WO1997025434A1

WO1997025434A1 - Genetic transformation of trees

Info

Publication number: WO1997025434A1
Application number: PCT/GB1997/000042
Authority: WO
Inventors: Martin Jack Maunders; Peter Richardson
Original assignee: Advanced Technologies (Cambridge) Limited
Priority date: 1996-01-13
Filing date: 1997-01-09
Publication date: 1997-07-17
Also published as: GB9600698D0; JP2000517162A; OA10709A; AR005443A1; AP9801292A0; CN1213404A; UY24435A1; EP0880592A2; BR9706942A; AU1388397A; NZ325384A; AU720610B2; ZA97192B

Abstract

The invention is an improvement in the transformation of trees and provides a method which is less stressful to the tree being transformed. Notoriously difficult trees such as Eucalyptus globulus can be successfully transformed. An incision is made into a site capable of shoot initiation, disarmed Agrobacterium cells carrying one or more exogenous DNA sequences are introduced into the incision and the site is subjected to a selection step for a time period sufficient to produce transformed shoots.

Description

flanetie Trans orm tion of Trees

This invention relates to the transformation of tree species by the introduction of alien genes, i.e. genes from a source other than the specific tree itself.

The introduction of novel genes into plant species has been discussed at length in the literature, and has become routine for a number of annual and perennial crops including tomato, potato, oilseed rape and rice. Introduced genes have been used to modify a wide variety of specific and general characteristics, including, for example, protein and carbohydrate quality, postharvest physiology, photosynthetic capability and pest resistance, expressed both in the whole plant and in specific plant parts. Depending upon the nature of the plant species, various methods of introduction of the novel genes have been developed, including both direct DNA transfer (microinjection, biolistics, microfibrils, chemical poration, electroporation) and quasi-natural infection by the micro-organisms Agrobacterium tumefaciens and Agrobacterium rhizogenes .

To date genetic transformation of forestry species has been largely limited to the use of marker genes, although some examples have introduced more commercially relevant traits including genes affecting insect resistance, herbicide tolerance, wound responses and lignification.

The genetic transformation of tree species has presented special problems (Jouanin et al 1993) , one of which is the recalcitrant nature of the tissue itself limiting the susceptibility to transformation. Many trees show poor growth in vi tro , including low rates of multiplication of in vitro shoot systems, and low shoot and root regenerability. In addition, trees usually show a wider range of in vitro behaviour according to individual genotype than do cultivated annual field crops. Furthermore, there is large inter- and intra-specific variation in susceptibility to genetic transformation, and some trees show marked decreasing susceptibility with increasing physiological age. Transient introduction of the novel DNA into the plant cell can be achieved, but without stable integration of the DNA into the plant genome, expression of the introduced genes is short¬ lived. A further problem is the difficulty in maintaining and propagating tissue in culture, prior to transformation, during selection of transformed cells and during regeneration of transformed whole plants. Tissue health and survival also has implications for the transformation and integration events described above and are relevant to an object of this invention.

The most commonly employed method of obtaining stable transformation is by means of modified strains of Agrobacterium. Many tree species are susceptible to infection by wild type Agrobacterium tumefaciens but stable transformation with additional genes has been achieved in only a few species, e.g. Brackpool et al 1990; DeBlock, 1990 and McGranahan et al , 1990. Success has been restricted to certain genotypes of these species, mainly those genotypes which are easily transformed and propagated. The methods employed generally entail a series of steps such as: i) excision of target explants from the plant tissue, e.g. leaf discs, root fragments, stem sections, hypocotyls, cotyledons, embryos or cell suspensions, ii) pre-culture of the explant on suitable media for a period of hours or days, iii) co-cultivation of the explants with bacterial cells containing the DNA vector to permit infection via the wounded plant cells, iv) transfer to a selective medium to (a) arrest the growth of residual bacterial cells, and (b) select those plant cells which have been transformed, and v) one or more transfers to media to initiate regeneration of complete plantlets from the explant- derived callus. The basic methodology involves subjecting the tissue explants to physical damage caused by the excision, to co- incubation with bacteria, and to chemical selection agents, each of which steps exerts severe stress on the transformed cells and tissue pieces, usually adversely affecting the powers of shoot regenerability possessed by the tissue. The effect of these treatments would be diminished if the necessity of using isolated tissue explants, which are therefore compromised in terms of health, survivability, and regenerative capacity, were removed.

