 80 x g, resuspended in protoplast culture medium MS-PPSI (MS salts and vitamins [16], 3 mM MES, 1 mg/1 2,4-D, 0.6 M glucose, pH 5.7) and plated at a density of 2 x 105 cells/ml. Transformed protoplasts were incubated 48 h in complete darkness at 26°C, and in dim light with photoperiod at 16 h light/8 h dark thereafter. Selection started 7 days after protoplast isolation, with dilution 1:1 with MS-PPSI medium 0.2 M glucose, supplemented with 100 mg/l kanamycin. After 14 days from transformation, the protocolonies were embedded in 0.8% agarose (Sigma type II-medium EEO) MS-PPSI medium, 0.2 M glucose, supplemented with 100 mg/l kanamycin. The agarose beads were transferred in bead-type culture, in which the liquid phase consisted of 10 ml conditioned El I medium containing 100 mg/l kanamycin. The conditioned Ell medium was taken from healthy growing 4-dayold cell suspensions [13] filtered and used. About 6 weeks after transformation, 3-5 mm 0 protoclones developing through the agarose beads in selection could be singly picked up and transferred for further growth onto 0.8% agar-gelled MSPPSII medium consisting of MS salts and vitamins, thiamine up to 1 mg/l, 30 g/I sucrose, 1 mg/1

P. Ajmone Marsan et a l . / Plant Sci. 93 (1993) 85-94

IAA, 0.5 mg/l zeatin, 100 mg/l kanamycin, pH 5.8. Indolacetic acid and zeatin were filter sterilized and added, together with kanamycin, after autoclaving. Protoclones were numbered and kept subcultured every 3 weeks onto the same medium in the presence of 100 mg/l kanamycin.

2.4. Characterization oJ" selected kanamycinresistant protoclones. Neomycin-phosphotransferase II (NPT-II) assay Kanamycin-resistant calli derived from individual events of transformation were maintained for further characterization, and indicated as ODZKn, that means kanamycin-resistant protoclones from wheat cv. Oderzo; for an easy description of the data reported, the 8 protoclones taken into consideration were numbered as ODZ-K1-ODZK8. Suspension cultures initiated from four ODZK protoclones (ODZ-K 1, ODZ-K2, ODZ-K6, and ODZ-K7) were characterized for their capability to grow in absence of selection and to retain the trait of resistance, and for growth in the presence of neomycin and geneticin (G418), other two antibiotics for which the resistance is conferred by the NPTII gene transfected. Cell growth was monitored by packed cell volume (PCV) after an 8day subculture. The active presence of the transfected gene was assayed in fresh tissues through the NPT-II assay performed according to McDonnell et al. [18]. Calli sustaining healthy growth on medium containing 100 mg/1 kanamycin were sampled at random for NPT-II activity. Protein content was determined by the BCA colorimetric assay (by Pierce) based on the Bradford assay [17]. Protein blots were perfomed in duplicate: one copy processed according to McDonnell et al. [18], the second copy treated with 1 mg/ml proteinase-k (Boheringer), in 1% (w/v) SDS, at 60°C for 30 min [19] followed by a 5-min rinse in distilled water at 80°C, in order to remove any unspecific background. Papers were exposed overnight at -80°C onto X-ray film, with intensifying screen. Spots were then cut off and their radioactivity measured by liquid scintillation. 2.5. Molecular analyses Total genomic DNA for PCR and Southern analyses was isolated from callus cultures accord-

