WO2000005941A1

WO2000005941A1 - A method of transformation

Info

Publication number: WO2000005941A1
Application number: PCT/AU1999/000602
Authority: WO
Inventors: Dacheng Tian; Kirby Ronald Siemering; Robert Dixon Teasdale; Johannes Bernard Wehr
Original assignee: Aat (Stba) R & D Pty. Ltd.; Redline (Stba) R & D Pty. Ltd.; Inworth (Stba) R & D Pty. Ltd.; Flair (Stba) R & D Pty. Ltd.; Colbina (Stba) R & D Pty. Ltd.
Priority date: 1998-07-24
Filing date: 1999-07-26
Publication date: 2000-02-10
Also published as: AUPP486498A0

Abstract

Many gymnosperms, particularly Pinus radiata, are refractory to Agrobacterium tumefaciens-mediated transformation. By sensitising shoots using non-conventional stress treatments the plant's pathogen defence response may be limited thereby rendering the plant material more susceptible to Agrobacterium infection. An excised shoot or portion of a plant may be subjected to stresses such as by metabolic inhibitors, high pH, heat, metabolic inhibitors, cold, darkness or combination therof prior or subsequent to infection.

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 transformation for pines and other conifers and for illustrative purposes reference will be made to such application. However, it is to be understood that this invention could be used in the transformation of other plant species, especially for those species that may be recalcitrant to transformation.

Pinus radiata, one of the most commercially important woody species, is a species recalcitrantto Agrobacterium

transformation. Overcoming poor transformation rates and low levels of gene expression in P. radiata using Agrobacterium-meό eAedι transformation is a common challenge. This difficulty results from the ability of pines, including P. radiata, to resist Agrobacterium infection. Thus, a method to successfully generate transgenic P. radiata plants was limited and even achieving a high frequency of transient expression has been reported for only a small number of cases using Agrobacter/utrj-rnediated transformation in pine.

The occasional successful transformation event is generally possible, however, the remaining major challenge has been to identify the conditions for a genotype independent stable transformation protocol that can be applied reproducibly and which results in regenerated transformed plants.

US Patent No. 5,164,310 discloses a procedure for plant transformation that uses shoot apex tissue as the target tissue for gene transfer. Shoot apices are transformed by excision from selected plants, culturing and inoculating with Agrobacterium containing the appropriate DNA vector common to the art. The application of this method is restricted to monocotyledonous and dicotyledonous species. There is no disclosure of applying the method to gymnosperms in which the production of transformed tissue has proven so far to be limited. Also, excising the apex can be time consuming and tedious limiting the commercial application.

Jean Gould and Ronald Newton (Plant Genome III Meeting - January, 1995) developed a method for transforming of Loblolly pine shoots using Agrobacterium by direct transformation of the shoot apex. The approach was based on the assumption that the cells of the shoot apical meristem are transformation competent and allow for continued development of a transformed shoot-meristem. The method disclosed is restricted to excised shoot apices. The present invention aims to alleviate at least one of the foregoing disadvantages and to provide a method of transformation for plants, in particular for pines and conifers which will be reliable and efficient in use. Other objects and advantages of this invention will hereinafter become apparent.

This invention in one aspect resides broadly in a method of transformation of a plant including the steps of: excising a regenerable portion of said plant; stress treating said excised portion; and transforming said stress treated portion with a transformation vector.

The regenerable portion may be excised from a whole plant or portion thereof, tissue culture or clonally propagated plantlets. The regenerable portion may be an explant, shoot, stem, apical and lateral meristem tissue, axillary buds, or the like. It is preferred that the excised portion is a shoot or stem selected from plant tissue cultured from shoot apices and stems of clonally propagated plantlets. The prior art teaches that the shoot apex allows continued development of a transformed shoot-meristem into a transformed plant. It should be appreciated that the above method is not limited to apical meristem tissue as it has been unexpectedly determined that the procedures as described herein maximise the plant tissues' transformation efficiency.