In tree crops in particular, the subsequent sexual or asexual propagation of improved germplasm is an established practice, and this propagation ability is an important characteristic to be maintained in improved material for the purposes of clonal forestry. The process of producing a transformed plant should not preclude subsequent propagation by any method by which the species which has been transformed is otherwise normally propagated.

It is an object of the present invention to provide a transformation method for trees which causes minimal stress level on the plant tissue and transformed cells.

It is also an object of the present invention to derive a transformation method for trees, which method is carried out on plant tissue which has a higher likelihood of survival and good regenerative capability, which properties help to ensure that the propagation capability of the transformed or improved germplasm is maintained.

It is a further object to target a zone of predetermined shoot initiation ability to maximise the likelihood of regeneration of shoots containing transformed cells.

This invention provides a means for the introduction of any DNA sequence into trees. More specifically, the method can utilise those genes or DNA sequences capable of affecting phenotypic characteristics, for example silvicultural traits, processing quality traits in the timber, fruit or leaves, or the reproductive phenology of the mature tree. Thus, the method will enable both the production of improved trees by clonal propagation and better control of the outcome of sexual propagation in seed orchards. Offspring of the transformed trees containing the novel genes can also be subjected to further transformation, if desired, to produce incremental improvement. A method has been described for the transformation of conifers by Ellis et al , 1992 which comprised introducing wild type Agrobacterium rhizogenes into a cut in the cotyledonary node of seedlings of Larix decidua . However, this method produced only a tumorous mass of shoot primordia, and regeneration of complete transformed plants was not achieved.

The present invention overcomes the problem of tumorous growth and provides a method suitable for the regeneration of transformed plants from at .least one other tree species which has hitherto been difficult to transform and for which there has been no published report of complete transformed plants.

The present invention provides a method of transformation of trees, rooted or otherwise, the method comprising the steps of providing an incision into a site capable of shoot initiation, introducing disarmed Agrobacterium cells carrying one or more exogenous DNA sequences into the incision, and a selection step in which the site is brought into contact with a medium containing a nutrient, a growth regulator and a selection agent to which the transformed cells will be resistant, for a time period sufficient to produce transformed shoots containing the exogenous DNA sequences.

Preferably the incision forms a cleft in the site of shoot initiation. The incision may be about 1mm deep or more, depending on the size of the tree being treated. As used herein the term 'tree¹ includes seedlings, shoots (derived from cuttings or otherwise) and cuttings, or larger plants thereof.

The site of shoot initiation may suitably be a cotyledonary node, an axillary meristem or an apical meristem. When the tree to be transformed is a seedling, preferably the site of shoot initiation is a cotyledonary node. Preferably the method for transforming tree seedlings further includes the step of removing all pre-existing shoot initials, before, during or after the introduction of the Agrobacterium cells. This introduction may be known as the inoculation step.

When the tree to be transformed is a tree shoot preferably the site of shoot initiation is an axillary meristem or an apical meristem. Removal of pre-existing meristematic cells before, during or after the inoculation step may not be required in this particular method.

Preferably the site of shoot initiation is brought into contact with a medium containing a nutrient and a growth regulator for a period of time before the selection step. This is known as a co-incubation step. When the site of shoot initiation is found . in a seedling, preferably the co- incubation step takes place with an upright seedling. Suitably the co-incubation step takes place in a test-tube.

Preferably the medium of the selection step also comprises an antibiotic to kill the Agrobacterium strain.

Preferably the tree exhibits epigeal plant germination. More preferably the method is applicable to the genera Eucalyptus, Populus, Maluε and Prunus , for example, and any one of tree species which is susceptible to infection by Agrobacterium tumefaciens or Agrobacterium rhizogenes . This method is particularly useful for transforming certain species of the Eucalyptus genus, for example Eucalyptus globulus , which have hitherto been notoriously difficult to successfully transform and then produce transformed plants from.

Preferably the trees have an intact root system when they are infected and/or co-incubated.

Preferably the tree species is Eucalyptus globulus or Eucalyptus grandis .

The present invention further provides a tree transformed according to the method described above.

