WO1999048355A1

WO1999048355A1 - A method of transformation

Info

Publication number: WO1999048355A1
Application number: PCT/AU1998/000195
Authority: WO
Inventors: Robert Dixon Teasdale; Ekaterina Mouradova; Yumin Yang; Ding G. He; Cecilia O'dwyer
Original assignee: Forbio Limited
Priority date: 1998-03-20
Filing date: 1998-03-20
Publication date: 1999-09-30
Also published as: AU6386498A

Abstract

Vacuum infiltration is an effective way of infecting Eucalyptus explants with Agrobacterium. It results in a high frequency of expression as shown by GUS histochemical assays. The procedure is highly time efficient and less damaging to the plant tissue, as wounding of cotyledon and leaf explants was not needed. Stably transformed plants could be regenerated from vacuum infiltrated explants. GUS histochemical assay and Southern hybridisation analyses confirmed the transformation. It has also been determined that the application of vacuum may be supplemented to the post application of pressure to create a transformationally effective pressure gradient.

Description

A METHOD OF TRANSFORMATION

This invention relates to a method of transformation.

This invention has particular but not exclusive application to a method of

transforming explants with Agrobactenum tumefaciens and for illustrative purposes reference will be made to such application. However, it is to be understood that this

invention could be used in other applications such as whole plant or seed transformation or with other infective vector systems.

Eucalyptus has been considered a difficult genus for transformation, with the main obstacle the low regeneration potential of explants. Agrobactenum mediated transformation is a commonly used method for transforming plants. To inoculate explants, the general practice is to incubate wounded explants with a suspension of Agrobactenum. Consequently, the transformation events occur mainly around the wound sites. In addition, there is the tedious, time-consuming, and painstaking work of wounding a large amount of explants. Further, wounding may cause tissue damage

due to callus formation at the wound sites. Wounding causes a particularly high failure

rate in regeneration of the explants.

New in planta transformation approaches have been developed, mainly in

Arabidopsis thaliana. Seeds have been imbided with Agrobactenum, and plants allowed to grow to maturity in soil. Transformed plants were selected out from the

harvested seeds. This method has had the advantage that no tissue culture is

involved, but the transformation frequency is low.

Vacuum infiltration has been used as a means of agroinfection for introducing

wheat dwarf virus into wheat embryos [Dale er al. Plant Sci 63: 237-245 (1989)]. This technique of inoculation has also been used for introducing Agrobactenum tumefaciens into adult plants of Arabidopsis, where selected transformed seeds can be obtained

after the infected plants reach maturity [Bechtold et al , C R Acad Soc Paris, Life Sci

316 1194-1199 (1993), Bent er a/., Science 265 1856-1860 (1994)]. More consistent results can be achieved using this new method However, this method can probably be described as indirect transformation of germ cells and will have limited use when

transformation targets are somatic cells For example, in transformation of trees the targets are frequently the selected superior trees themselves, not the second generation from seeds, which are of different genotypes to the superior parent trees. Further, it may a substantial period of time for some plant species to reach maturity to enable selection of transformed seeds

Recently, Norel et al [HortSci 31. 1026-1027 (1996)] tried to use vacuum infiltration as a way to inoculate explants with Agrobactenum They found, however, that it did not enhance transformation frequency, nor change the distribution of the blue

GUS pigmentation on apple leaf explants when compared with wounded explants

infected in an Agrobactenum bath Norelli et al concluded that vacuum infiltration did

not have a significant effect on transformation Kapila et al., [Plant Sci 122: 101-108 (1997)] has reported that up to 90% of the area of unwounded Phaseolus vulgaris

leaves showed transient GUS expression following vacuum infiltration However, there was no regeneration of the leaves after vacuum infiltration to obtain a transformed

plant

In each case, it is presumed that plant tissue cannot remain viable after being

subjected to vacuum for extended periods of time In the case of Kapila et al. above, regeneration was not the point It has now been surprisingly determined that stable

transformation of certain plant tissue can be achieved with good transformation efficiency under conditions which would ordinarily be condemned as destructive of regeneration capacity This has been established especially for Eucalyptus spp

