GB2298205A - Genetic transformation of eucalyptus - Google Patents

Abstract

A process for producing genetically-engineered eucalyptus, comprising the co-cultivation of explant cells of Eucalyptus species such as E. grandis, E. globulus, E. nitens or E. dunnii with Agrobacterium tumefaciens microorganism including one or more DNA sequences which are functional in plants, in order to achieve a genetic modification by which the cells are stably transformed with the DNA. The explants may be derived from seedling hypocotyls or from petioles, leaves or stems.

Description

GENETIC MODIFICATION OF PLANTS Field of the Invention This invention relates to the genetic modification of eucalyptus, primarily for the commercial production of wood and wood products.

Backaround to the Invention At present, commercial-scale planting of eucalyptus forests uses planting stock which is produced either directly from seed or from rooted cuttings. In both of these production systems, traditional plant-breeding techniques are used to produce superior planting stock, with the potential for increased productivity of wood with desirable properties.

Recent advances in plant genetic engineering have now made it possible stably to incorporate heterologous DNA, or reincorporate homologous DNA into plants, with the potential for further rapid improvements in current superior Eucalvptus planting stock. The specific strategies and methods that may be employed to achieve genetic modification are usually dependent on the biological properties and attributes of the Eucalyptus strain, cultivar, variety or hybrid involved. The overall efficiency of genetic modification is a function of the efficiencies of both the introduction of the heterologous DNA into a cell (dependant on cell type and method of transformation used) and the subsequent regeneration of viable plants from transformed cells.

Adam et al, AFOCEL ed. Complement de Annals 1991 ISSN 0398-494X (1992), and Chriqui et al IUFRO Symposium on Intensive Forestry. The role of eucalypts, Durban S.A.

1991, ed. Schonau, vol. 1:70-80 (1992), report that successful genetic modification of seedlings of several Eucalyptus species, including E. globulus, transformed by Aprobacterium rhizosenes or tumefaciens, is dependant on age. The greater level of tissue differentiation and an increased ability to produce polyphenolics are thought to be responsible for the reduction in susceptibility to genetic modification observed in older seedlings.

Furthermore, seedlings over 6 weeks old were found to be incapable of forming adventitious shoots, tissues and cells, i.e. to undergo regeneration, unlike their younger counterparts.

Consequently, most of the prior art is focused on genetic modification of Eucalyptus sources that exhibit juvenile characteristics, such as seeds or young seedlings of E. aunnii, E. arandis, E. camaldulensis and E. saliana.

Although these make good planting stock, they suffer from the major disadvantage of needing a lengthy field-trial period before the subsequent exploitation of superior trees can occur. Other sources can be obtained by subjecting a mature tree to physical damage, chemical agents or a change in environmental conditions, in order to induce the production of juvenile shoots.

Field-trials of juvenile genetically-modified Eucalyptus plants are subject to a number of difficulties.

For instance, superior Eucalyptus trees cannot be selected on phenotype until marked changes in the Eucalyptus morphology and physiology have occurred. These changes, from a juvenile to a mature state, can take up to 3-4 years to materialise. The mature state is characterised by the ability of the Eucalvptus to produce flower buds and seeds.

Furthermore, the resulting progeny of any sexual crosses may also have to be field-trialled after each step of the breeding programme. This extends the time taken to commercially exploit genetically-modified Eucalvptus trees.

To maximise commercial yields and improve product quality, it is desirable to establish clonal Eucalyptus forests that are selected on the basis of one or a number of superior phenotypic properties after their identification within a population of trees. This population may be a natural population or a superior population derived by traditional breeding techniques or genetic modification. Suitable sources for propagation can then be removed from the superior tree for use as clonal material that can subsequently undergo genetic modification. The process of propagation is serially reproduced until there is sufficient genetically-modified material to enable a plantation to be established.

The prior art also describes many ways by which heterologous DNA could be stably incorporated into Eucalyptus seedling explants, indirectly or directly. For example, Hope et al, VIII International Congress of Plant Tissue Culture, Firenze, Italy, June 12-17, 1994, Abstract 57-106 (1994), report successful genetic modification in E.

arandis seedlings mediated by an A. tumefaciens vector; Teulieres et al, in: Biotechnology in Agriculture and Forestry 29, ed. Bajaj, Springer-Verlag, Berlin (1994), bombarded embryos of E. slobulus with DNA-coated particles and recovered viable genetically-modified shoots.

Trees which have been field-trialled and identified as having superior properties may be the product of a significant investment of effort in breeding and selection.