The present invention even further provides a cell which harbours a gene transformed according to the above method. The invention also provides propagules of a tree transformed according to the method, seedlings of a tree transformed according to the method, seeds of a tree transformed according to the method, and germplasm of a tree transformed according to the method.

In order that the invention may be easily understood and readily carried into effect reference will now be made, by way of example, to the following Example.

The transformation of Eucalyptus globulus seedlings was effected by the following procedure:

Exam^ple l. Preparation of Eucalyptus σlobuluε seedlings for transformation.

E . globulus seed was surface-sterilised using a solution of household bleach containing sodium hypochlorite and detergent. The concentration and length of time of sterilisation was not critical. Normally seed was incubated in a 10% v/v dilution of household bleach in sterile reverse-osmosis water, with continuous agitation on an orbital shaker. The sterilising solution was renewed after 30 minutes and a second incubation of 60 minutes was performed. During the first incubation period, the bleach solution often became very dark in colour. Finally, the seed was shaken in clean sterile water, with as many repeated washes as necessary to remove the smell of chlorine and the foaming of residual detergent.

Sterilised seed was sown by laying the seed on the surface of sterile 0.4% water agar medium containing 5g/l sucrose in Magenta culture vessels. The seed was sown well- spaced on the surface of the medium, at a density of 25 seed per culture vessel. The culture vessels were placed in a growth cabinet at 20°C under low light intensity with a 16- hour day-length, and the seed allowed to germinate and grow for 10 days. At this stage the majority of the seedlings should have reached the stage where the cotyledons are fully unfurled and the plumule is just visible between them.

The seedlings are of an age such that they are able to provide a robust environment in which the transformed cells will survive and proliferate. The seedlings are also of a sufficient size to permit relatively easy manipulation.

Example 2. Preparation of Agrobacterium tumefacien cells or co-cultivation

Co-cultivation was performed using Agrobacterium tumefaciens strain EHA105 carrying the plasmid p35S-GUSint which encodes the β-glucuronidase (GUS) gene and also the NPTII gene conferring resistance to the antibiotic kanamycin. Transformation of the E. globulus tissue with this plasmid vector would result in the expression of these two marker genes in the plant cells, thus enabling the selection of tissue comprising solely or predominantly transformed cells as exhibited by healthy growth in the presence of kanamycin, and subsequent identification of those particular transformed cells by the reaction catalysed by β-glucuronidase on addition of the chromogenic substrate x-gluc (5-bromo-4-chloro-3- indolyl-β-D-glucuronide) . The cells were prepared by inoculation of 50ml of L-broth medium containing 50mg/l kanamycin with a stock culture of the bacterium. After overnight growth at 28°C the cells were collected by centrifugation and resuspended in 10ml of 1% glucose prior to use.

L-broth lOg/1 bactotryptone

5g/l yeast extract

5g/l sodium chloride

Example 3. Co-cultivation of .g.σloJuJus and Aσrobacteri um tumefaciens

The germinated seedlings prepared by the method of Example 1 were sampled under sterile conditions for co- cultivation with the bacterium harbouring the transformation vector. To determine the optimal method of co-cultivation for transformation of E . globulus three different methods of infection with the AgroJacterium tumefaciens cells prepared by the method of Example 2 were assessed.

Method l. Seedlings were cut approximately halfway along the hypocotyl and the lower halves plus the roots were discarded. The plumules were excised using a scalpel tip, and the petiole blades were removed. The excised hypocotyls were then laid horizontally on solid Medium 1 in a petri dish. The tissue was inoculated with A. tumefaciens by dripping the bacterial suspension onto the horizontal hypocotyl from a disposable pipette.

Method 2. Seedlings were cut approximately halfway along the hypocotyl and the lower halves plus the roots were discarded. The plumules were excised using a scalpel tip. Then a cleft 1mm deep was cut with a scalpel longitudinally through the cotyledonary node into the top of each hypocotyl. Inoculation with the A . tumefaciens cells was achieved simultaneously by immersing the scalpel blade in the bacterial suspension immediately prior to performing each incision. The inoculated hypocotyls were then stood erect by inserting the base of each into solid Medium 1 in a petri dish. Twenty (20) hypocotyls were so arranged per petri dish.