The present invention aims to alleviate at least one of the foregoing

disadvantages and to provide a method of transformation which will be reliable and efficient in use Other objects and advantages of this invention will hereinafter become apparent

With the foregoing in view, this invention in one aspect resides broadly in a method of transformation of plant including the steps of immersing plant tissue in a medium including an infective transformation vector, reducing the pressure on said tissue to -10 to -100 kPa gauge, maintaining said pressure for 10 to 60 minutes, and raising said pressure to

atmospheric pressure or above, said transformation vector being selected to provide

integrating transformation of said plant tissue

The aforementioned mentioned method has found particular application in the

transformation of eucalypts Accordingly in a further aspect, this invention in one aspect resides in a method of transformation of eucalypts including the steps of

immersing eucalypt tissue in a medium including an infective transformation

vector, reducing the pressure on said tissue to -10 to -100 kPa gauge,

maintaining said pressure for 10 to 60 minutes, and raising said pressure to

atmospheric pressure or above said transformation vector being selected to provide 4 The eucalypt tissue may be selected from E grandis, E tereticornis or E camaldulensis The eucalypt tissue may be cell culture, a whole plant, an explant or

germ material In order to provide commercial material for prolongation of transformed

plants it is preferred that the plant material be explants suitable for prolongation after transformation The explants may be selected from shoots, cotyledons, hypocotyls, leaves, seedlings, or the meπstem

The infective transformation vector may be selected from any known suitable infective system to mediate transformation, such as a virus or bacterium Preferably, the vector is Agrobactenum Agrobactenum is the most common type of bacterium used for bacterially mediated-transformation of plants However, it is envisaged that any other infective microorganism which may be proved to be effective in transformation of plant cells may be used It is to be appreciated that with the rapidly growing development in biotechnology that other appropriate bacterium or other infective agent may be developed which might also be applicable The medium may be any liquid medium used to support the bacterial suspension

without affecting the explant tissue The medium may include chemicals which assist

in transformation and regeneration with less tissue damage For example,

acetosyπngone may be added to the medium The transformation culture of

Agrobactenum is preferably established at a population in the range 1-5 x 10⁸ cfu ml^"1 The reduced pressure may be achieved in any vacuum chamber or desiccator

It may be advantageous to use a vacuum vessel which is capable of maintaining a

pressure above atmospheric as is described hereinafter The vacuum vessel may be

adapted to cycle the contents of the vessel through a range of pressures It was found It was found that prolonged exposure of plant tissue leads to hyperhydricity in the explant The duration of the exposure is in the range of 10 to 60 minutes, the longer periods being preferred for recalcitrant species of eucalypt Preferably, the duration of vacuum treatment is in the range of 15 to 20 minutes

The pressure may be raised to atmospheric pressure or above slowly or

stepwise Preferably, the pressure is raised to atmospheric pressure or above very rapidly This may be achieved by opening the vacuum chamber while still under negative pressure Not being bound by theory, it is surmised that the vacuum (negative pressure) causes the air spaces between the cells in the plant tissue to

decrease The longer the duration and the lower the pressure of the vacuum the less air space within the plant tissue The increase in pressure allows the infiltration medium including the infective transformation vector to relocate into the plant tissue This is supported by the evidence of hyperhydricity from prolonged exposure in the

vacuum Selection and regeneration of transformed explants may be performed by any

method commonly used in the field tissue culture For example, regeneration may

include any of the steps of co-cultivation, incubation, callus induction, shoot induction and subculturing Selection mediums or other selection methods typically used may

be used for selecting successful transformants

In a further embodiment, the multiple application of vacuum infiltration may also

lead to improved effects in transformation and regeneration For example, the reduced pressure effect may be provided by cyclically applying vacuum to the tissue

rather than applying a vacuum for an extended period WυO 9^9^;/4w8^j5-5³ PCT/AU98/00195

6 hitherto been unsuited to vacuum infiltration. It has been unexpectedly determined that the application of vacuum may be supplemented by the post application of pressure

to create a transformationly effective pressure gradient.

