Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8205—Agrobacterium mediated transformation
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation

Definitions

• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 'tree 1 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.
• the site of shoot initiation is a cotyledonary node.
• 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.
• 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.
• 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.
• a co-incubation step is known as a co-incubation step.
• the co-incubation step takes place with an upright seedling.
• the co-incubation step takes place in a test-tube.
• the medium of the selection step also comprises an antibiotic to kill the Agrobacterium strain.
• the tree exhibits epigeal plant germination. More preferably the method is applicable to the genera Eucalyptus, Populus, Malu ⁇ 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.
• the trees have an intact root system when they are infected and/or co-incubated.
• 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.
• globulus seed was surface-sterilised using a solution of household bleach containing sodium hypochlorite and detergent.
• 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.
• Co-cultivation was performed using Agrobacterium tumefaciens strain EHA105 carrying the plasmid p35S-GUSint which encodes the ⁇ -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 ⁇ -glucuronidase on addition of the chromogenic substrate x-gluc (5-bromo-4-chloro-3- indolyl- ⁇ -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.
• 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.
• One hundred (100) seedlings were prepared by Methods 1 and 3 and two hundred seedlings (200) were prepared by Method
• 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.
• 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.
• 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 trans ⁇ enes
• ⁇ -glucuronidase activity (GUS+) by x-gluc assay as follows. 3mg x-gluc dissolved in lOO ⁇ l 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. ⁇ -glucuronidase activity was detected by the presence of blue-stained regions in the tissue.
• GUS+ ⁇ -glucuronidase activity
• 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.
• Example 3 Method 1
• One hundred and twenty (120) seedlings were prepared and co-cultivated as described in Example 3.
• 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.
• 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.
• sixteen (16) hypocotyls sampled for x-gluc assay all but one exhibited blue-staining areas.
• 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.
• a shoot initiation site on a tree shoot in in vitro culture such as an axillary meristem or apical meristem
• a shoot initiation site on a tree shoot in in vitro culture such as an axillary meristem or apical meristem
• the transformed cells can be removed as explants and cultivated to produce transformed plantlets.
• Selection Medium 1 is g/i glucose
• Selection Medium 4 2 ⁇ M 6-benzyl amino purine 1 ⁇ M indolyl-3-acetic acid 250 mg/1 claforan 30 mg/1 kanamycin 7 g/1 agar

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• 1996
  • 1996-01-13 GB GBGB9600698.6A patent/GB9600698D0/en active Pending
• 1997
  • 1997-01-09 BR BR9706942A patent/BR9706942A/pt unknown
  • 1997-01-09 AU AU13883/97A patent/AU720610B2/en not_active Ceased
  • 1997-01-09 WO PCT/GB1997/000042 patent/WO1997025434A1/en not_active Application Discontinuation
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  • 1997-01-09 CN CN97192880A patent/CN1213404A/zh active Pending
  • 1997-01-09 EP EP97900294A patent/EP0880592A2/en not_active Withdrawn
  • 1997-01-09 JP JP09524975A patent/JP2000517162A/ja active Pending
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  • 1997-01-10 UY UY24435A patent/UY24435A1/es not_active Application Discontinuation
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