In another aspect, this invention resides in a method of transformation of a plant including the steps of: culturing shoots from tissue of said plant; excising a shoot or portion thereof; stress treating said excised shoot, and transforming said stress treated shoot with a transformation vector. The plant tissue may be derived from a whole plant or portion thereof, seedling, tissue culture or clonally propagated plantlets. The shoots may be cultured from an explant, shoot, stem, apical or lateral meristem tissue, axillary buds, or the like of the plant tissue. For example, the shoots may be cultured from explants selected from propagated plantlets. It is preferred that the plant tissue selected to culture shoots are shoot apices or stems selected from propagated plantlets that have been grown on a culture medium including charcoal.

The cultured shoots may be cultured under conditions which render the shoot material more susceptible to infection by the transformation vector. It is to be appreciated that the terms "susceptible" or "receptive" are used in a relative sense as to a final result of higher transformation efficiency as compared to shoots that may be grown under typical culture conditions for a target plant species. The physiological state of the shoot material may be less resistant or more receptive to infection and may provide significantly higher levels of gene transfer and expression. The terms "susceptible" or "receptive" are not intended to limit the invention by defining any particular parameter/s. A person skilled in the art could determine if a given plant species was more susceptible or receptive to transformation and would be able to determine appropriate conditions to induce susceptibility or receptiveness to transformation.

Variations in growth media, light, temperature, humidity and density of growth may affect the susceptibility of the cultured shoot. For example, the growing conditions may include high humidity, low light intensity and low plant culture density. It is preferred that the culture medium includes charcoal. The charcoal concentration may be in the range of 0.03% - 0.3%, though preferably around 0.1 %.

Growth rate, firmness, colour, shape, and size of the shoot may indicate the suitability of the shoot. The selected shoot is preferably a healthy shoot, which may be relatively fast growing, soft, relatively large and elongated. It is expected that a person skilled in the art would be able to select a suitable shoot or portion thereof from their knowledge of the growth of tissue culture of the target plant species. Ideally, the needles of the shoot are cut off to allow improved access to the apical and lateral meristem.

The excised shoot may be treated with one or more physical or chemical stresses. Although not wishing to be bound by theory, it is believed that physically stressing the excised portion limits the plant's pathogen defence response thereby diminishing or weakening resistance to infection by the transformation vector.

The treatments may include one or more of a variety of stresses to place the excised shoot in an environment that is outside typical growth parameters. The stress treatment may include means selected from one or more of heat, cold, high pH, water treating, metabolic inhibitors, cobalt, nitrogen, sinapyl alcohol and any other chemicals which may limit the plant's pathogen defence response. Stress treatment may be conducted preceding or subsequent to transformation or a combination thereof. For example, stress treatments may include:

^■ The excised shoot may be subjected to high pH (pH 10-14) treatment. This may include subjecting the excised shoot at an appropriate pH not to cause deterioration of the tissue. For example, this may be achieved by washing the excised portion in about 1.5% to 5% KOH for no more than 5 mins. More suitably the alkali treatment is 2.5%

KOH. Alkali washing also has the advantage of assisting in erosion or removal the cuticle of the plant which may act as a barrier to infection during transformation.

^■ Immersion of the excised portion in water. ^■ Ensuring adequate nitrogen nutrition to minimise the production of resin which is used by the plant as a defence to pathogens. This may be achieved by determining the optimum tissue culture medium and subculture intervals so that the plants do not become nitrogen deficient.

^■ The excised shoot may be subjected to heat treatment in a range of above normal growing temperatures to an upper limit where the plant tissue becomes non-viable. For example, the heating range may be in the order of 25°C to the limiting viability temperature of the tissue. Preferably, the excised shoot is treated in the range of 30°C - 45°C. Most preferably, the excised shoot is treated at a temperature of 37°C to 40°C. It is expected that a person skilled in the art could determine the ideal heating temperature to maximise transformation efficiency for the target plant species. The heat treated shoot is then subjected to transformation. Alternatively or subsequently, the excised shoot may be treated during co-cultivation and transformation. For example, after alkali washing and application of the transformation vector, shoots may be placed in darkness at 25°C - 30°C for about 40 hours. ^■ Subjecting the shoot to low temperature (-20°C).