In a further aspect, this invention resides in a method of transforming plants including the steps of: immersing plant tissue in a medium including an infective transformation vector; reducing the pressure on said tissue;

increasing said pressure to a pressure of at least 10 kPa above said reduced pressure to effect infiltration, and regenerating said tissue.

The plant tissue may be cell culture, a whole plant, an explant or germ material.

In order to provide commercial material for propagation of transformed plants it is preferred that the plant material be explants suitable for propagation after transformation. The explant may be selected from shoots, cotyledons, hypocotyls,

leaves, seedlings, or the meristem.

The medium may include any infiltration media or bacterial suspensions typically

used in vacuum infiltration procedures, or as described above.

The pressure differential between the reduced pressure and the over-pressure may in a suitable range so as not to cause damage or excessive hyperhydricity to the

plant tissue. Particularly, the reduced pressure and time of maintenance at the

reduced pressure is preferably selected to avoid hyperhydricity of the tissue. The

corresponding over pressure is preferably selected to provide sufficient pressure

differential between the reduced pressure and the over pressure to promote infiltration. 7 pressure may be in the range of 10 to 500 kPa The plant material may be subjected

to alternating cycles of said reduced and over pressures For hardy species the pressure differential is preferably about 90 kPa or higher

When the plant material is to be subjected to alternating positive and negative

pressure, this may be achieved in any suitable vessel including a vacuum/pressure chamber or desiccator modified to allow the apparatus to function as a pressure vessel

Alternatively, the negative and positive pressure may be built by manual pulling and pushing of a piston of a syringe containing the plant material and medium The piston may be pulled to a scale to create negative pressure or a vacuum In opposition, the pushing of the piston creates a positive pressure The pulling and pushing may performed repeatedly

The inoculated plant material may be co-cultivated and normal selection and regeneration procedures used to obtain a transgenic plant

In a further aspect, this invention resides in a method of transforming plants

including the steps of immersing plant tissue in a medium including an infective transformation vector,

increasing said pressure to a pressure of at least 10 kPa to 500 kPa above the

starting pressure to effect infiltration, and

regenerating said tissue In order that this invention may be more readily understood and put into

practical effect, reference will now be made to the accompanying drawings which

illustrate a preferred embodiment of the invention and wherein

FIG 1 is the control (E grandis) seedlings, where the wounded explant FIG. 2 is the high frequency GUS expression in vacuum-infiltrated E. camaldulensis seedling as assayed 5-6 days after inoculation;

FIG. 3 is the high frequency GUS expression in vacuum-infiltrated E. grandis seedlings as assayed 5-6 days after inoculation;

FIG. 4 is the high frequency GUS expression in vacuum-infiltrated E. tereticornis shoot as assayed 5-6 days after inoculation;

FIG. 5 is the regeneration of multiple Gl/S-positive shoots from E. camaldulensis callus obtained from vacuum-infiltrated explants;

FIG. 6 is a GL/S-positive E. grandis shoot obtained from vacuum-infiltrated

explants;

FIG. 7 is a GL S-positive E. camaldulensis plantlet obtained from vacuum-

infiltrated explants;

FIG. 8 is a transgenic E. grandis plant in soil obtained from vacuum-infiltrated

explants, and FIG. 9 is a southern blot analysis of transformed E. grandis lines EG66 and

EG77 that were obtained from vacuum infiltrated explants. The control was from non-

transformed E. grandis. 35SG /SINT was used as the probe for hybridization, (a)

undigested DNA samples. The absence of low molecular weight fragments indicated

that there was no contamination of plasmid DNA. (b) DNA samples digested with

HindlW.

EXAMPLE 1

Medium

The media used in this study were G22 and KG. G22 medium was used in a modified 9 were omitted. The phytohormone regimes in inductions medium were also different in using 1 μM BA, 0.03-.01 μM TDZ (Sigma) and 1 μM NAA for callus induction of E. grandis and E. tereticornis and 1 μM BA and 0.5 μM NAA for E. camaldulensis. The differentiation medium was a G22 medium supplemented with 2-5 μM BA and 0.5 μM

NAA. A KG medium contain 0.2μM BA was used as the subculture medium for clone materials. All media was solidified with 0.25% Gelrite (Kelco, San Diego), and the pH was adjusted to 5.7 - 5.8 using KOH before autoclaving for 15 minutes at 121 °C.