87

ing to the CTAB method [20], using approx. 1 g fresh weight tissue for each callus clone to be analyzed. To conduct the PCR assay, 100 ng DNA resuspended in 1 x TE buffer were used. PCR analysis was carried out in 30-/~1 final volumes in standard conditions according to the supplier in an MJ Programmable Thermal Controller (MJ Research Inc.) for45 cycles of 1 min at 94°C, 1 rain at 55°C and 2 min at 72°C. Twenty #1 of the PCR product were electrophoresed in 0.8% agarose gels and visualized by staining with ethidium bromide. The sequences of the PCR primers used for diagnostic analysis of the presence of the NPT-II gene were 5 ' - A G G C T A T T C G G C T A T G A C T G G - 3 ' and 5'-GCGGTCCGCCACACCCAGCCG-3'. These primers are complementary to sequences within the SmaI sites cutting the NPT-I1 coding region, and amplify an expected fragment of 592 bp. For Southern analyses [21], digested and undigested DNA samples (8 /zg) were subjected to electrophoresis in 0.8% agarose gels and blotted onto Hybond N ÷ nylon membranes according to the manifacturer's alkaline blotting protocol (Amersham). Hybridization and washing were according to Motto et al. [22]. The DNA fragments used as hybridization probes were produced by restriction enzyme digestions of plasmid pCGN778 derived from each of the three regions composing the chimaeric gene: the HindIII-EcoRI 35S CaMV promoter region of about 700 bp; the Smal-SmaI NPT-II coding region of about 1 kb; the SmalSmaI terminal polyadenilation region of about 500 bp. Restriction endonucleases were used according to the manufacturer's instructions. A lc and 10c value, 1 x and l0 x DNA content of the unreplicated haploid chromosomal content, respectively, were used for copy number reconstruction. 3. Results

3.1. Protoplast transformation and selection Untreated protoplast cultures showed a maximal plating efficiency of 34% at day 7 after plating, whilst after PEG treatment the plating efficiency was reduced at 10-15%. After 1 week culture in liquid MS-PPS! medium, protocolonies of

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4-8 cells were observed in the liquid. At this point selection started by 1:1 dilution with lower osmoticum MS-PPSI medium containing 100 mg/1 kanamycin. The bead-type culture in agarose beads started 1 week later till the development of 2-3 mm in diameter protoclones growing through the agarose beads. A selective pressure of 100 mg/1 kanamycin was maintained by replacing the liquid phase around the beads every other week. In this way the transformation efficiency, evaluated as kanamycin-resistant protoclones rescued over the total number of treated protoplasts, varied between 1-2.25 x 10 -6 treated cells (Table 1). There was clearly a variation with different isolates in the ability of protoplasts to sustain divisions after the transformation procedure. Isolated protoclones were subsequently grown on solid MS-PPSII medium containing 100 mg/1 kanamycin for 10 months without losing the transfected trait. The NPT-II assays performed on calli stably growing on kanamycin-containing medium were positive, thus confirming that resistance was due to the activity of the foreign gene. Dose response experiments with bead-type culture of non-treated protoplasts gave indications on the level to be used for a stringent selection of the events of transformation. Kanamycin was selective at 100 mg/l, in analogy to neomycin, whilst G418 was more active, and 30 mg/1 were sufficient to stop the growth of untransformed cells.

3.2. Characterization of selected calliclones Suspension cultures in E11 medium were easily established from four single protoclones: ODZKI, ODZ-K2, ODZ-K6, and ODZ-K7. Suspen-

sions were grown in the presence of 100 mg/1 kanamycin, 100 mg/1 neomycin, or 30 mg/1 G418, and their growth monitored as PCV at the end of an 8-day subculture (Fig. IA). PCV triplicated after 8 days for control unlransformed cultures (Fig. 1A, condition a), whilst it was totally inhibited in the presence of each antibiotic (Fig. 1A, condition b, c, and d). As expected, transformed cultures grew in control conditions (condition a) as well as in the presence of the three antibiotics (conditions b, c and d). The trait of resistance was maintained also after a 3-month period of culture in absence of selective pressure (Fig. 1A, condition e). The expression of the NPT-II gene was confirmed by NPT-II assay as shown in Fig. lB. The enzyme activity, measured as counts/min/ug reacted proteins, was very similar in the four cases.

