Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-05-06
2001-07-31
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C800S278000, C800S284000, C800S298000, C800S307000, C800S308000
Reexamination Certificate
active
06268552
ABSTRACT:
THE BACKGROUND OF THE INVENTION
The invention relates generally to genetic engineering and, more particularly, to a means and method for making plants which produce substantially seedless fruit, wherein the seedless fruit has desirable taste and size characteristics, rendering it more appealing than naturally occurring fruit to the consumer.
Parthenocarpy, the production of seedless fruits, can be achieved by the addition of the plant growth regulators auxin, cytokinin or gibberellin in many crop species (see, e.g., Naylor (1984) in
Hormonal Regulation of Development II: the functions of hormones from the levels of the cell to the whole plant
, Scott, T., ed., pp. 172-218, Springer-Verlag). Applications of these hormones to the unfertilized flowers of tomato, pepper, tobacco, holly, fig, cucumber, watermelon, avocado, eggplant, pear, blackberry and many other species, induced fruit set in the absence of pollen.
It has been shown that the exogenous application of auxin or gibberellin to unfertilized flowers in a number of plant species, including tomato (
Lysopersicon esculentum
) induces fruit set in the absence of pollination, resulting in the production of parthenocarpic fruit [Wareing and Phillips (1981)
Growth and Differentiation in Plants
, Pergamon Press, Oxford, UK]. By contrast, the exogenous application of cytokinin to ovaries or developing fruits is less effective for the production of seedless fruits. It is believed that exogenously applied cytokinin cannot reach the site of action for fruit development because the hormone is immobile within the plant.
In previous efforts to produce seedless fruits, traditional plant breeding and exogenous application of hormones have been used with some success. However, the exogenous application of plant hormones is a labor-intensive process, and traditional plant breeding is a long term process. Moreover, at least some of the previous attempts to produce certain seedless fruits have resulted in low numbers of seedless fruits and/or in relatively small seedless fruits as compared with the normal, seeded fruits.
There is a long felt need in the art for an effective and economical means and methods for the production of seedless fruit, particularly in good yield and quality as compared with prior art seedless fruits.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide compositions and methods for the production of seedless fruit by transgenic means. This is accomplished by the stable introduction into the plant genome of an expression cassette in which a gene encoding an enzyme involved in the biosynthetic pathway of a plant developmental regulator (cytokinin, auxin or gibberellic acid) is operably linked to transcription control sequences which mediate expression of the linked gene in the proper plant part at the appropriate time during development. As specifically exemplified herein, the gene encodes tryptophan oxygenase (iaaM gene) or isopentenyl transferase (ipt gene), and the transcriptional regulatory sequences are those from the GH3 gene, directing tissue-specific expression of a downstream coding sequence in the ovary and developing fruit. The nucleotide sequence of a specifically exemplified GH3 regulatory region from
Glycine max
is given in SEQ ID NO:1. Other regulatory sequences which mediate selective expression in the ovary and/or developing fruit can be substituted for the GH3 regulatory and promoter sequences, such as the AGL5 or PLE 36 transcriptional control sequences.
Also provided by the present invention is an expression cassette can be expressed in plant tissue after the introduction of the cassette into plant tissue. A preferred coding sequence of interest is that for an auxin biosynthetic enzyme, a gibberellin biosynthetic gene or a cytokinin biosynthetic enzyme. The specifically exemplified coding sequence and deduced amino acid sequence for the auxin biosynthetic enzyme (tryptophan oxygenase), are given in SEQ ID NOs:2 and 3, respectively. The specifically exemplified coding sequence and deduced amino acid sequences of the cytokinin biosynthetic enzyme (isopentenyl transferase) are given in SEQ ID NO:4 and 5, respectively. Transcription is regulated by an ovary and developing fruit specific and auxin-inducible transcriptional regulatory sequence (GH3, from
Glycine max
), as specifically exemplified herein. The AGL5 promoter (See SEQ ID NO:7) (from
Arabidopsis thaliana
) operably linked to an iaaM or ipt coding sequence, also functions in the present invention. It is understood that other tissue-specific regulatory sequences which direct expression of an operably linked coding sequence in the developing ovary or developing fruit can be substituted for the GH3 sequence disclosed herein.
A further aspect of the present invention are transgenic plant cells, plant tissue and plants which have been genetically engineered to contain and express a nucleotide sequence encoding a cytokinin or auxin biosynthetic enzyme under the regulatory control of the tissue-specific transcription regulatory element, such that elevated gibberellin(s), auxin or cytokinin (as compared with normal plant tissue) are produced in the developing ovary or developing fruit and such that the fruit so produced is substantially seedless and is increased in solids content as compared with wildtype fruit. Preferably the tissue-specific transcription regulatory element is associated with the GH3 promoter and promoter-associated sequences (e.g., having the specifically exemplified nucleotide sequence given in SEQ ID NO:1) or the tissue-specific promoter is an AGL promoter (active in the ovaries of flowers), as exemplified by the sequence in SEQ ID NO:7.
The present invention provides a method for the production of substantially seedless fruit, said method comprising the steps of constructing an expression cassette in which a coding sequence for an auxin biosynthetic anzyme, cytokinin biosynthetic enzyme, or gibberellin biosynthetic enzyme(s) is operably linked to a transcriptional regulatory sequence which transcription regulatory sequence mediates the expression of a downstream coding sequence in a developing ovary and/or fruit, stably incorporating the expression cassette into a plant cell to produce a stably transformed plant cell and regenerating a transgenic plant from the stably transformed plant cell, whereby substantially seedless fruit having a higher solids content than wildtype fruit are produced when the transgenic plant is cultivated. The auxin biosynthetic coding sequence can be a tryptophan oxygenase coding sequence, for example, with an amino acid sequence as given in SEQ ID NO:3. The cytokinin biosynthetic coding sequence can be an isopentenyl transferase coding sequence, for example, having an amino acid sequence as given in SEQ ID NO:5.
The transcriptional regulatory sequence mediates tissue-specific expression of an operably linked downstream coding sequence in ovary and developing fruit tissue; the regulatory sequence can be an auxin-inducible transcriptional regulatory sequence, for example, the GH3 transcription regulatory sequences given in SEQ ID NO:1, the AGL5 transcriptional regulatory sequences as given in SEQ ID NO:7, 2A11, pTPRPF1, PLE36 or PZ130 transcription regulatory sequences.
The present invention further provides a transgenic plant which has been genetically engineered to contain and express an auxin biosynthetic enzyme coding sequence, a cytokinin biosynthetic enzyme coding sequence or gibberellin biosynthetic enzyme's coding sequence under the regulatory control of a tissue-specific transcription regulatory sequence which is selectively expressing in developing ovary tissue or developing fruit tissue. Seeds and embryos containing the genetically engineered DNA construct are within the intended definition of “plant,” as are progeny containing the DNA construct. The auxin biosynthetic coding sequence can be a tryptophan oxygenase coding sequence, or the cytokinin biosynthetic coding sequence can be an isopentenyl transferase coding sequence. Transgenic plants descri
Greenlee Winner and Sullivan P.C.
Kansas State University Research Foundation
Nelson Amy J.
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