Such trees can therefore represent a valuable asset which can be rapidly exploited without the need for further plant-breeding steps, as the trees can be directly propagated by the rooting of cuttings or by the techniques of micropropagation, and used as planting stock for the establishment of commercial forests.

The ability to introduce heterologous DNA (or reintroduce homologous DNA) into the cells and tissues of explants derived from field-grown trees would therefore allow additional commercially-significant properties to be introduced into these (already valuable) plants, and hence facilitate the commercial exploitation of the resultant trees in clonal Eucalyptus forests. However, these explants have proved recalcitrant to genetic modification and regeneration.

Laine et al, Plant Cell Reports 13:473-476 (1994), report the successful regeneration of E. grands clones from callus-tissue initiated from micropropagated shoots, that had previously been field-trialled, using a particular tissue culture medium. Furthermore, they clearly state that this medium could be used to promote regeneration in genetically-modified E. grandis clones. Our work (which incorporated this medium into a method used to regenerate over 5,000 genetically-modified E. arandis leaf explants) failed to substantiate this. The leaves showed an abnormal response, in that the cells around the cut edge produced phenolic compounds, and many subsequently died. This is consistent with observations made by Adam et al (1992) and Chriqui et al (1992), supra.Callus which contained the heterologous gene(s) was rarely produced, and the frequency of regeneration was too low to give rise to geneticallymodified shoots. This adverse response was attributed, at least in part, to contact with the Aqrobacterium strain used to perform the modification.

There have been a number of reports of the stable introduction of heterologous gene(s) into EucalvDtus species and, in some cases, viable genetically-modified plants have subsequently been regenerated. The CSIRO Cooperative Research Centre for Temperate Hardwood Forestry, Hobart, Australia, issued a press release in August 1992 indicating that heterologous gene(s) had been inserted into E. camaldulensis using seedling explants as the starting material. This work used Aqrobacteriummediated gene transfer to produce these plants but few other details of this work have been released. Hope et al (1994) reported using Agrobacterium tumefaciens-mediated gene transfer for the production of genetically-modified E.

srandis seedlings via two different tissue culture methods.

Edwards (1993) reported the production of geneticallymodified E. grand is seedlings. Some of the resultant genetically-modified E. arandis plants were subsequently field-trialled in the UK and some details of the technique used were published by the Department of the Environment of the UK (Public Register of H.M. Pollution Inspectorate, 1993). Dwevidi et al (1993) also report using Asrobacterium tumefaciens-mediated gene transfer for the production of genetically-modified E. arandis seedlings, but no details of the technique used have been published.

Brasileiro et al (1991) and Machardo et al (1993) have reported using a method involving the use of two different Aprobacterium strains to produce shooty callus from E.

arandis seedlings. Genetically-modified E. arandis shoots were subsequently produced from this callus. Young and Chandler (1990) have described the use of Aarobacterium tumefaciens strains to transfer heterologous gene(s) into embryos of E. alobulus. The strains used contained the wild-type Aprobacterium tumefaciens gene(s) which direct the expression of phytohormones in plant cells. Callus containing the heterologous gene(s) was produced, but no shoots were regenerated from this callus.

US-A-4795855 describes the transformation of poplar shoot cultures using Aprobacterium tumefaciens, and regeneration of the transformed cells. This technique is not satisfactory when eucalyptus is used instead of poplar.

Summarv of the Invention The present invention provides a process by which explants from mature Eucalyptus trees exhibiting superior phenotypic properties are genetically-modified and regenerated into viable plants. To date, no such process exists, despite the commercial desirability of the results, and the numerous reports of genetic modification of Eucalyptus seedlings.

The novel process can overcome the problems encountered in the prior art with respect to the regeneration of genetically-modified explants that are derived from EucalvDtus plants. The process involves two distinct stages, the genetic modification of the explant and subsequent regeneration into a viable plant.

In addition, the invention provides a process for the genetic modification of E. alobulis, E. nitens and E.

dunnii seedlings. No such process yet exists.

Description of the Drawings Figure 1 is a map of a plasmid identified herein as pSCV1. Figure 2 shows the T-DNA of a plasmid identified herein as pSCV1.6.

Description of the Invention In the process of the invention, explant cells are cultivated, in a suitable medium, with a microorganism including a DNA sequence that is functional in plants, and which can be transferred to the explant cells. For example, the DNA may function to impart a phenotypic property, e.g. resistance to a herbicide such as glyphosate, to the eucalyptus.

Suitable media and conditions for culture are known.

The medium may contain glutamate and/or ascorbic acid, in order to promote regeneration of shoots at high efficiency.

The starting pH is preferably 5.0-5.6.