Liquid medium, used for selecting transformed shoots, was KG supplemented with 0.01 μM BA and antibiotics.

Plant Materials

Seedlings of the two species (E. grandis and E. camaldulensis) used in this study were obtained by germinating sterile seeds on hormone-free KG medium.

Cultures were maintained at 23°C under dim light (5-10 μMol m^"2). Seedlings aged 8- 15 days were used as starting material for regeneration and transformation

experiments. E grandis and E. tereticornis clones maintained on KG medium were

also used as starting material. Wounding of explants, when necessary was performed

using the tip of a scalpel blade or a sharpened needle. The general protocol is as follows:

a. Clones are sub-cultured every 3-5 weeks on KG media [Laine E and David A:

Plant Cell Rep. 13: 473-476 (1994) supplemented with 2 μM BA (6-benzlaminopurine).

b. At subculture, a piece of shoot consisting of one or two nodes are cut off from

the young shoots and used for the initiation of new cultures. If the leaves are too big, 10 c. Eight to ten node cultures are kept in a 250 ml jar (bunzel) containing 40 ml of

medium. d. The jars are kept at 23°C under dim light (5-10 μMol m^"2) with a 12-hour photo- period. Under such growth conditions, the shoot cultures are generally clusters

consisting of 3 - 10 short shoots (5-20mm) with small leaves (~ 5mm). e. For transformation, single shoots were excised and the leaves at the base are removed. The young leaves, generally the top 2 - 3 pairs are retained for transformation.

This particular protocol was optimised for use with Eucalyptus, but it is not restricted to said genus and is generally envisioned to be applicable to other plant genus/species, particularly those which lend themselves to clonal micro-propagation. One aspect of this method is designed to clonally propagate large numbers of suitable plant starting material for use in transformation. In doing so, this methodology is

genotype independent, as it is capable of generating as much starting material as

desired from a given genotype. Even very low regeneration frequencies can be accomodated. From this material, different plant tissues can be isolated for

transformation (e.g. stem or leaves). Transformation into the clonal plant starting

material may be by several transformation methods, including, but not limited to,

vacuum infiltration.

AQrobacterium and vacuum infiltration 11 INT. This vector carries a GUS gene interrupted by a plant intron. Consequently, GUS expression is exclusively from plant cells, and contamination from stained bacteria are

excluded.

Agrobactenum were grown overnight in a liquid YEP medium at 28°C until the

OD₆₀₀ value was around 1. The bacterium suspension was diluted to the desired density of 1-5x10⁸ ml^"1 (6-30 times) using a liquid KG medium. Acetosyringone was

added at a final concentration of 50 μM. For vacuum infiltration, the plant materials were placed in an Agrobactenum suspension. The mixture was put into a desiccator, and vacuum infiltrated -95 kPa for 20 minutes in a BioHood. As the control, explants were immersed in an Agrobactenum bath for 20 minutes. After inoculation, the explants were blotted dry with sterile filter paper before transfer to an MG22 co- cultivation medium. The cultures were kept on the co-cultivation medium, in the dark for 2-5 days, depending on the degree of Agrobactenum overgrowth.

Selection

Following 2-5 days incubation on the co-cultivation medium, cotyledon and leaf

explants were placed on a solid callus induction medium containing 250 mg I^'1

cefotaxime or 100 mg I^"1 Timentin (SmithKline Beecham), and 0-30 mg I^"1 geneticin

(Sigma). The cultures were subcultured onto fresh medium every 2-3 Weeks. When

shoots emerged, they were transferred to a liquid medium containing 0.01 μM BA and

antibiotics (50 mg I^"1 cefotaxime or 100 mg I^"1 Timentin) and 2.5 or 5 mg I^"1 geneticin for further selection. Shoots which survived the liquid selection were subcultured in fresh

liquid medium containing 5 mg I^"1 geneticin. During the first 3 weeks of callus induction 12 under light (5-10 μMol m^"2).