^■ Phenolics are produced by gymnosperms as a pathogen defence response. A plant may be more susceptible to infection by inhibiting the phenolics biosynthetic pathway. For example, O-benzylhydoxylamine (OBHA) is a phenylalanine ammonia lyase inhibitor. Shoots may be grown on Modified Lepoivre medium containing 0-benzylhydroxylamine (OBHA) pre- and post- infection.

^■ Gymnosperms also produce terpenoids as a response to pathogen infection. An example of an inhibitor of the terpenoid biosynthetic pathway is mevinolin (HMG CoA reductase inhibitor). Shoots may be grown on medium that includes mevinolin pre- and post- infection.

^■ Cobalt is an agent that reduces lignification, phenolics and ethylene synthesis, being an ACC oxidase and peroxidase inhibitor. Shoots may be grown on medium that includes cobalt pre- and post- infection with the vector. - A combination heat shock and hot alkali washing may be used to stress the plant. For example the excised shoot may be washed in solution with a pH of about 11 -14 at 25°C - 40°C for 5 min. The alkali wash may be 1.5% - 4.0% KOH, more suitably 2.5% KOH.

^■ Sinapyl alcohol may be incorporated into the cell wall, mimicking the presence of angiosperm lignin which may either be more easily degraded or more readily recognised by a transformation vector such as Agrobacterium.

^■ The infected excised shoots may be co-cultivated in the darkness which may minimise formation of phenolics and terpenoids. Depending on the stress treatment, the excised shoot may be washed in appropriate wetting agents prior or subsequent to stress treatment. Most plants have a waxy cuticle that retards loss of water from the plant body. Gymnosperms have a thick cuticle which may repel aqueous solutions and may act as a barrier to infection by the transformation vector. Wetting agents may assist in making aqueous solutions, such as those containing the transformation vector, to be attracted or attach to the plant surface. Wetting agents may include any suitable detergent well known in the art. Detergents may even remove some of the outer wax layers or the entire cuticle if the cuticle is not well developed on the shoot. For example, the detergents included in the products sold under the trade names Silwet or Tween 20 may be suitable. Alternatively, the wetting agent may be an alkali solution which may partially or wholly erode or remove the cuticle to allow improved delivery of the transformation vector. For example the alkali solution may be a solution with a pH of about 10-1 1 such as a 2.5% KOH solution.

The transformation vector may be selected from any known suitable system capable of transformation, such as bacterium, viral, or plasmid. Variation in the transformation vector is dependent on the target plant species and the characteristic to be transferred to the plant. Preferably, the vector is a suitable infective transformation vector such as Agrobacterium tumefaciens. Agrobacterium is the most common genus of bacterium used for bacterially- mediated transformation. 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 agents may be developed which might also be applicable.

Transformation may be achieved by any suitable method known in the art appropriate to the target plant species and transformation vector used. For example, suitable methods of Agrobacterium-med^'aXed transformation may include direct inoculation at a wound site, dropwise inoculation at the apical tip, placing a cut portion on or in medium containing Agrobacterium, immersion of the shoot in an Agrobacterium suspension, or vacuum infiltration. In one embodiment, the treated shoot may be wounded by slicing down the shoot apex and placing the cut side onto medium containing Agrobacterium. Excessive Agrobacterium may induce a hypersensitivity response in the plant. To reduce the defence response the shoot may be kept intact with only the needles trimmed to allow access of the inoculation media to the meristem tissue. Transformation may be performed by dropwise inoculation on the meristem.

The aforementioned method may be suitable for any plant species capable of regenerating from tissue culture or explants. In particular, this method has application to plants that are generally recalcitrant to Agrobacterium-medϊated transformation, such as pines and other conifers. It is preferred that the target plant species is Pinus radiata.

The difficulty in successfully transforming pines, including P. radiata, is due to reactions to Agrobacterium infection. Hypersensitivity of plants to Agrobacterium can result in tissue necrosis and death, precluding recovery of transformed cells.

In another aspect, this invention resides in a method of minimising the hypersensitivity response to infection in plant tissue following Agrobacterium- mediated transformation including culturing said transformed plant tissue in a medium containing charcoal.

It has been found that culturing the transformed plant tissue in a medium containing a high charcoal concentration may be important in the neutralisation of potentially toxic compounds produced by the plant tissue in response to treatments and infection. The transformed plant tissue may be cultured on media containing charcoal until shoot regeneration occurs.