It is preferred to incorporate the substituted phenylurea CPPU, i.e. N-(2-chloro-4-pyridyl) -N'-phenylurea, into the tissue culture medium, e.g. as the sole cytokinin.

CPPU can induce bud formation at high frequencies and, unlike some other phytohormones and plant growth factors, a further effect is that the buds produced are capable of further development into shoots.

Preferably, the explant cells are vegetatively derived, directly or indirectly, from vegetative tissue of trees that have been selected, or are selectable, for favourable characteristics. The tissue may be from a plant grown in the field or a greenhouse; it may be used in nonsterile form, i.e. without the use of an intervening micropropagation step, for the introduction of heterologous (or homologous) gene(s). The tissue may be leaves, stems or, petioles. In the case of E. globulus, E. nitens or E.

dunnii, the explant cells may be derived, directly or indirectly, via vegetative propagation, from seedlings. In general, the tissue may be taken from a plant that is less than 6 months old.

In the case of clonal material at least, the eucalyptus species is not restricted. It is preferably E.

arandis, E. nitens, E. dunnii or E. alobulus, or a variety, cultivar or hybrid thereof. Eucalyptus species of the subgenus Symphomyrtus may be used.

Any suitable vector may be used to mediate genetic modification of Eucalyptus explants. The Aqrobacterium tumefaciens strain used to transform E. slobulus and E.

nitens seedlings, and E. arandis clones and E. grandis/E.

camaldulensis hybrid clones as described in the Examples is the disarmed strain EHAlOlA containing the binary Ti plasmid pSCVl.6. Suitable binary Aprobacterium-Ti plasmid vector systems have been fully described elsewhere, e.g. in EP-A-0120516.

Successful transformation may be determined, by conventional methods, using any suitable characteristic as a marker. For example, the presence of the NPTII gene may be detected using a phytotoxic agent such as G418.

The present invention is readily adaptable to the production of a single clone, or of a variety of clones.

A mono-clonal or multi-clonal forest can thus be grown, based on this invention.

Improved yields and better quality product can and are obtained from forests grown from seed, but the overall gains are typically smaller when compared to those achieved in forests planted with genetically identical trees ("clones") derived from a single superior tree.

Hence, clones which have been grown in the field are the optimal source of starting material for further genetic improvement, by inserting additional genes using the techniques of genetic modification. However, in some cases, such as with species such as E. nitens, E. alobulus and E. dunnii, or hybrids thereof, improved seed may be employed as the starting material. This may be necessary where clones of these species (or hybrids thereof) are not available, or do not root with high efficiency.

The following Examples illustrate the invention.

EXAMPLES A. Aarobacterium strain, binary Ti plasmid vector and gene construct a). Disarmed Anrobacterium strain The construction of A. tumefaciens strain EHA101 has been described by Hood et al., 1986. The strain consists of a derivative of the of nopaline A. tumefaciens strain C58 in which the native Ti plasmid has been removed and replaced with the disarmed Ti plasmid pEHAlOl in which the wild-type T-DNA (ie opine synthesis and phytohormone genes) has been deleted from the Ti plasmid and replaced with a bacterially-expressed kanamycinineomycin resistance gene.The disarmed plasmid pEHA101 is a derivative of the wild-type Ti plasmid pTiBo542 isolated from A. tumefaciens strain Bo542 (AT4) which is a L,L-succinamopine producing strain (Hood et al. 1986). Strain EHA1O1A is a chloramphenicol resistant mutant of strain EHA101 which was isolated by Olszwelski et al., 1988.

b). Binary vector construct The strain used in the transformation also contains the binary Ti plasmid pSCV1 .6, which is a derivative of pSCV1. Genetic manipulations involving these plasmids were performed using standard techniques (Sambrook et al., 1989).

The component parts of pSCV1 are derived from the following (gram-negative) plasmids: the sequence used for the right DNA border and overdrive sequence was synthesised using sequence information from from the TL right border of the octopine Ti plasmid pTiA6 (Peralta et al., 1986). The left border was synthesised using sequence information from the TL of the same Ti- plasmid (Simpson et al., 1982). and is identical to the TL left border of the octopine plasmid pTiACH5 (Holsters et 1983). Octopine-type border sequences were used as these have been shown to promote more efficient tumour formation when used in conjunction with the hypervimlent strain EHA101 (Hood et al.,1986). The 97bp polylinker containing restriction enzyme sites for cloning genes into the T-DNA was derived from pUC19 (Yannish-Penron et al., 1985). The high copy number origin of replication which is active in E. coli cells but not Aprobacterium cells was derived from pUC19 (Yannish Perron et al., 1985). The origin of replication of pUC 19 which was itself originally derived from the plasmid ColE1, a plasmid isolated from E. coli.The actual pUC sequence used has been extensively deleted to remove some non-functional (superfluous) DNA sequences.