Method 3. Plumules were excised from seedlings using a scalpel tip, but the seedlings were left otherwise intact with complete root systems. A cleft 1mm deep was cut with a scalpel longitudinally through the cotyledonary node into the top of each hypocotyl. Inoculation with the A. umefaciens cells was achieved simultaneously by immersing the scalpel blade in the bacterial suspension immediately prior to performing each incision. The inoculated deplumuled seedlings were transferred into individual 25mm diameter test-tubes and stood erect with their root systems inserted into 20ml of Medium

2.

One hundred (100) seedlings were prepared by Methods 1 and 3 and two hundred seedlings (200) were prepared by Method

2.

Co-cultivation was continued for 10 days at 24°C under low light intensity with a 16-hour daylength. After this period, the explants prepared by Methods 2 and 3 were removed from their respective culture vessels or test-tubes and received further manipulation.

The laminae of the cotyledons were excised from the hypocotyls prepared by Method 2. The laminae of the cotyledons, roots and lower half of the hypocotyl were removed from the seedlings prepared by Method 3.

Any axillary shoots which had appeared on any of the explants prepared by either of the three treatments were also removed. The truncated hypocotyls from all three methods were then lain horizontally on Selection Medium 1 containing the antibiotics claforan (cefotaxime) to remove residual

A . tumefaciens cells and kanamycin to select for transformed plant cells expressing the NPTII gene. Twenty (20) excised hypocotyls were arranged per petri dish. These were then transferred to Selection Medium 2 after 10 days, and onto fresh plates of the same medium after a further 4 weeks.

Incubation was performed at 24°C under low light intensity with a 16-hour daylength. After a further 4 weeks, those explants exhibiting green

(unbleached) regions of tissue were transferred to new plates of the same medium. Selected tissue was maintained by further transfers to fresh plates at regular intervals.

Results

Treatment 1 2 3

Hypocotyls 100 200 100

Surviving at 8 weeks 0 9 7

Surviving at 15 weeks 0 4 4

Surviving at 22 weeks 0 3 1

Established Regenerants 0 1 1

Low numbers of regenerants were expected from the transformation of Eucalyptus globulus . No regenerants were obtained _from excised hypocotyls co-cultivated horizontally in contact with the culture medium (Method 1) , and the tissue rapidly declined in health. Regenerants were obtained with both Methods 2 and 3. The decline in survival with time probably indicates a number of regeneration loci formed by untransformed cells which subsequently succumbed to kanamycin selection. The efficiency of regeneration from tissues prepared and co-cultivated using Method 2 was slightly lower than that of tissues prepared by Method 3.

Method ₃ was characterised by minimal but precise wounding of the tissue, maintenance of the vascular system between root and hypocotyl, minimal direct contact between the hypocotyl and the culture medium containing exogenous growth substances, and precise inoculation of the bacterial cells, minimising bacterial overgrowth. Method 3 was selected as the method of choice for transformation of E. globulus tissues. Example 4. Modifications of transformation method and expression of transσenes

■E.grlojbulus seedlings and the A . tumefaciens suspension were prepared as in Examples 1 and 2. Three modifications of the optimal inoculation method of Example 3 were assessed as follows:

1. Decapitated and cleft (i.e. entire cotyledonary node and cotyledons removed) .

2. Deplumuled and cleft (as Example 3, Method 3).

3. Deplumuled and scored (Hypocotyl surface scored along a portion of its length with a scalpel blade) .

In addition, three further modifications were also investigated:

4. Deplumuled and stabbed with a hypodermic needle.

5. Deplumuled, scored and crushed at the apex using forceps.

6. Deplumuled and crushed.

Following inoculation, all seedlings were transplanted to individual tubes containing Medium 3, and placed on an unlit shelf in the growth room to co-cultivate.

After 3 weeks, seedlings from the three main treatments were sampled for β-glucuronidase activity (GUS+) by x-gluc assay as follows. 3mg x-gluc dissolved in lOOμl dimethyl fornamide was made up to 10ml with 50mM sodium phosphate buffer (pH 7.0) containing 0.5% Triton XI00 and lmM EDTA. The sample tissue was incubated in a small volume of this reagent at 37° for 16 hours in darkness. Subsequently the tissue was decolourised by incubation in isopropanol at room temperature for 4 hours to several days. β-glucuronidase activity was detected by the presence of blue-stained regions in the tissue.