3.3. Molecular analysis Genomic DNAs were subjected to polymerase chain reaction (PCR) amplification using primers designed to amplify an internal NPT-II fragment of 592 bp. Eight callus lines in which the expected fragment was amplified in the PCR assay (Fig. 2), were further analyzed by Southern blot analysis. Southern blot analyses of chromosomal DNAs purified from protoclones are shown in Figs. 3, 4 and 5. The untransformed control callus (NT) did not show any hybridization signal when probed with the NPTII 1000 bp SmaI-SmaI fragment of pCGN778 (Fig. 3, lanes 4 and 5; Fig. 4, lane 2). The same probe evidenced the expected 1500 bp (NPT-II and 3' polyA tail) and 2200 bp (35S CaMV promoter, NPT-II and 3' poly-A tail) restriction fragments when hybridized to the trans-

Table 1 Transformation frequency in five experiments of PEG-mediated DNA uptake into wheat protoplasts Expt. No.

No. of protoplasts treated

Plating efficiency at day 7 untreated treated

No. of resistant protoclones rescued

NPT-II positive assay

Frequency of transformation

1 2 3 4 5

4 6 4 4 6

25.0 31.1 34.6 18.3 26.4

4 10 7 9 12

4/4 10/10 7/7 9/9 12/12

1 1.7 1.75 2.25 2.0

x x × × ×

106 106 106 106 106

10.0 11.2 15.0 11.1 12.0

x X x × ×

10 -6 10 6 10 -6 10 -6 10 -6

P. Ajmone Marsan et al./ Plant Sci. 93 (1993) 85-94

89

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Fig. 1. Characterization of four transformed wheat protoclones. (A) Growth of suspension cultures derived from a non-transformed (NT) and four transformed protoclones (ODZ-K1, ODZ-K2, OKZ-K6, ODZ-KT); a, Ell medium without selection; b, 100 mg/I kanamycin; c, 100 mg/I neomycin; d, 30 mg/l geneticin (G418); e, condition b after a period of 3 months culture in absence of selective pressure. Resistance to the three antibiotics and stability of the trait was confirmed for the four cultures. (B) NPT-II assay for the four transformed protoclones. The activity of the enzyme was assayed as dot-blot protein assay as described in Materials and methods. In the upper lane the results of the autoradiography on X-ray film are shown, while the lower lane reports the quantitative data obtained after counting of dots by liquid scintillation. Results are expressed in counts/min/~g of protein reacted.

formed D N A s digested with EcoRI (Fig. 3, lanes 6, 8, 10, 12, 14, 16, 18 and 20) or with HindlII and BamHI (Fig. 4, lanes 5, 8, 11,14, 17, 20, 23, 26), respectively. Bands at higher and lower than expected molecular weight were also present, presumably indicating integration and alteration o f restriction site(s) in one case, and integration o f fragments o f the chimaeric construct in other cases.

Integration o f the chimaeric construct in the chromosomal D N A o f O D Z - K calli was confirmed by hybridization in a region o f high molecular weight when undigested D N A s were probed (Fig. 4, lanes 3, 6, 9, 12, 15, 18, 21). Probing the D N A s digested with enzymes that have no restriction sites within the sequence o f the foreign gene gave a random pattern o f integration, as BamHI (Fig. 3, lanes 7, 9, 11, 13, 15, 17, 19 and 21) or HindlII

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P. Ajmone Marsan et al./ Plant Sci. 93 (1993) 85-94

bp

K1 K2 K3 K4 K5 K6 K7 K8

(Fig. 4, lanes 4, 7, 10, 13, 16, 19, 22, 25), thus providing a further indication of the integration and an estimation of the NPT-1I gene copy number in the transformed calli. All the assayed callus lines showed several bands indicating multiple integration at different locations in the genome. The estimated intact unit copy number ranged from one or two, for ODZ-K5, 6, 7 and 8 to at least twenty for ODZ-K2, 3, and 4. Six hybridization patterns were unique, likely representing independent events of integration, whereas the hybridization patterns of ODZ-K2, K3, and K4 were very similar. DNAs from ODZ-K1 and ODZ-K2 were further probed with the 700-bp EcoRI-HindlII (35S CaMV promoter) and the 500 bp SmaI-SmaI (3' polyA tail) fragments of pCGN778. Restric-

2000

1600~ 1000--

* 592

500--

12

3

4

5

6

789

Fig. 2. Ethidium bromide stained agarose gel of PCR amplification products from eight independent kanamycin resistant protoclones of wheat cv. Oderzo. Lane 1, untransformed wheat negative control; lanes 2-9, transformed protoclones in the order from ODZ-KI to ODZK-8. Each transformant was shown to contain the amplified expected band of 592 bp,

NT |

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K8 !