The low copy number origin of replication which is active in both E. coli cells and Aprobacterium cells was derived from the the broad host-range Inc P plasmid RK2. The origin used is a minimal 4.3kb origin which was constructed by deleting most of the nonfunctional sequences originally present in the wild-type RK2 plasmid (Thomas et al., 1980).

The minimal origin therefore contains only two genes (trf A and trf JB and associated noncoding sequences needed for replication in bacteria. The bacterially-expressed gentamycin/kanamycin resistance gene was derived from the plasmid pSa (Edwards, 1988) and is probably an aminoglycoside acetylase (Valantine and Kato, 1989). It has no apparent homology to the neomycin phosphotransferase ll coding region (Edwards, 1988). The bacterially-expressed ampicillinlcarbenicillin resistance (B-lactamase, bla) gene was cloned from pUC19 (Yannish-Penron et al., 1985). A genetic and restriction map of pSCV1 is shown in Figure 1.

pSCV1.6 is a derivative of pSCV1, into which a plant-expressed B-glucuronidase (GUS) gene and a plant-expressed kanamycin resistance gene were cloned between the T-DNA borders. The CaMV-NPTII was derived from the construct of Fromm et al., 1986. However, it has been reported that several of the most common NPTII genes used in plant geneticmanipulation encode a mutant enzyme that has a reduced ability to detoxify kanamycin (Yenofsky et al., 1990). The mutation involves a single base change, resulting in the replacement of a glutamic acid residue by an aspartic acid at the active site of the neomycin phosphotransferase (NPTII) enzyme (originally isolated from the bacterial transposon Tn5).

While the stability of the mRNA and the protein appeared unaffected by the mutation, the enzyme activity towards kanamycin is significantly reduced. The presence of the mutation in a gene can be identified by checking for the loss of a site for the restriction endonuclease Xholl in the NPTII coding sequence. This mutation was found to be present in the CaMV NPTII gene of Fromm et al., 1986 and was repaired in the following manner. The plasmid pSUP2021 (Simon et al, 1983) is approximately 10kb in size and includes a complete copy of the transposon Tn5. Digestion of this plasmid with Pst 1 and Sma 1 gives a 788bp fragment that extends from position 1730 to 2518 within Tn5 (Beck et al., 1982).This fragment was isolated and restricted with Sph 1 (giving fragments of 352 and 436 bp) or Xholl (giving fragments of 120, 246, 394 and 28 bp), and is therefore wild-type' with respect to the mutation at position 2096. The Pst 1/Sma 1 fragment was subcloned into Pst 1/Sma 1 cut pUC19 to give pTn5sub. This was then digested with Sma 1 and ligated with 8mer phosphorylated Bam H1 linkers. A clone in which the Sma 1 site had been converted to a Bam H1 site (pTn5subA) was then digested with Sph 1 and Bam H1 and the 436bp fragment (from position 2082 to 2518) isolated.This was used in a tripartite ligation with the 542 bp Bam H1/Sph 1 fragment from pCaMVNeo (positions 1540 to 2082) and Bam H1 digested pUC19. Recombinants were restricted with Bam H1 and Sph 1 to ensure that they contained both the 436 and 542 Bam H1/Sph 1 fragments, and Xho II to confirm that the site at position 2096 had been restored. This construct has a Bam H1 fragment which contains the NPTII gene coding sequence which is essentially identical to the Bam H1 fragment used by Fromm et al., (1986) to make pCaMVNeo, except that the mutation has been corrected. This construct was designated pneoNeo.The Bam H1 insert of pneoNeo contained the NPTI I coding sequence was then isolated and religated with the large (approx. 3 kb) fragment isolated from Bam H1 restricted pCaMVNeo, this fragment containing the vector plus CaMV promoter and nopaline synthase gene 3' termination sequence. Recombinants were checked against pCaMVNeo for the correct orientation using both Pvu it (2 sites) or Eco R1/Sph 1 (both unique), giving pCaMVneoNeo. This was again checked for the correct number of Xho II sites.

The Hind Ill fragment from pCaMVneoNeo containing the restored plant-expressed kanamycin resistance gene was cloned into the Hind Ill site of pSCV1 to give the plasmid pSCV1.2. pSCV1.2 was partially digested with Hindlll and the linear 10.2kb product isolated. This was dephosphorylated with calf intestinal alkaline phosphatase and ligated with a 2.8kb Hind III DNA fragment containing a plant expressed Oglucuronidase gene (CaMV-GUS INT gene) isolated from the plasmid pGUS INT which has been described by Vancanneyt et al., 1990.