Other conditions identified to minimise the hypersensitivity response include reducing damage due to wounding, lowering exposure during transformation (such as lessening time and contact area with Agrobacterium and lowering concentration of Agrobacterium), lowering the concentration of 1 - naphthylacetic acid (NAA) in the media and using only healthy plant material. It is preferred that all the aforementioned conditions are followed.

In another aspect, this invention resides in a method of obtaining a transgenic plant including the steps of: excising a regenerable portion of said plant; stress treating said excised portion; transforming said stress treated portion with a transformation vector, and regenerating said transformed portion. Culturing of the transformed plant tissue in a medium containing a high charcoal concentration may neutralise potential toxic compounds produced by the plant in response to said treatment and infection. Other conditions, as identified above, which minimise the hypersensitivity response, may further assist in regeneration of the transformed plant tissue.

The cuticle of a plant, especially if it is thick, may act as a physical barrier to infection repelling aqueous solutions.

In a further aspect, this invention resides in a method of transformation of a plant including the steps of: culturing shoots from tissue of said plant; excising a shoot or portion thereof; improving the wetting of said excised shoot, and transforming said stress treated shoot with a transformation vector.

Improved wetting of the excised shoot may be achieved by use of wetting agents. Wetting agents may assist in making aqueous solutions such as those containing the transformation vector attach or be more attracted to the plant.

Wetting agents may include any suitable detergent well known in the art.

Detergents may even remove some of the outer wax layers or the entire cuticle if the cuticle is not well developed on the shoot. For example, the detergents included in the products sold under the trade names Silwet or Tween 20 may be suitable. Alternatively, the wetting agent may be an alkali solution which may partially or wholly erode or remove the cuticle to allow improved delivery of the transformation vector. For example the alkali solution may be a solution with a pH of about 10-14 such as a 1.5% - 4.0% KOH solution. More suitably, the alkali solution is 2.5% KOH. It is to be appreciated that subjecting an excised shoot to wetting agents may place the plant under stress as the cell wall and cell wall pH may be modified.

The excised shoot may also be stress treated with one or more of the above stress treatments as described above, preceding or subsequent to transformation.

In order that this invention may be more readily understood and put into practical effect, reference will now be made to the following example and accompanying drawing which illustrate a preferred embodiment of the invention, and wherein:

FIG. 1 illustrates transient GUS expression in Pinus radiata shoot apices five (5) days after infection with Agrobacterium. The two shoots on the left are untreated controls. The boxed area shows the meristem regions of one shoot;

FIG. 2 illustrates stable GUS expression in the shoot apex 35 days after infection, and

FIG 3 illustrates stable GUS expression in the shoot apex 35 days after infection. A transformed axillary bud is indicated by the larger arrow. The strategy in the following examples overcomes poor transformation rates and low transient gene expression in P. radiata by improving delivery of

Agrobacterium and sensitising shoots using non-conventional stress treatments that render the plant material more susceptible to Agrobacterium infection by limiting the plant's pathogen defence response. These treatments largely increased transient gene expression and expression was observed in meristem tissue or the shoot apex as illustrated in FIGS 1 -3 by the darker areas indicated by the smaller arrows. This success has paved the way for the production of stable transformed axillary tissues and shoots. Axillary buds may be cultured under selection pressures such as geneticin resulting in the generation of transgenic P. radiata plants. This system may be a genotype-independent method for the transformation of elite clonal material.

Hypersensitivity of plants in response to Agrobacterium infection results in tissue necrosis. A majority, if not all, of the shoots several weeks post-inoculation with Agrobacterium die or exhibit necrosis in the wounding site even without selection pressure (i.e. without antibiotics added to the medium), which precludes recovery of positively transformed cells. Hence, it is important to minimise hypersensitivity to recover the transformed cells for the production of transgenic plants. To minimise the hypersensitivity response, the following conditions were identified: ^• reduced damage during wounding (i.e. more suitably no wounding except for trimming needles to allow access to apical and lateral meristem, no vacuum infiltration, except for particular clones), • lower pressure during inoculation of particular clones (shorter time, lower concentration of Agrobacterium, and less area of shoots in contact with Agrobacterium) ,

• low concentration of NAA in the medium for a short time then recovery of the culture in a charcoal containing medium,

• ideally use of healthy plant material.