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12 13 14 15 16 17 18 19 20 21

start

70O0 8000

3600

1500

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10c lc Fig. 3. Southern blot analysis of genomic DNA extracted from kanamycin-resistant calli of wheat. Lane 1, MW marker (lambda Hindill digested); lane 2 and 3 designated 10c and lc, respectively representing EcoRl digestion of pCGN778 corresponding to 10 and 1 copy of the target gene for wheat haploid genome. Eight DNA samples (8t*g each) were digested with EcoRl (lanes 6, 8, 10, 12, 14, 16, 18, 20) and BamHl (lanes 7, 9, 1 I, 13, 15, 17, 19, 21). Lanes 4 and 5 represent untransformed DNA (NT) digested with EcoRl and BamHI, respectively, The blot was hybridized with the 32p-labelled Smal fragment of pCGN788. The arrow at 1500 bp indicates the position of the fragment containing the coding region of the NPT-II gene and polyA tail.

91

P. Ajmone Marsan et al./ Plant Sci. 93 (1993) 85-94

K1 •

bp

K2 g

K3 l

K4 |

K5 l

K6 |

K7 |

K8 i

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I 2 3 4 5 6 7 8 9 1011 12 1314151#718 19 202122 23 24 2526 start

5700 4100

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Fig. 4. Southern blot analysis of DNA isolated from kanamycin-resistant calli. Lane I, pCGN778 digested with HindlII and BamHI; lane 2, untransformed control DNA cut with HindlI1. DNA samples (8-t~g each) from ODZ-K1 to ODZ-K8 were undigested (lanes 3, 6, 9, 12, 15, 18, 21, 24), digested with Hindlll (lanes 4, 7, 10, 13, 16, 19, 22, 25) or double digested with HindllI and BamHl (lanes 5, 8, 11, 14, 17, 20, 23, 26). The blot was hybridized with the 32p-labelled Sinai fragment of pCGN778, the arrow at 2.2 kb indicates the position of the fragment containing the complete chimaeric gene.

tion fragments identified by the NPT-II probe were also recognized by 35S CaMV promoter and 3' polyA tail confirming that intact copies of the construct were integrated. PolyA tail probe evidenced also additional bands in sites were integration of the remaining part of the construct did not occur.

4. Discussion Although the biolistic approach is becoming more and more extensively utilized for transformation, still direct gene transfer can offer some advantages, such as the ease of methodology and the independence from the particle gun apparatus.

Stable integration of foreign genes via PEGmediated DNA uptake into cereal protoplasts has been demonstrated in various species. In few cases, plant regeneration was also accomplished. The advantages as well as the disadvantages of the direct gene transfer methodology have been extensively reviewed [23]. The transformation efficiencies obtained in our experiments, between 1 and 2.25 × 10.6 of treated protoplasts, are lower than those reported for other cereal species, such as maize (5 × 10 -5) [24], and rice (9 × 10 -5) [25], but are comparable to the transformation frequencies obtained in barley (0.4-3 × 10 -6) by Lazzeri et al. [26]. Kanamycin efficiently selected events of transfor-

92

P. Ajmone Marsan et al./Plant Sci. 93 (1993) 85-94

Smal EcoRI

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p35SCa~

probes

KI

bp

.