A map of the T-DNA in the resultant construct (pSCV1.6), indicating the orientation of the genes and the region of DNA for transfer to plants are shown in Figure 2.

c). Introduction of the binary plasmid vector pSCV1.6 into the disarmed A. tumefaciens strain Cells of Aprobacterium tumefaciens strain EHA1O1A were transformed by electroporation using a Biorad Gene Pulser as described by Wen-jun and Forde (1989).

B. Example 1: Transformation of E. alobulus and E. nitens seedlings.

a). Plant material Seedlots of E. alobulus collected from Curanilahue, Chile were obtained from Forestal Y Agricola Monte Aguila. S.A., Nacimiento, Chile. Seeds of E. nitens provenance Errinundra S.F. were obtained from CSIRO Division of Forestry, Australian Tree Seed Centre, Queen Victoria Terrace, Canberra, Australia (suppliers reference number 16341).

b). Seed germination and preparation of explants for transformation Seeds of E alobulus were surface sterilised in a solution of 15% vlv Milton fluid containing 0.1% vlv Tween 20 for 30 minutes with gentle agitation followed by three ten minute rinses in sterile double distilled water. Seeds of E. nitens were surface sterilised in a solution of 25% v/v Milton fluid containing 0.1% vlv Tween 20 for 10 minutes with gentle agitation followed by three ten minute rinses in sterile double distilled water.Sterilised E. alobulus seeds were sown on a plant culture medium consisting of half-strength macro and micro elements as described by Murashige and Skoog (1962), vitamins as described by Morel and Wentmore (1951), 10 g t1 sucrose and 2 grphytagel (Sigma), pH adjusted to 5.8 with KOH.The seedlings were germinated and grown in a controlled environment growth room at a temperature of 23 OC and a 16 hour photoperiod with a light intensity of 40 ;jmol m'2 5-1. Sterilised E. nitens seeds were sown and germinated on a plant culture medium and under the conditions described for E. alobulus except germination was conducted at 16 OC prior to transfer to the 23 OC growth room for subsequent growth of the seedlings as described for E. alobulus.

Hypocotyls were prepared from 8-26 day-old E. alobulus and E. nitens seedlings and explants 2.5 -5mm long were excised from the apical end (upper one third) of the hypoctyl.

The hypocotyl explants were transferred to a reservoir of liquid seedling shoot induction medium (see later) until required.

c). Preparation of Anrobacterium inoculum Ovemight liquid cultures of Anrobacterium tumefaciens strain EHA1O1A containing the binary plasmid pSCV1.6 were grown on YEB medium ((tryptone 5 9 1-1, yeast extract 1 g 1 1 beef extract 5 g 1-1, magnesium sulphate 0.46 g 1-1, pH 7.2 and sucrose 5 g 1-1 added after autoclaving) containing 50 mg r1 chloramphenicol, 25 mg 1 neomycin and 15 mg t1 gentamycin at 28 OC with vigorous shaking. 10 pl of a fresh ovemight liquid culture was inoculated into 25ml of fresh media and grown for 24 h.The cells were harvested by centrifugation at 60009 for 10 minutes, resuspended in 2mM MgSO4 and repelletted. The cells were washed once more in 2mM MgSO4 and once in liquid seedling shoot induction medium. The cells were finally resuspended in liquid seedling shoot induction medium (see later) and diluted to a density of 109 cells mr1 ready for co-cultivation with the explants.

d). Inoculation of explants with Aarobacterium and regeneration of putative transgenic shoots.

Hypocotyl explants were incubated with the Anrobacterium suspension, prepared as previously described, for 15 minutes in a sterile 9 cm petri dish. The dish was placed on an orbital shaker and gently shaken at 230C during the incubation. After incubation, excess bacterial suspension was removed from the explants by blotting with filter papers and the hypocotyl explants were transferred to solid seedling shoot induction medium (half strength macroelements and iron as desribed by Murashige and Skoog (1962), microelements as described by Bourgin and Nitsch (1967), vitamins as described by Nitsch and Nitsch (1965), 30 g t1 sucrose, 1 mg t1 CPPU (N-(2-Chloro-4pyridyl)-N'-phenylurea), 0.1 mg r1 NAA (napthaleneacetic acid), pH adjusted to 5.8 with KOH) containing 3 g r1 phytagel (Sigma) as gelling agent. The inoculated hypocotyls were incubated for 48 h at 230C in the dark.