Assays for Transformation

For GUS histochemical assays explants were incubated in an X-gluc solution

(0.5mg I^"1 X-gluc in Phosphate buffer, pH 7.0) overnight at 37°C. NPTII enzyme assay was performed. For Southern blot analysis, DNA was extracted from frozen eucalypt leaves using Genomic-tip 100G Kit (QIAGEN). Purified DNA (20 μg) was digested with Hind\\\, separated by electrophoresis on 0.7% agarose gel, blotted onto Hybond N+ membrane (Amersham) and hybridised using a labeled, random-primed internal Hind\\\

fragment of 35SGCSINT containing the GUS gene as a probe.

Transient Expressions

Factors affecting transformation efficiency, including density of Agrobactenum, vacuum pressure and duration of vacuum treatment, were studied. Two pressure settings were tested. Transient expression was high when vacuumed at -95 kPa rather

than at -45 kPa. Using Agrobactenum at a density of 1 x 10⁸ or 5 x 10⁸ ml^"1 had no

obvious effects on the level of transient expression. Time of vacuum treatment in the

tested range (10 to 60 minutes at -95 kPa) appeared to have very little effect on the

level of transient expression of E grandis. However, prolonged exposure at the lower

pressure resulted in more severe hyperhydricity of explants following the vacuum

treatment. Therefore, the time for vacuum treatment was chosen as 20 minutes for E.

grandis, and 15 minutes for E. camaldulensis.

Distribution of blue cells on vacuum infiltrated Eucalyptus explants was notably 13 wounded explants in an Agrobactenum bath for 20 minutes. On wound explants inoculated an Agrobactenum bath, blue areas or single blue spots were usually observed around the wound sites. Sometimes, single blue spots were seen away from

the wound sites, but generally still within a close distance, as seen in Fig. 1. In contrast, there were many single blue spots or blue areas distributed on the whole vacuum infiltrated leaf or cotyledon explant, in addition to the blue areas and cells around the wound sites, as seen in Figs. 2 - 4. These randomly distributed blue spots and areas were also observed at similar frequencies on vacuum infiltrated cotyledons and leaves that had not been wounded. In addition to the individual blue spots, large continuous blue areas were frequently observed, which could cover 50-

90% of the area of the cotyledon or leaf, as also seen in Figs 2 - 4. Occasionally, when whole shoots of E. grandis and E. tereticornis were vacuum infiltrated, the whole meristem area was stained blue by GUS pigmentation. For hypocotyls and internodes, the pattern of GUS expression of vacuum infiltrated explants was similar to those

inoculated in an Agrobactenum bath, i.e. the expression was predominantly at the

wound sites (the cut ends).

Regeneration of Transformed Plants

On induction medium, vacuum infiltrated leaves and cotyledons were darker

green than control explants. They were also slightly swollen, with an appearance

similar to vitrificated (hyperhydric) cultures. However, regeneration potential of the

explants appeared not to be affected. In one experiment, explants from E. grandis

seedlings were vacuum infiltrated under -95 kPa for 20 minutes before being placed 14 and 14 of the 30 hypocotyls formed shoots, a similar regeneration frequency (50%) to the average regeneration experiments.

Transgenic plants, referring to Figs. 5-8, were obtained from explants vacuum infiltrated for 20 minutes under -95 kPa in a liquid containing 1-5x10⁸ ml^'1 Agrobactenum. The explants were co-cultivated in MG22 medium for 2-3 days before

subculturing onto medium containing 250 mg I^'1 cefotaxime or 100 mg I^"1 Timentin for 4-5 days. They were then transferred onto medium containing 10 mg I^"1 geneticin for 10 days, following by 15mg I^"1 geneticin for 2 weeks, and finally kept on 30 mg I^"1 geneticin until plant formation.

Four putatively transformed (GUS positive) plants of E. grandis were regenerated from 278 cotyledon (1.4%), 3 putatively transformed plants of E. camaldulensis were regenerated form 150 cotyledons (2.2%), and a one putatively transformed plant of E. camaldulensis was regenerated from 100 hypocotyls (1.0%). These frequencies were similar to those for wounding followed by inoculation in an Agrobactenum bath.