EXAMPLE 1

PRODUCTION OF SUSCEPTIBLE SHOOTS The culturing of young and strong explants

The shoots were cultured using the standard propagation protocols, Modified Lepoivre medium (Genetic Manipulation of Woody Plants, JW Hanover and D E Keithley, Plenum 1988 pg 413-432) with 0.05% charcoal in ForBio VITRON™ robotic tissue culture containers under light intensity of 70-80 μmol.m^" ².s ^~1 at 23° C for eight weeks. Selection of explants

The top two to three cm of young and strong stems without shoot apex or 1.5 - 2.0 cm good shoot apexes were used as explants. Culture of susceptible shoots

The explants were cultured on Modified Lepoivre or P24 (tissue culture medium as disclosed in AU Patent No. 660432) agar medium with a high charcoal concentration (0.1 %), low light intensity (30-40 μmol.m^"2.s^"1), low plant culture density and high humidity in 8 cm pots with 0-2 mg/l benzylaminopurine (BA) for four to eight weeks. Charcoal is important for production of susceptible shoots. Selection of transformation plant material

Fast growing, soft, yellowish, elongated (not tufted), healthy and relatively big shoots were selected for transformation. Good starting material is the most important factor for higher GUS expression. SUSCEPTIBILITY TREATMENT OF SHOOTS FOR AGROBACTERIUM INFECTION

One challenge of P. radiata transformation by Agrobacterium is the resistance of P. radiata to infection. The following treatments overcoming this challenge include the steps of:

Stress treatment

The shoots were heated at 37° C for about 20 hours in small pots (with a few drops of sterile water to keep moist). This has been shown to be an important factor to induce susceptible shoots for infection of Agrobacterium.

Shown in table 1 , the percentage of GUS stained shoots increased 3.8 fold, and the average number of stains per shoot increased 5.6 fold.

Table 1

Wash

The shoots were then washed with 0.01% Silwet (10 μl/100ml) or 0.005% Tween 20 solution for 5 minutes, and then rinsed once or twice with sterile water. Wounding

The shoot was sliced down through the middle of shoot apex, following removal of the top part of the needles. The shoots were kept wet at all times during wounding (with even minimal water loss GUS expression decreases dramatically). Vacuum infiltration

Vacuum infiltration is not favourable for the recovery of Agrobacterium infected shoots when the plant material is of good health. However, vacuum infiltration of the shoots at 100 kPa for 30 seconds to 2 minutes with Agrobacterium is generally beneficial for infection of less healthy clones and may be applied when the desired starting material is limited.

INDUCTION OF AGROBACTERIUM Stock plate establishment According to strain and construct, the stock plate was subcultured under selection on yeast extract peptone media (YEP) medium every one to two months.

Work plate establishment

Mixed colonies were picked from the stock plate onto YEP agar selection medium and cultured for one to two days, and subcultured on the same agar medium overnight. All of the Agrobacterium on the plate was harvested by suspending the cultures in YEP liquid medium without antibiotics and transferred to a flask.

Virulence induction After three to four hours culturing at 28° C, the Agrobacterium suspension was diluted to 2 x10⁸ in Modified Lepoivre medium and acetosyringone was added at the concentration of 50 μg/l. Following one to two hours, the

Agrobacterium was ready for inoculation and co-cultivation.

INOCULATION AND CO-CULTIVATION Inoculation

Inoculation comprised placing the cutting side of the susceptible shoots, as described above, on an agar plate containing Agrobacterium. This agar plate was prepared by pouring liquid YEP onto the work plate and removing all of the Agrobacterium and most liquid medium. The plate was incubated containing the remaining Agrobacterium for two to four hours at 28°C, and diluted liquid Agrobacterium medium was applied, prepared as above for virulence induction and containing acetosyringone, to the plate, forming a very thin layer. The susceptible shoot was placed onto the plate for 10-20 minutes without vacuum at 28°C.