The hypocotyl segments were then washed twice (3 hours per wash) in liquid regeneration medium containing 400 mg t1 augmentin (Beechams) at 230C with gentle shaking. Excess liquid was then removed by blotting the explants with filter paper and the explants transferred to solid seedling shoot induction medium containing 300 mg 1-1 augmentin and 10 mg t1 S418 (geneticin). The hypocotyls were incubated for 4 weeks (with one subculture onto fresh medium after 2 weeks) at 230C in the dark.The explants were then subcultured onto seedling regeneration medium No. 1 (half strength macroelements and iron as desribed by Murashige and Skoog (1962), microelements as described by Bourgin and Nitsch (1967), vitamins as described by Nitsch and Nitsch (1965), 30 g t1 sucrose, 0.5 mg 1'1 BAP (6-benzylaminopurine), 0.1 mg U1 NAA, 50 mg r1 argenine, 50 mg rng serine, 50mg t1 glycine, 500 mg t1 glutamine, 50 mg t1ascorbic acid, 300 mg r1 augmentin, 10 mg r1 G418, pH adjusted to 5.8 with KOH, 3 g t1 phytagel).The explants were incubated for two weeks at 230C with a 16 hour day illumination regime (40 pmol m'2 s-1). Explants with regenerating callus were then subcultured onto seedling regeneration medium No. 2 (as seedling regeneration medium No. 1 except that the BAP concentration is 0.2 mg t1 BAP) and subcultured at intervals of two weeks using the same environmental conditions described for incubation on seedling regeneration medium No. 1.

e). Multiplication and rooting of putative genetically modified E. alobulus and E. nitens shoots Putative genetically modified shoots developing on the seedling regeneration medium containing G418 were excised from the callus and transfered to E. qlobulus/E. nitens micropropagation medium (full strength macroelements and microelements as described by Murashige and Skoog (1962), 20 g r sucrose, 0.01 mg 1-13-indolebutyric acid (IBA), 0.1 mg r1 BAP, pH adjusted to 5.6 with KOH, 2 g t1 phytagel) containing 300 mg 1 augmentin and propagated at 230C using 16 hour day illumination regime (50-70 pmol m'2 s-1.The multiplying shoots were divided and subcultured onto fresh E. globulusiE nitens micropropagation medium at 4 weekly intervals.Putative genetically modified shoots were rooted by removing shoots from the micropropagated cultures, removing any callus from the stems and transferring the shoots to E. qlobuluslE. nitens root induction medium (quarterstrength macroelements and microelements as described by Murashige and Skoog (1962), 20 g r1 sucrose, 40 mg r1 IBA, pH adjusted to 5.6 with KOH, 2 g l-1 phytogel) and incubating the shoots for 24h under the environmental conditions used for micropropagation.Following the root-induction step, shoots were transferred and the stems inserted into a polypropylene fibre substrate (Milcaps, Milcap France, S.A., Chemin de Montbeau, 49340 Nuaille, France) soaked in liquid E. alobuluslE. nitens rooting medium (quarter-strength macroelements and microelements as described by Murashige and Skoog (1962), 20 g I-1 sucrose, pH adjusted to 5.6 with KOH) and incubated under the environmental conditions used for micropropagation and root induction. When actively growing roots were visible growing through the polypropylene plug, the plants were transferred to 3" square plant pot filled with coco-peat. The plants were placed inside a mist propagator and slowly hardened off by reducing the humidity over a period of a week. After three to four weeks, the plants were transferred to 7" pots and placed in a glasshouse facility.The plants were grown under natural daylight and were watered daily.

C. Example 2: Transformation of E. arandis clones and E. arandislE. camaldulensis hybrid clones.

a). Plant material E. arandis clone 9114 and E. arandis/E. camaldulensis hybrid clone 1 it5 were supplied to Shell South Africa by the South African Forestry research institute, PO Box 727, Pretoria 0001, South Africa (now FORESTEK, Private Bag X11 227, Nelspruit 1200, South Africa).

b). Preparation of explants for transformation.