Treatment 1 2 3 4 5 6

No. 120 200 200 40 40 40

Surviving at 3 68 123 88 10 0 2 weeks (57%) (62%) (44%) (25%) (0%) (5%)

GUS+ (3wk) 8/12 10/11 21/31

(67%) (91%) (68%)

An x-gluc assay was also performed on seedlings sampled after only one week. This indicated GUS activity in most samples selected from all six methods.

The method of choice established in Example 3, which in this instance is Treatment 2, gives both the highest survival rate, and the greatest, or most persistent, transformation response. This is indicative of stable, rather than transient, expression of the transgenes.

In all cases the major area of blue staining presumably demarking transformed cells were located around the cleft. With Treatment 1, this took the form of discreet "speckles" of isolated cells, whereas in Treatment 2 there was a greater predominance . of larger heavily stained blue areas, suggesting that the regeneration of shoots from these areas would have resulted in transformed plants. The transformation of scored hypocotyls was generally much poorer than in those where an apical cleft had been made.

As a further comparison, a number of refinements were also made to the excised hypocotyl transformation method

(Example 3, Method 1). These included scoring the hypocotyl with a scalpel to produce varying numbers of wounds of various lengths, and also puncturing the hypocotyl tissue with a sterile wire brush. None of these treatments resulted in successful regeneration of transformants.

Example 5. Regeneration of Shoots expressing transqenes

One hundred and twenty (120) seedlings were prepared and co- cultivated as described in Example 3. At the end of the co- cultivation period, sixteen (16) hypocotyls were selected at random for x-gluc assay. The remaining hypocotyls were prepared for selection and regeneration. The lower half of the hypocotyl plus the root and the laminae of the cotyledons were removed along with any cotyledonary axillaries which had sprouted. The remaining hypocotyl sections were then plated horizontally on plates of Selection Medium 1 at a density of 10 per plate. Plates were sealed in clingfilm and placed on an unlit shelf in the growth room. After 4 weeks the hypocotyls were transferred to fresh plates of the same medium, and were examined for growth of green callus. Any green calli were then transferred to Selection Medium 3, and those which appeared to be close to differentiating shoots were placed on Selection Medium 4. Of sixteen (16) hypocotyls sampled for x-gluc assay, all but one exhibited blue-staining areas.

After l week, all hypocotyl explants appeared totally bleached and no cotyledonary axillaries were observed. After 4 weeks on selective medium, eight (8) of the one hundred (100) hypocotyls exhibited callus which was of a very dark green and healthy appearance, and a further ten (10) had mid- green coloured callus. Thirteen (13) calli were transferred to Selection Medium 3 and five (5) to Selection Medium 4.

After transfer to Selection Medium 4, four (4) calli regenerated shoots. After a further 6 weeks these were shown to be GUS+ by x-gluc assay.

The invention places no restriction on the DNA sequence or combination of sequences employed and so it can be applied to introduce any range of characteristics, as mentioned earlier. The invention can also be applied to material which has already been transformed in the same or earlier generations.

In another alternative to the seedling method above a shoot initiation site on a tree shoot in in vitro culture, such as an axillary meristem or apical meristem, can be treated by incising the meristem area in the same way and infecting with the genetically modified Agrobacterium strain. After a period of time the transformed cells can be removed as explants and cultivated to produce transformed plantlets. References

Brackpool A.L., Ward M.R. & Weir A.F. (1990) "Optimisation of conditions for regeneration and transformation of adventitious shoots from seedling hypocotyls of Eucalypts . " 7th

International Congress of Plant Tissue and Cell Culture, Amsterdam, 24-26 June, 1990. Poster Al-23

DeBlock M. (1990) "Factors influencing the tissue culture and the Agrobacterium tumefaciens mediated transformation of hybrid aspen and poplar clones." Plant Physiol. 93 1110-1116.

Ellis D.D., Diner A.M. & Huang Y. (1992) "Regeneration of the genetically engineered conifer - the importance of the biological system." Southern Regional Information Exchange Group Biennial Symposium, Huntsville, AL, 8-10 July, 1992

Jouanin L. , Brasileiro A.CM., Leple J.C., Pilate G. & Cornu

D. (1993) "Genetic transformation: a short review of methods and their applications, results and perspectives for forest trees." Ann. Sci. For. 50 325-336.

