Methods for producing genetically modified plants,...

Details Methods for producing genetically modified plants,... Methods for producing genetically modified plants,...

1998-09-15

2001-07-03

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S279000, C800S281000, C800S283000, C800S284000, C800S290000, C800S288000, C800S289000, C800S300000, C800S302000, C800S303000, C800S319000, C800S323000, C800S294000, C435S418000, C435S419000, C435S422000, C435S430000, C435S468000, C435S469000

Reexamination Certificate

active

06255559

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to methods for producing genetically modified plants, particularly woody plants, and most particularly plants of the Eucalyptus and Pinus species, as well as plants, plant materials and plant products produced by or from such genetic modification. This invention relates, more specifically, to techniques for producing genetically modified plants, including transgenic woody plants and interspecies hybrid woody plants, particularly of the Eucalyptus and Pinus species.
BACKGROUND OF THE INVENTION
Recent advances in plant genetic engineering have made possible the transfer of DNA into plants, including commercially important forestry tree species. The application of genetic engineering to commercially important forestry varieties provides opportunities to incorporate new or improved traits of commercial interest, such as disease resistance, male sterility, increased productivity, rooting ability, wood quality, and others, in forestry varieties.
Commercial scale planting stocks of forestry varieties are generally produced directly from seed or from rooted cuttings. In both of these production systems, traditional plant-breeding techniques are used to produce superior planting stock. The application of genetic engineering techniques to stably incorporate homologous and/or heterologous genetic material into plants offers the potential of improved planting stocks compared to those developed using traditional breeding techniques.
The overall efficiency of techniques for genetically modifying plants depends upon the efficiency of the transformation technique(s) used to stably incorporate the homologous and/or heterologous genetic material into plant cells or tissues, and the regeneration technique(s) used to produce viable plants from transformed cells. In general, the efficiency of transformation and regeneration techniques adapted for genetically modifying forestry plants, such as plants of the Eucalyptus species, is low.
Publications report the successfiul transfer of DNA into commercial varieties of tree species, including Eucalyptus. 1997 Biological Sciences Symposium, “Prospects For Eucalyptus Tranformation,” TAPPI Press, pp.313-326. Genetic transformation has generally been achieved through Agrobacterium-mediated transformation. Transformation techniques have been demonstrated using reporter genes such as GUS ((-glucuronidase), nptli and cat (chloramphenical acetyl transferase). A reproducible and reliable tissue culture regeneration system is required for regenerating plants from transformed cells. Regeneration systems developed for use with forestry varieties have generally demonstrated very low levels of reproducibility and efficiency. Regeneration is generally the limiting factor in the production of transgenic forestry species.
Techniques for plant tissue culture have been developed and used extensively for micropropagation of various Eucalyptus species. J. J. LeRoux and J. Van Staden, “Micropropagation and tissue culture of Eucalyptus—a review,” Tree Physiology 9, 435-477, 1991. Techniques used for micropropagation generally involve axillary bud multiplication. The axillary bud is induced form the leaf axils of stem segments, the bud is allowed to elongate into a shoot, and it is then allowed to multiply in the same manner, producing more axillary shoots. When sufficient copies of a clone are produced, the shoots are rooted and then transplanted. This system has been widely used for commercial production of clones for reforestation because it reliably produces stably cloned propagules that are true to type.
Although the axillary bud multiplication system is well developed, it is not a preferred regeneration system for regenerating genetically modified plants. Transgenic plants produced using axillary bud multiplication regeneration techniques are often chimeric because the axillary buds are generated from preformed buds that may carry a mixture of transformed and non-transformed cells. Only portions of transgenic plants produced from chimeric tissues are transformed and carry the introduced genetic material.
Applicants are aware of two published protocols for regeneration of Eucalyptus. In a protocol published in Plant Cell Reports 13:473-476, 1994, an organogenesis pathway using leaf explants was described. In a protocol published in Suid-Afrikaanse Bobboutydskrif 157:59-65, 1991, a somatic embryogenesis pathway using leaf explants was described. These reported systems demonstrated a low efficiency and, additionally required an impractically long time period for the regeneration process. The long duration (six months) of the regeneration process is not commercially feasible. Furthermore, neither of these systems was successfully reproduced by applicants.
The success and efficiency of methods for producing genetically modified plants thus depends on the selection and optimization of a tissue culture regeneration system that provides de novo origination of plant material from transformed cells, and development of the genetically modified plant material to produce a genetically modified plant. Techniques developed to date for genetically modifying forestry species such as Eucalyptus generally demonstrate low reproducibility of the regeneration protocol, long duration of regeneration, low efficiency of plant regeneration (0-5%), and low transformation efficiency. The present invention is directed to improved methods for producing genetically modified plants, particularly forestry species, and most particularly plants of the Eucalyptus and Pinus species.
SUMMARY OF THE INVENTION
The present invention involves methods for producing genetically modified plant material, particularly woody plant material of the Eucalyptus or Pinus species. The methods of the present invention involve introduction of genetic material using transformation techniques. Agrobacterium-mediated transformation techniques whereby one or more genetic construct(s) comprising a reporter gene and the genetic material desired to be introduced is transformed into an Agrobacterium strain using well known techniques.
Preferred tissue explants of the target plant comprise stem segments from micropropagated shoot cultures. The target plant stem segments may be pretreated in a multiplication medium and then transferred to a shoot elongation medium to promote formation of mature shoots. Nodes may be excised from the target plant stem segments and leaves from the stem segments and/or selected nodes of the shoot explants are preferably removed. The stem segments and/or nodes may be additionally wounded, such as by cutting. The stem segments and/or nodes may then be incubated with a transformed Agrobacterium culture to inoculate the target plant explants with the desired genetic material. Following inoculation, regeneration of adventitious shoot buds from the Agrobacterium infected stem segments is promoted in tissue culture using a combination of regeneration agents. The system of the present invention is advantageous for producing genetically modified plants because it employs transformation and regeneration techniques that provide de novo shoot origination from transformed cells.
Following a suitable period for adventitious shoot bud formation, putative transformed adventitious shoots may be excised from the stem segments. Selection techniques may then be used to identify successfully transformed adventitious shoot buds. The selection technique may vary, depending upon the reporter construct used. According to preferred embodiments, the reporter construct introduced to the Agrobacterium and, then, the explants, includes an antibiotic-resistance gene. In this system, suitable selection agents comprise antibiotic agents. A two stage selection technique is preferably employed, whereby the adventitious shoot buds are exposed to a first selection medium having a first concentration of the selection agent, preferably an antibiotic, and the surviving adventitious buds are then are exposed to a second selection medium having a second concentration of the selection agent,

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