Leaf, petiole or stem explants from E. arandis clone 9114 and E. arandislE. camaldulensis hybrid done 11125 were prepared directly from axenic micropropagated shoot cultures or rooted micropropagated shoots without disinfection (protocols for micropropagation and susequent rooting of shoots are given later). Altematively, leaf, petiole or stem explants were prepared from ramets (either produced via micropropagation or by cuttings) grown in the greenhouse or in the field and disinfected prior to cocultivation with Anrobacterium tumefaciens.In this case, young scions with healthy leaves less than 3cm in length were harvested from the upper portion of the crown from vigorous plants of less than 1.5 metres in height, and disinfected by immersion in a 20% vlv Milton solution containing 0.1% v/v Tween 20 for 10 minutes with gentle agitation. The scions were then rinsed three times in sterile distilled water prior to dissection. 3-5mm diameter leaf explants or 2-4mm long sections of stem or petioles were prepared from the scions and placed in liquid E. arandis co-cultivation medium (see later) until required for cocultivation with the A. tumefaciens strain.

c). Preparation of Aarobacterium tumefaciens inoculum Aprobacterium tumefaciens strain EHA1O1A containing the binary plasmid pSCV1.6 was prepared for inoculation of the zonal explants as has been described in the previous example except that the final wash and subsequent resuspension of the cells was conducted in liquid E. arandis co-cultivation medium (see later) containing 1 mg 1-1 CPPU and 0.465 mg r1 NAA d). Inoculation of explants with Aarobacterium and regeneration of putative transgenic shoots.

Leaf, petiole or stem explants of E arandis clone 91/4 and E. arandislE. camaldulensis hybrid done 11/25 were incubated with the Anrobacterium suspension, prepared as previously described, for 15 minutes in a sterile 9 cm petri dish. The dish was placed on an orbital shaker and gently shaken at 230C during the incubation.After incubation, excess bacterial suspension was removed from the explants by blotting with filter papers and the hypocotyl explants were transferred to solid E. arandismybrid co-cultivation medium (750 mug 1 KNO3, 250mg l-1 MgSO4.7H2O, 250 mg U1 NH4H2PO4,100mg r1 CaCI2 2H2O, 20 g 1- sucrose, 600 mg t1 2-[N-morpholino]ethanesulphonic acid (MES), half-strength Murashige and Skoog basal salt micronutrient solution (Sigma catalogue number M0529), vitamins as described by Morel and Wentmore (1951), 1 mg r1 CPPU, 0.465 mg r1 NAA, pH adjusted to. pH 5.5 with KOH, 3 g 1-I phytogel). The explants were co-cultivated with the Aarobacterium strain for 48 h in the dark at 230C. After incubation, excess bacterial suspension was removed from the explants by blotting with filter paper and the explants were then washed twice (3 hours per wash) in liquid E. arandislhybrid co-cultivation medium containing 400 mg 1-1 augmentin at 230C with gentle shaking.The explants were then transferred to E. arandis/hybrid shoot induction medium (as for E. arandislhybrid cocultivation medium but containing 500 mg l-1 glutamine, 50 mg r ascorbic acid, 300 mg t1 augmentin and 10 mg r1 G418. The explants were incubated in the dark at 230C for 4 weeks with subculture to fresh medium after 2 weeks and at the end of the period of incubation in the dark. The cultures were then transferred to continuous light (40 pmol m'2 s-1) and incubated at 230C for a further two weeks.The explants were subcultured to E.qrandis/hybrid shoot elongation medium (as E. qrandis/hybrid shoot induction medium) but with the CPPU ommitted, the NAA concentration adjusted to 0.112 mg t1 and containing 1.16 mg t1 BAP and incubated at 230C under continuous light (40 pmol m-2s 1) e). Multiplication and rooting of putative genetically modified shoots Putative genetically modified shoots were excised from the cultures and transferred to E.

arandis\hybrid shoot multiplication medium (1900 mg 1-1KNO3, 1650 mg t1 NH4NO3, 440 mg r CaCl2.H2O, 370 mg r1MgSO4, 170 mg t1 KH2PO4, half-strength Murashige and Skoog basal salt micronutrient solution (catalogue number M0529), vitamins as described by Morel and Wentmore (1951), 10 g r1 sucrose, 0.04 mg t1 BAP, 300 mg 1 augmentin, pH adjusted to 5.6 with KOH, 2 g t1 phytagel).The cultures were propogated at 230C using a 16 hour day illumination regime (50-70 pmol m'2 s-l). The multiplying shoots were divided and subcultured onto fresh E. arandis\hybrid shoot multiplication medium at 4 weekly intervals. Once rapidly growing shoot cultures had been established, individual shoots were transferred to rooting medium (as for E. arandis\hybrid shoot multiplication medium but with the BAP ommitted and containing 0.2 mg r1 IBA) and retumed to the growth room. Shoots with developing roots were transferred to a sterile Jiffy-7 peat pellet (Jiffy Products (UK) Limited, 14116 commercial Road, March, Cambridge, UK) in a Magenta pot (Sigma) for root establishment.