Transformation of plants was confirmed by positive Gl/S-histochemical staining (Figs. 1 - 8), NPTII enzyme assay (not shown), and Southern blot hybridisation analysis (Fig. 9). Southern hybridization analysis was performed to confirm the

presence of the 35SGL/SINT transgene into the plant genome in two putatively

transformed E. grandis lines EG66 and EG67, and a non-transformed control.

Hybridisation, with ³²P-labeled GUS gene from 35SGL SINT vector as a probe, shows

strong signals corresponding to high molecular weight DNA (> 23kb) in lines EG66,

EG67. The DNA extracted from the control, non-transformed plant, did not hybridise 15 the genomic DNA bands. These results indicate the integration of the 35SGI/SINT

plasmid into the Eucalyptus genomic DNA, and that no contamination with plasmid DNA had occurred.

The DNA samples were digested with Hind\\\, which released a 2.8 kb internal fragment of the T-DNA, containing the GUS gene sequence with 35S promoter and terminator sequences. A comparison of the intensities of the bands indicates that

EG66 and EG67 have multiple (three to four) copies of 35SGL SINT transgene, as

seen in Fig. 9. No detectable signal was observed in the control.

Eucalyptus has been considered a difficult genus for transformation, with the main obstacle the low regeneration potential of explants. Methods in accordance with the foregoing embodiment results in an increase in regeneration capacity by vacuum infiltration, due to the minimisation of tissue damage by avoidance of wounding. It is

to be appreciated that better results than those obtained in Eucalyptus could be expected if this method is applied to other plant species whose leaf and cotyledon explants have a greater potential for regeneration. It is to be appreciated that factors

such as the density of the bacterium suspension, the vacuum pressure, and the

duration of the vacuum treatment will vary in relation to the type of plant material used

and the type of plant species.

One feature of vacuum infiltration inoculation is the occurrence of infection is not

limited to the wound sites, but is widely spread across the whole leaf blade. Transient expression on cotyledons and leaves was frequently so high that it was impossible to

count the blue spots to quantitatively present transformation results. This high level

of transformation is advantageous in plants where whole leaf or cotyledon explants 16 from a larger tissue area.

EXAMPLE 2

Negative/Positive Pressure Inoculation

Two experiments were conducted trying to use a syringe to produce positive/negative pressure to introduce Agrobactenum into eucalypt plants.

10 day old seedlings of E. camaldulensis were used. After cutting off the root ends, the explants (the intact cotyledons attached to the hypocotyls) were put into a 50 ml plastic syringe to which 20 ml of Agrobactenum (AGL1 ) suspension was added. The negative and positive pressure was built by manual pulling and pushing of the piston. The piston was pulled to the scale of 50 ml to create the negative pressure (vacuum). The pushing to give positive pressure was difficult to perform and impossible to measure. Pulling and pushing were performed repeatedly for 5 minutes and the explants were allowed to stay in the syringe for an extra 10 minutes before

blotting dry using a filter paper and transferred to co-cultivation medium. Seedlings

inoculated in an Agrobactenum bath for 30 minutes were used as the experimental

control. The inoculated seedlings were allowed to stand on a KG medium 0.2 μM BA)

for 3-5 days before the cotyledon and hypocotyls were excised. The excised

cotyledons and hypocotyls were then transferred to a selection/regeneration medium and subcultured as in routine transformation experiments.

Transient Expression

Transient expression of the GUS gene was assayed in the first experiment. 15 of 18 17 cells. Only 2 out of 20 cotyledons (10%) and 6 out of 11 (55%) from the control had blue cells.

Stable Transformation

GUS expression in calluses was assayed in the second experiment using cultures

maintained on selection medium for 3 weeks. Two of six assayed cotyledon calluses and two of three hypocotyl calluses were found to be Gl/S-positive. Four hypocotyl calluses from the control were all negative.

Transformed Shoot

One GL/S-positive shoot was obtained following 6 weeks' incubation on selection medium. The shoot was obtained from the first experiment, where 30 seedlings were used as starting material. Ten seedlings were assayed for transient expression and

the shoot was obtained from one of the remaining 20 hypocotyls.