Alternatively, shoots can be inoculated for 10-20 minutes with vacuum infiltration or 20-40 minutes without vacuum in Agrobacterium containing medium, prepared as above for virulence induction. Following inoculation, the plant material was blotted using sterile paper. Co-cultivation

The shoots were inserted vertically for improved recovery, especially in the case of using vacuum infiltration, into Modified Lepoivre medium containing 5mg/l BA and 0.2 mg/l NAA for 3-5 days in the dark.

CULTURING AND SELECTION CONDITIONS Recovery and activation The shoots were transferred to Modified Lepoivre medium with 5 mg/l BA,

5 mg/l geneticin, and 150 mg/l timentin for two to three weeks to activate meristem tissue into growth. The shoots were recovered on the charcoal medium (0.05% charcoal, 10 mg/l geneticin and 200 mg/l timentin) for two to four weeks. Selection and regeneration The survivors were activated again in medium with 5 mg/l BA, 10 mg/l geneticin and 150 mg/l timentin for three to four weeks, or cultured directly in charcoal medium without BA (0.05% charcoal, 15-20 mg/l geneticin and 200 mg/l timentin) until shoot regeneration occurred.

EXAMPLE 2

Agrobacterium tumefaciens AGL1 containing a DNA vector including the marker GUS intron was grown in YEP broth overnight at 30°C on an orbital shaker using a single colony picked off YEP plate. Acetosyringone was added to a final concentration of 50 μM in the YEP broth. The optical density (at 600 nm) was adjusted to 0.4-0.42. Shoots of P. radiata (several clones) were grown on Modified Lepoivre medium with 0.1 % charcoal. The plants were grown at 30°C and a light intensity of 30-40 μmole/m2/s with cool-white (fluorescent tubes) and incandescent (25W light bulbs) light. The shoot tips were harvested (approx. 1 cm long) and needles trimmed to expose the meristem. The shoots were washed with 2.5% KOH for a maximum of 5 min, then rinsed extensively (3-5 times) with sterile water and blotted dry. The glassy part of the shoot base was re-cut and the apical needles cut back to expose the shoot meristematic region. The prepared shoots (2-5 mm long) were transferred to fresh Modified

Lepoivre medium with 0.1 % charcoal and 2 μl of the Agrobacterium was applied to the meristem dropwise. Containers with shoots were placed for 40 hours in darkness at 23°C.

After the co-cultivation, the shoots were removed from the medium and placed in a vial with 150 mg/l Timentin or sterile water and shaken. The solution was replaced several times and the shoots blotted dry. The shoots were transferred to Modified Lepoivre medium containing 0.1 % charcoal and 200 mg/l Timentin. The containers with shoots were placed in 30-40 μmole/m²/s light intensity (approximately one cool-white:one incandescent light) at 30°C. It will of course be realised that while the foregoing has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to person skilled in the art are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims

CLAIMS:

1. A method of transformation of a plant including the steps of: excising a regenerable portion of said plant; stress treating said excised portion; and transforming said stress treated portion with a transformation vector.

2. A method of transformation of a plant according to claim 1 , wherein the regenerable portion is excised from a whole plant or portion thereof, tissue culture or clonally propagated plantlets.

3. A method of transformation of a plant according to claim 1 or claim 2, wherein the excised portion is an explant, a shoot, a stem, apical and lateral meristem tissue or an axillary bud.

4. A method of transformation of a plant according to any one of the preceding claims, wherein the excised portion is a shoot or stem selected from plant tissue cultured from shoot apices and stems of clonally propagated plantlets.

5. A method of transformation of a plant including the steps of: culturing shoots from tissue of said plant; excising a shoot or portion thereof; stress treating said excised shoot, and transforming said stress treated shoot with a transformation vector.

6. A method of transformation of a plant according to claim 5, wherein the cultured shoots are cultured under conditions which render the shoot material more susceptible to infection by the transformation vector.

7. A method of transformation of a plant according to claim 6, wherein the culture conditions include high humidity, low light intensity and low plant culture density.

8. A method of transformation of a plant according to any of the claims 5 to 7, wherein the cultured shoots are cultured in a medium including charcoal.

9. A method of transformation of a plant according to claim 8, wherein the charcoal concentration may be in the range of 0.03% - 0.3%.