When actively growing roots were visible growing through the peat pellet, the plant was transferred to a 3" square plant pot filled with coco-peat. The plants were placed inside a mist propagator and slowly hardened off by reducing the humidity over a period of a week.

After three to four weeks, the plants were transferred to 7" pots and placed in a glasshouse facility. The plants were grown under natural daylight and were watered daily.

D. Biochemical and genetic analysis of genetically-modified Eucalvptus plants.

a). Histochemical ss-glucuronidase (GUS) assays Histochemical GUS assays were performed on the leaves of putative genetically modified E. globules and E. nitens seedlings and E. arandis clones and E. arandislE. camaldulensis hybrid clones as described by Draper et al. (1988). Leaf explants were transferred to a petri dish containing fixation solution (100 ml double distilled water containing 750 pl 40% formaldehyde, 2 ml 0.5 M MES and 5.46 g 1-1 Mannitol). The petri dish was placed in a vacuum desiccator and the vessel was evacuated several times until all of the explants were submerged in the fixation solution. The explants were incubated for 45 minutes at room temperature and then washed twice in 50mM sodium phosphate buffer (pH 7.0).The explants were then transferred into a 2mM 5-bromo-4-chloro-3-indoyl glucuronide (X-GLUC) solution made up in 50mM sodium phosphate buffer (pH 7.0). The X-GLUC solution was vacuum infiltrated into the explants several times, the dish sealed with Nescofilm and then incubated at 37 OC ovemight. The reaction was stopped by transferring the explants to 70% ethanol. GUS activity could be detected by the presence of an insoluble blue stain.

b) Detection of genes transfered to transgenic Eucalyptus plants by Southem blotting and hybridisation.

DNA extraction was carried out as described by Keil and Griffin (1994). 10 micrograms of DNA of each sample were digested with with Hind 111 (60 units per sample) in the appropriate restriction buffer supplied with the enzyme (Northumbrian Biologicals). To aid the digestion of DNA, Casein was added to the restriction mixture at a final concentration of 0.1 mglml (Drayer and Schulte-Holthausen, 1991). The restrictions were canied out at 370C ovemight at 370C. Electrophoresis of the samples, Southem blotting and hybridisation were performed as described by Sambrook et al. (I 989). 10 micrograms of the plasmid pJlT65 (Guerineau, 1990) was digested with the restriction enzyme Eco RV and the resulting restriction fragments were separated by electrophoresis on a 1.5% agarose gel (Sambrook et al., 1989). A 2kb (approximately) DNA fragment containing part of the coding sequence of the GUS gene, and the Cauliflower Mosaic Virus 35S gene terminator region, was eluted from the gel by the method of Heery et al. (1990). The eluted fragment was radiolabelled by the method of Feinberg and Vogelstein (1983), using the random primer labelling kit supplied by Boehringer Manheim.

Claims (13)

1. A process for producing genetically-engineered eucalyptus, which comprises the co-cultivation of explant cells of the eucalyptus species with a microorganism including one or more DNA sequences which are functional in plants and which are transferable to the eucalyptus cells, in order to achieve a genetic modification by which the cells are stably transformed with the DNA.

2. A process according to claim 1, in which the plants comprise Eucalyptus species of the subgenus Symphomyrtus.

3. A process according to claim 1 or claim 2 in which the eucalyptus species is E. srandis, or a variety, cultivar or hybrid thereof.

4. A process according to claim 1 or claim 2 in which the eucalyptus species is E. slobulus, or a variety, cultivar or hybrid thereof.

5. A process according to claim 1 or claim 2 in which the eucalyptus species is E. nitens, or a variety, cultivar or hybrid thereof.

6. A process according to claim 1 or claim 2 in which the eucalyptus species is E. dunnii, or a variety, cultivar or hybrid thereof.

7. A process according to any preceding claim, in which the explant cells are derived vegetatively, directly or indirectly, from vegetative tissue of trees that have grown in the field.

8. A process according to claim 7, wherein the tissue comprises petioles, leaves or stems.

9. A process according to any of claims 4 to 6, in which the explant cells are derived, directly or indirectly, via vegetative propagation from seedlings.

10. A process according to claim 3, in which the explant cells are derived, directly or indirectly, via vegetative propagation from seedlings of E. arandis, variety, cultivar or hybrid, which are more than 6 months old.

11. A process according to any preceding claim, wherein the function of at least one of the DNA sequences is to impart a phenotypic property to the eucalyptus.

12. A mature adult plant or tree obtained by a process according to any preceding claim.

13. Timber obtained from a mature tree according to claim 12.