In use, vacuum infiltration is an effective way of infecting Eucalyptus explants

with Agrobactenum. It results in a high frequency of expression as shown by GUS

histochemical assays. The procedure is highly time efficient and less damaging to the tissue, as wounding of cotyledon and leaf explants was not needed. Stably

transformed plants could be regenerated from vacuum infiltrated explants. GUS

histochemical assay and Southern hybridisation analyses confirmed the transformation.

It will of course be realised that while the foregoing has been given by way of 18 thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims

19 CLAIMS

1. A method of transformation of plant tissue including the steps of: immersing plant tissue in a medium including an infective transformation vector; reducing the pressure on said tissue to -10 to -100 kPa gauge;

maintaining said pressure for 10 to 60 minutes, and raising said pressure to atmospheric pressure or above, said transformation

vector being selected to provide integrating transformation of said plant tissue.

2. A method of transformation according to Claim 1 , wherein said plant tissue comprises Eucalyptus explants.

3. A method of transformation of eucalypts including the steps of: immersing eucalypt tissue in a medium including an infective transformation vector; reducing the pressure on said tissue to -10 to -100 kPa gauge;

maintaining said pressure for 10 to 60 minutes, and

raising said pressure to atmospheric pressure or above, said transformation

vector being selected to provide integrating transformation of said eucalypt tissue.

4. A transformation method according to Claim 3, wherein eucalypt tissue is

selected from E. grandis, E. tereticornis or E. camaldulensis.

5. A transformation method according to any one of claims 3 or 4, wherein the 20 material.

6. A transformation method according to Claim 5, wherein the tissue is explants selected from shoots, cotyledons, hypocotyls, leaves, seedlings, or meristem.

7. A transformation method according to any one of the preceding claims, wherein the infective transformation vector is selected from a bacterial or viral infective system.

8. A transformation method according to Claim 7, wherein the infective transformation vector is Agrobactenum.

9. A transformation method according to Claim 8, wherein the medium includes chemicals which assist in transformation and regeneration with less tissue damage.

10. A transformation method according to Claim 9, wherein acetosy ngone is added

to the medium.

11. A transformation method according to any one of claims 8 to 10, wherein the transformation culture of Agrobactenum is established at a population in the range 1-5

x 10⁸ cfu ml^"1.

12. A transformation method according to any one of the preceding claims, wherein

said reduced pressure is achieved in a vacuum chamber or desiccator. 21

13. A transformation method according to Claim 12, wherein said vacuum chamber comprises a vacuum vessel which is capable of maintaining a pressure above

atmospheric.

14. A transformation method according to Claim 13, wherein said vacuum vessel is adapted to cycle the contents of the vessel through a range of pressures.

15. A transformation method according to Claim 3, wherein said reduced pressure

is -95 kPa gauge.

16. A transformation method according to Claim 4, wherein the duration of vacuum treatment is in the range of 15 to 20 minutes.

17. A transformation method according to Claim 13, wherein the pressure is rapidly

raised to atmospheric pressure or above.

18. A transformation method according to any one of the preceding claims, wherein

said reduced pressure comprises cyclically applying vacuum to said tissue.

19. A method of transforming plants including the steps of:

immersing plant tissue in a medium including an infective transformation vector;

reducing the pressure on said tissue; increasing said pressure to a pressure of at least 10 kPa above said reduced 22 regenerating said tissue.

20. A transformation method according to Claim 19, wherein said reduced pressure and a time of maintenance at said reduced pressure is selected to avoid hyperhydricity

of said tissue.

21. A transformation method according to Claim 20, wherein said over pressure is selected to provide sufficient pressure differential between said reduced pressure and said over pressure to promote infiltration.

22. A transformation method according to Claim 21 , wherein said pressure gradient between the reduced pressure and the over-pressure is in the range of 10 to 500 kPa.

23. A transformation method according to any one of claims 19 to 22, wherein said plant material is subjected to alternating cycles of said reduced and over pressures.

24. A method of transforming plants including the steps of: immersing plant tissue in a medium including an infective transformation vector;

increasing said pressure to a pressure of at least 10 kPa to 500 kPa above the

starting pressure to effect infiltration, and

regenerating said tissue.