McGranahan G.H., Leslie CA. , Uratsu S.L. & Dandekar A.M. (1990) "Improved efficiency of the walnut somatic embryo gene transfer system." Plant Cell. Rep. 8 512-516.

Linsmaier E.M. & Skoog F. (1965) "Organic growth factor requirements of tobacco tissue cultures." Physiol Plant. 18 100-127 APPENDIX

Culture Media Used - All based upon the medium of Linsmaier and Skoog (1965) at full strength, with the exception of Medium 2 which used half-strength medium and Medium 3 which used quarter-strength medium. Media contained the following additions:

Medium 1 15 g/1 glucose

10 μM zeatin

5 μM indolyl-3-acetic acid

0.01% pluronic F-68

4 g/i agar

Medium 2 10 g/1 sucrose

4 g/1 agar Medium 3 10 g/1 sucrose

4 g/i agar i g/i activated charcoal

Selection Medium 1 is g/i glucose

10 μM zeatin

5 μM indolyl-3-acetic acid

250 mg/1 claforan

30 mg/1 kanamycin

7 g/1 agar

Selection Medium 2 15 g/1 glucose

4 μM zeatin

1 μM indoly1-3-acetic acid 250 mg/1 claforan

35 mg/1 kanamycin 7 g/1 agar

Selection Medium 3 5 μM zeatin

2 μM indolyl-3-acetic acid 250 mg/1 claforan

30 mg/1 kanamycin 7 g/1 agar

Selection Medium 4 2 μM 6-benzyl amino purine 1 μM indolyl-3-acetic acid 250 mg/1 claforan 30 mg/1 kanamycin 7 g/1 agar

Claims

1. A method of transformation of trees, rooted or otherwise, the method comprising the steps of providing an incision into a site capable of shoot initiation, introducing disarmed Agrobacterium cells carrying one or more exogenous DNA sequences into the incision, and a selection step in which the site is brought into contact with a medium containing a nutrient, growth regulator and a selection agent to which the transformed cells will be resistant, for a time period sufficient to produce transformed shoots containing the exogenous DNA sequences.

2. A method according to Claim 1, wherein said incision forms a cleft in the site of shoot initiation.

3. A method according to Claim 1 or 2, wherein said incision is about 1mm deep, or more.

4. A method according to Claim 1, 2 or 3, wherein said site capable of shoot initiation is a cotyledonary node, and axillary meristem or an apical meristem.

5. A method according to Claim 4, wherein when the tree to be transformed is a seedling, the site of shoot initiation is a cotyledonary node.

6. A method according to Claim 5, wherein all per- existing shoot initials before, during or after the introduction of the Agrobacterium cells are removed.

7. A method according to Claim 4, wherein when the tree to be transformed is a tree shoot, the site of shoot initiation is an axillary meristem or apical meristem.

8. A method according to Claim 5, wherein the co- incubation step utilises an upright seedling.

9. A method according to any one of the preceding claims, wherein the trees have an intact root system when they are injected and/or co-incubated.

10. A method according to any on of the preceding claims, wherein the medium of the selection step also comprises an antibiotic to kill the Agrobacterium strain.

11. A method according to any one of the preceding claims, and applied to any one or more of the genera Eucalyptus, Populus, Malus and Prunus and any one of tree species susceptible to infection by Agrobacterium tumefaciens or Agrobacterium rhizogenes.

12. A method according to any one of the preceding claims wherein said tree is Eucalyptus globulus or Eucalyptus grandis.

13. A tree transformed according to the method described in any one of Claims 1 to 12.

14. A cell which harbours a gene transformed according to the method described in any one of Claims 1 to 12.

15. Propagules of a tree transformed according to the method described in any one of Claims 1 to 12.

16. Seedlings of a tree transformed according to the method described in any one of Claims 1 to 12.

17. Seeds of a tree transformed according to the method described in any one of Claims 1 to 12.

18. Germplasm of a tree transformed according to the method described in any one of Claims l to 12.

19. A method of transformation of trees substantially as hereinabove described with reference to the Examples hereof.