Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1999-12-10
2004-11-02
Grunberg, Anne Marie (Department: 1661)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S430000, C435S420000, C435S410000, C435S468000, C435S469000, C435S470000, C800S278000, C800S293000, C800S294000, C800S299000
Reexamination Certificate
active
06812028
ABSTRACT:
The present invention relates to methods for producing embryos and regenerating plants from cultured microspores.
BACKGROUND OF THE INVENTION
Doubled haploid plants can be generated from hapioid plants or cells in which the chromosome number is doubled to the normal somatic number(2n) by duplication. Methods for production of haploid and doubled haploid plants have application to plant breeding and transgenic plant production.
The induction of microspore division leading to embryogenesis has application to large-scale haploid and doubled haploid plant production. Methods are known which induce formation of embryo-Like structures from microspores in which genotypes within a species can be induced to produce large numbers of haploids. However, the regeneration of green plants from such embryos has been problematic using known methodology. Genotypes of some monocotyledonous species such as cereals produce albino plants from embryo-like structures. Conventional isolated microspore culture techniques have not led to consistent embryo regeneration, and disadvantageously result in the production of a high proportion of albino plants.
U.S. Pat. No. 4,840,906 (Hunter) teaches a method of plant regeneration from barley microspores incubated at 25° C. for 28 days in a sugar-containing culture medium following 28 days of cold pre-treatment. The method disclosed results in wide variation in the rate of green plantlet formation from microspores, ranging anywhere from about 30 to about 200 green plantlets formed per 100 anthers cultured. There is clearly a need for an improved method that consistently produces high yields of green plantlets from isolated microspores.
U.S. Pat. No. 5,445,961 (Genovesi et al.) discloses a method for embryogenesis of microspores using a pretreatment of sugar alcohol and cold (about 10° C.) which also requires colchicine, a chromosome doubling agent. This pretreatment is followed by microspore isolation and growth on culture medium. From 2 to 115 embryoids were produced for every 10
4
microspores incubated according to this method. Transformation of microspores after treatment with a chromosome doubling agent would be less likely to result in homozygous transformants than if transformation were to occur prior to chromosome doubling. However, no transgenic transformation of microspores is disclosed in this document.
The haploid single-celled microspore is an attractive target for mutation, selection, and transformation. When transformation is performed at the G1 phase of the nuclear cycle, genetically homozygous plants are produced which include transgenes introduced prior to division. Yao et al. (
Genome
1997;40:570-581) treated highly inducible barley microspores of the genotype Igri with mannitol, followed by biolistic bombardment to transform the microspores. However, the regenerated plants were largely heterozygous for the transgene.
Jähne et al. (
Theor Appl. Genet
. 1994;89:525-533) describes a method of using barley isolated microspores for particle bombardment. From cold pretreated spikes, microspores were quickly isolated and maintained at about 24C for 1 hour prior to bombardment with a transgene. An average of 82 green plants per 10
5
microspores were produced according to the disclosed method, and only one transgenic plant formed for every 2.8×10
6
microspores.
Methodological conditions which contribute to successful methodology for embryogenesis, and plant regeneration include donor plant growth, pre-treatment, culture medium components, such as sugar type, nitrogen source and balance, hormones, and medium density and osmolality. However, a method resulting in a high rate of embryogenesis, and successful production of high numbers of green plants has not been disclosed. A methodology is required which employs a optimal and synergistic combination of the above-noted conditions, resulting in a high production rate of green plants from microspores. Such an improved method would be labor saving, and of higher efficiency than conventional methodology, thereby reducing production costs.
Arabinogalactan proteins (AGP) are plant proteoglycans present in a diverse number of plant tissues which may have regulatory functions in plant cell reproduction processes. Previous reports have illustrated positive effects of arabinogalactan proteins on somatic embryogenesis in
Picea abies
(Norway Spruce) and in
Daucus carota L
. (see, for example Egertsdoner et al.
Physiol Plant
1995;93:334-45; and Kreuger et al.
Planta
1993;189:243-248). Kawaguchi et al. (Plant Journal, 1996;9(6):777-785) describe a tetrasaccharide similar in structure to arabinogalactan protein, which accumulates in rice anthers in a stage-specific manner. The effects of AGP on embryogenesis and plant regeneration from microspores has not been evaluated.
European Patent Publication No. 0 455 597 A1 (Sandoz Ltd) discloses a method for stimulating growth of
Daucus carota L
cells plants in in vitro culture. This publication describes the stimulation of growth by adding AGP to culture medium at a level of from 0.01 to 100 mg/L. However, AGP was not used or assessed for efficacy in inducing embryogenesis of microspores. Additionally, no transformation methodology or staging of cells in culture is taught.
There is a need for a method of embryogenesis which results in the production of green plants with a high success rate.
There is also a need for an effective method of plant transformation from embryogenic induction of microspores, which provides an effective pre-treatment to synchronize microspores and holds them at the G1 phase of the cell cycle prior to transformation, thereby increasing the production of homozygous transformants.
It is an object of the invention to overcome disadvantages of the prior art. The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
SUMMARY OF THE INVENTION
The present invention relates to methods for producing embryos and regenerating plants from cultured microspores.
According to this invention, there is provided a method of embryo production comprising the steps of (a) harvesting a microspore-containing plant segment from a donor plant; (b) incubating the segment under pretreatment conditions to maintain a substantial portion of microspores at a uninucleate cell cycle G1 phase; (c) isolating microspores from the segment; and (d) incubating said isolated microspores in an induction medium comprising arabinogalactan protein to induce embryogenesis, thereby producing embryos.
Further, the invention provides a method of plant regeneration from micro spores comprising the steps of (a) harvesting a micro spore-containing plant segment from a donor plant; (b) incubating the segment under pre-treatment conditions to maintain a substantial portion of microspore uninucleate cell cycle G1 phase; (c) isolating microspores from the segment; (d) incubating the isolated microspores in an induction medium comprising an auxin to induce the production of embryos; (e) incubating the embryos in a differentiation medium to produce differentiated embryos; and (f) regenerating plants from the differentiated embryos.
The present invention is also directed to the production of an embryo that is prepared by the above method, and to a plant produced from this embryo.
This invention pertains to a method of introducing a gene of interest into a microspore comprising introducing a genetic construct comprising the gene of interest into the microspore following the steps of pretreatment and isolation. Methods for introducing the genetic construct into the microspore comprise particle bombardment or Agrobacterium mediated transformation. Furthermore, the present invention is directed to a transgenic microspore prepared by this method, and to a transgenic embryo and transgenic plant produced from this transgenic microspore and transgenic embryo, respectively.
Advantageously, the method is efficient and labor saving as compared with known microspore culture and anther culture procedures. The hi
Kasha Kenneth J.
Simion Ecaterina
Grunberg Anne Marie
Quarles & Brady LLP
University of Guelph
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