Methods for producing parthenocarpic or female sterile...

Details Methods for producing parthenocarpic or female sterile... Methods for producing parthenocarpic or female sterile...

1999-09-21

2002-11-19

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters plant part growth

C800S278000, C800S287000, C800S288000, C435S069100, C435S320100, C435S419000, C435S468000, C536S023600, C536S023700

Reexamination Certificate

active

06483012

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the use of the promoter region of the DefH9 gene of
Antirrhinum majus
and of promoter regions of homologous genes in other species for the highly specific expression of genes in the placenta and/or ovules of plants or tissues derived from placenta or ovules in order to achieve, for example, parthenocarpy, female sterility and for an enhancement in fruit setting and development. The present invention also relates to DNA constructs in which said promoter controls the expression of a DNA sequence which upon expression leads to the above mentioned effects. Furthermore, the present invention relates to transgenic plants genetically modified with such constructs which can develop fruits also in the absence of fertilization (i.e. with parthenocarpic development) or which are female sterile as well as to the fruits of these plants and to the propagation material of these plants.
In the field of crop plants grown for the commercial value of their fruits, there is a great demand for plants able to develop fruits in the absence of fertilization. This is due not only to the absence of seeds (e.g. table grape, melon), but most prominently, to obtain fruits in environmental conditions not favorable for fertilization (e.g. eggplant, tomato; Lipari and Paratore, Acta Hort. 229 (1988), 307-312; Savin, PHM Revue Horticole 374 (1996), 50-52). Methods to achieve parthenocarpic development essentially consist either in using chemically active ingredients, in using mutants conferring parthenocarpic development to the species where they have been selected or by using alterations in chromosome number (i.e. polyploidy). Thus, plants which are suitable for breeding plants with the mentioned desired properties have so far been available only to a limited degree. Indeed, it is common practice for some horticultural plants to treat (i.e. spray) flower buds with synthetic growth factors to cause parthenocarpic development (references above, and: La Torre and Imbroglini, Informatore Agrario 16 (1992), 71-78; Roberts and Hooley, Plant Growth Regulators, Chapman and Hall (New York (1988)). The success of exogenous application of phytohormones relies on their even action on the plant organ, it is labor-intensive and adds extra costs to the production process. Secondly, the chemicals can be transported from the site of application, and so they can affect other parts of the plant or the whole plant.
Plant genetic engineering has recently been applied in order to circumvent the above-mentioned drawbacks connected with the use of mutants or the exogenous application of phytohormones to the plants. For example, Barg, Acts of the III I.S.H.S. Symposium on “In vitro culture and horticultural breeding” Jerusalem, Jun. 16-21 (1996), 13, disclosed the generation of transgenic tomato plants which contain a rolB gene under the control of the TPRP-F1 promoter (from tomato). The use of this promoter leads to the expression of the rolB gene preferentially in the ovary and young fruit. As a result the plants showed parthenocarpic development. However the promoter used displays also a well detectable level of expression in vegetative tissue (i.e. 1.8%, 3.5%, 0.1% in root, stem and leaf, respectively) as compared to expression in the ovary (=100%; Salts et al. (Plant Mol. Biol. 17 (1991), 149-150). Due to this basal level of expression, the plant can also be altered in its physiological processes in vegetative tissues. In a second example, the same TPRP-F1 promoter was used by Szechtman et al., Acts of the III I.S.H.S. Symposium on “In vitro culture and horticultural breeding” Jerusalem, Jun. 16-21 (1996) 32, to drive the expression of the bacterial iaaH gene coding for an indoleacetamide hydrolase able to hydrolyse a number of indoleacetamide analogs, and thus to convert the inactive indolacetamide (IAM) and naphthalenacetamide (NAM) to the active phytohormones indoleacetic acid (IAA) and napthalene acetic acid (NAA), respectively. The resulting transgenic plants showed parthenocarpic development when sprayed with NAM. This disclosure represents an improvement of the efficiency of parthenocarpic development, but still depends on the exogenous application of chemicals such as NAM. The transgene caused no adverse pleiotropic effects per se, though young plants sprayed with 25 ppm NAM have been reported to exhibit a slight epinastic response (Szechtman et al., loc. cit.). In the same communication Szechtman et al. (1996) proposed that the TPRP-F1-iaaH system will have to be combined with TPRP-F1-iaaM to enable endogenous auxin biosynthesis in the fruit.
However, the TPRP-F1 promoter used in the disclosed chimeric genes, has the drawback that due to its basal level of expression also in vegetative tissue it is also active after transformation and during the regeneration process of transformed cells. Since the expression of the iaaM gene or of genes leading to a higher sensitivity for auxins (like rolB) interferes with the regeneration process, the TPRP-F1 promoter is not suitable to obtain optimal plants transgenic for the iaaM or rolB gene. The reason for this is its constitutive basal level of expression in vegetative tissue. This feature of the promoter hinders the efficient regeneration of plants with an optimum level of expression and unaltered in their vegetative growth. As a consequence either transgenic plants are regenerated which do not express the iaaM gene and/or plants are regenerated with a level of constitutive expression of the iaaM or rolB gene so low to be compatible with regeneration. It is known that the constitutive expression of the iaaM gene has deleterious effects in transgenic plants (Gaudin et al., Plant Physiol. Biochem. 32 (1994), 11-29). Thus, these plants do not represent optimal products since i) they might be altered in their vegetative growth (auxin affects many physiological processes including interactions with environmental and microbial factors) and ii) their level of expression in the ovary might be curtailed and not strong enough to promote parthenocarpic development efficiently. In the case of plants obtained using transformation methods not involving manipulations in tissue culture, the constitutive level of expression in vegetative tissues would furthermore interfere with seed germination and seedling growth. The above described experiments for the generation of transgenic plants are based on the well known fact that developing ovules are a good source of auxins (Archbold and Dennis, J. Amer. Soc. Hort. Sci. 110 (1985), 816-820), and that exogenous auxin can substitute the developing ovules (e.g. the achenes of strawberries) to support growth of the receptacle (Nitsch, Amer. J. Bot. 37 (1950), 211-215; Archbold and Dennis, 1985, loc. cit.) thereby leading to parthenocarpic development. Thus, to mimic the hormonal effects of pollination by plant genetic engineering, the expression of a chimeric gene able to alter auxin content and activity should take place specifically in cells of the female reproductive organs, preferably in the ovules and most preferably also in tissue derived therefrom. This requires a promoter which is highly specific for expression in such cells.
With regard to female sterility there are several reports in the art how to obtain female sterile plants by genetically engineering plants with genetic information which leads to the killing or disabling of cells of the female reproductive organs. These approaches are mainly based on the concept that a highly toxic agent is produced in the cells, such as an RNase, a protease or a bacterial toxin. Alternatively, antisense RNA or a ribozyme against transcripts of essential genes are produced. All these approaches require the highly specific expression of the introduced construct in cells of the female reproductive organ and the absence of expression in other tissues since the expression in other tissues would be deleterious for the development of the plant.
Thus, the technical problem underlying the present invention is the provision of methods and means for the

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