10. A method of transformation of a plant according to claim 8 or claim 9 wherein the charcoal concentration is about 0.1 %.

1 1 . A method of transformation of a plant according to any one of claims 5 to 10, wherein the needles of the excised shoot are cut off.

12. A method of transformation of a plant according to any one of the preceding claims, wherein the stress treatment is performed preceding and/or subsequent to transformation.

13. A method of transformation of a plant according to any one of the preceding claims, wherein the stress treating includes means selected from one or more of heat, cold, high pH, water treating, metabolic inhibitors, cobalt, nitrogen, sinapyl alcohol and any chemicals which may limit the plant's pathogen defence response.

14. A method of transformation of a plant according to claim 13, wherein the excised shoot is washed in an alkali solution with a pH of 10-14.

15. A method of transformation of a plant according to claim 14, wherein the alkali solution is 1.5% -5% KOH.

16. A method of transformation of a plant according to claim 14, wherein the alkali solution is 2.5% KOH.

17. A method of transformation of a plant according any one of claims 14 to 16 wherein the excised shoot is washed no more than 5 minutes.

18. A method of transformation of a plant according to any one of claims 14 to 17, wherein the temperature of the alkali solution is 23┬░C - 40┬░C.

19. A method of transformation of a plant according to claim 13, wherein the excised shoot is immersed in water.

20. A method of transformation of a plant according to claim 13, wherein the excised shoot is heated in a range of above normal growing temperature to an upper limit where the plant tissue becomes non-viable.

21. A method of transformation of a plant according to claim 20, wherein the shoot is treated at a temperature of about 30┬░C - 45┬░C.

22. A method of transformation of a plant according to claim 21 , wherein the excised shoot is treated at a temperature of 37┬░C - 40┬░C.

23. A method of transformation of a plant according to claim 13, wherein the excised shoot is treated at temperatures of 23┬░C - 40┬░C during co-cultivation.

24. A method of transformation of a plant according to claim 13, wherein the excised shoot is treated at temperatures of about -20┬░C.

25. A method of transformation of a plant according to claim 13, wherein the cultured shoots or excised shoots are grown on media including one or more of O-benzylhydoxylamine, mevinolin or cobalt.

26. A method of transformation of a plant according to claim 13, wherein the excised shoot is subjected to sinapyl alcohol.

27. A method of transformation of a plant according to claim 13, wherein the excised shoot is washed in wetting agents prior or subsequent to stress treatment.

28. A method of transformation of a plant according to claim 27, wherein the wetting agent is a detergent or an alkali wash.

29. A method of transformation of a plant according to claim 28, wherein the detergent is Silwet or Tween 20.

30. A method of transformation of a plant according to claim 28, wherein the alkali wash is a 2.5% KOH solution.

31. A method of transformation of a plant according to any one of the preceding claims, wherein the transformation vector is selected from bacterium, viral, or plasmid.

32. A method of transformation of a plant according to claim 31 , wherein the transformation vector is Agrobacterium tumefaciens.

33. A method of transformation of a plant according to any one of the preceding claims, wherein transformation is performed by direct inoculation at a wound site, dropwise inoculation at the apical tip, placing a cut portion on or in medium containing the transformation vector, immersion of the shoot in a suspension of the transformation vector, or vacuum infiltration of the excised portion or shoot.

34. A method of transformation of a plant according to claim 33, wherein the transformed shoots are co-cultivated in darkness.

33. A method of transformation of a plant according to claim 32 wherein the transformed shoot is at a temperature of 25┬░C - 30┬░C for about 40 hours.

34. A method of transformation of a plant according to any one of the preceding claims wherein the transformed shoot or portion is cultured in a medium containing charcoal.

35. A method of transformation of a plant according to any one of the preceding claims, wherein the plant is a gymnosperm.

36. A method of transformation of a plant according to claim 35, wherein the plant is Pinus radiata.

37. A method of obtaining a transgenic plant including the steps of: excising a regenerable portion of said plant; stress treating said excised portion; transforming said stress treated portion with a transformation vector, and regenerating said transformed portion.

38. A method of obtaining a transgenic plant, wherein the transformed portion is cultured in a medium charcoal.