1

2

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4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 I I II \\~ I I I I/ II I I t /

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5700 4100

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Fig. 5. The same membrane utilized in Fig. 4 was stripped and rehybridized with (a) 700 bp HindlII-EcoRl fragment of pCGN778 corresponding to 35S CaMV promoter; (b) 1000 bp Smal-Smal fragment of pCGN778 corresponding to the coding region of the NPT-II gene; (c) 500-bp SmaI-Smal fragment of pCGN778 corresponding to the 3' polyA tail. Genomic DNA samples (8 ~g) of ODZ-KI and ODZ-K2 were undigested (lanes 1,4, 9, 12, 15, 18), digested with Hindlll (lanes 2, 5, 10, 13,16, 19), and double digested with HindllI and BamHl (lanes 3, 6, 11, 14, 17, 20). Lane 7, pCGN778 digested with Hindllland BamH1; lane 8, untransformed control wheat DNA digested with Hindlll.

mation and no escapes were rescued after the selection procedure, as confirmed by enzymatic and P C R analyses. We therefore support the results obtained with L o l i u m m u h i f l o r u m [27], T r i t i c u m m o n o c o c c u m [28], maize [10,29]; rice [25,30] and barley [26], in which kanamycin-resistant calli were successfully produced and selected. Furthermore, the direct gene transfer we adopted with protoplasts o f wheat, allowed efficient selection on 100 mg/1 kanamycin while much higher concentra-

tions (200-400 mg/1) were required for selection o f transformation events by using particle b o m b a r d ment on wheat cells [5]. Even, the absence of escapes might suggest that the selection level was too stringent, as discussed in the case o f barley [26], and this may consequently have stopped the growth of other events of transformation in which lower N P T - I I activities had negatively affected slow growing calli. The number of functional copies of a gene and

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P. Ajrnone Marsan et al. / Plant Sci. 93 (1993) 85-94

the pattern of integration are important factors in the evaluation of the results obtained via direct gene transfer. In our experiments we have shown that the NPT-II gene was stably-integrated into the genomic D N A of each transformed wheat protoclone. Various patterns of integration of the NPT-II gene were observed. The presence of multiple bands of molecular weight higher or lower than expected even when genomic D N A s were digested with enzymes cutting at the external edges of the complete chimaeric gene, represents an indication of multiple insertion events and, in some cases, rearrangements of the integrated gene. This is a common feature in the analysis of chimaeric genes inserted into plant material [24]. In spite of this, in most cases 35S C a M V promoter and the NPT-II coding region remained linked on the same restriction fragment. The different intensity of some of the bands at higher molecular weight suggests the possible presence of head-to-tail tandem insertions of plasmid D N A as hypothized in the case of PEG-mediated transformation of rice protoplasts [31]. Shorter sequences of the chimaeric gene were also further integrated into the host genome, as was evidenced by probing the digested DNAs with polyA tail probe. Either complete or partial chimaeric genes resulted inserted into the genomic DNAs, in a variable number of copies and in different locations. However, the resistance to selection and the positive NPT-II assay for enzymatic activity confirmed that at least one copy of the functional gene was present and expressed in each transformed protoclone. In reconstitution experiments we estimated that the copy number of the gene inserted accounted from 1-20 per haploid genome, which was a relatively small number compared to the copy number estimated for example in rice (50-100 copies [32]) but comparable to the values reported for sorghum (1-4 copies [33]). In this respect, we cannot explain that the N P T - I I activity detected through the dot blot protein assay as described was high in any case, independently from the copy number of the gene inserted. Three protoclones resulted characterized by a very similar pattern of gene integration, which suggested that they might be the same event of transformation, each of them deriving from the fragmentation of one single pro-

tocolony very early during culture in liquid medium. The procedure described here was developed on the protoplast system established for wheat cv. Oderzo. Meantime, suspension cultures derived from other cultivars of wheat characterized by higher regenerative capability in vitro have been obtained, so that the methodology is now being transferred on those cultures in order to regenerate plants fi'om the transformed calliclones. 5. Acknowledgements

This work was developed within the project 'Sviluppo di Tecnologie Avanzate Applicate alle Piante' supported by the Ministry of Agriculture and Forestry, Rome. We acknowledge Calgene, CA, USA, for the gift of plasmid pCGN778. We wish to thank Prof. F. Sala, Dept. of Genetics and Microbiology, University of Pavia, Italy, and Dr. S. Datta, Institute of Plant Sciences, E T H Zentrum, Zfirich, Switzerland, for helpful discussions during the work and critical reading of the manuscript. 6. References

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