Transgenic plants belonging to the species Cucumis melo

Details Transgenic plants belonging to the species Cucumis melo Transgenic plants belonging to the species Cucumis melo

1998-08-03

2001-03-06

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C435S069100, C435S469000, C536S023720, C800S294000, C800S301000, C800S309000

Reexamination Certificate

active

06198022

ABSTRACT:

This invention describes a process for the genetic transformation of plants belonging to the genus Cucumis, and in particular of the melon (
Cucumis melo
). This process involves the transformation of explants by
Agrobacterium tumefaciens
and the in vitro regeneration of the transformed plants.
This process of transformation and regeneration can be used to introduce a gene for resistance to the cucumber mosaic virus, for example, in plants belonging to the species
Cucumis melo.
In many cases, the transfer of genetic characters from one plant species to another is limited by incompatibility barriers.
Such problems are encountered particularly in the genera Cucumis (the melon and the cucumber) and Cucurbita (the squash) of the family Cucurbitaceae. Thus, the transfer of agronomically beneficial characters such as resistance to viral diseases or insects, present in wild-type species, cannot be transferred to cultivated species.
Among the cultivated species of the genus Cucumis, sexual crosses are only possible between
C. sativus
(the cucumber) and the closely related species
C. hardwickii
and
C. Sikkimensis
(Deakin et al., 1971; Van Raamsdonk, 1989). In most cases, the crosses of
C. sativus
with other wild-type species give only sterile fruits; however, the melon (
Cucumis melo
) appears to be a refractory species which cannot be crossed with any other species (Kho et al., 1980; Van Raamsdonk, 1989).
The applications of the novel techniques of genetic engineering offer a promising alternative for the introduction of new characters with a view to improving plant species. These techniques include genetic transformation by the introduction of one or more foreign genes, somatic hybridization by fusion of protoplasts and the induction of somaclonal variations of mutations in order to induce genetic modifications.
The transfer of foreign genes into plant species is quite commonly done by using strains of Agrobacterium tumefaciens containing a disarmed Ti plasmid (Fraley et al., 1986) (Klee et al., 1987) (Horsch et al., 1985). Hitherto, a large variety of transgenic plants has been obtained with
Agrobacterium tumefaciens.
This bacterium enables a foreign gene to be transferred to plant cells which can regenerate transformed plants. Genes coding for profitable agronomic characters have been introduced into plant species. Thus, it has been possible to obtain plants resistant to herbicides (Degreef et al., 1989) and insects (Hilder et al., 1987); Vaeck et al., 1987).
The transfer of genes can be made either from a disarmed Ti plasmid, after homologous recombination, by using intermediate vectors (Fraley et al., 1985) or from a binary vector with the aid of a disarmed Ti plasmid (Bevan, 1984; Fraley et al., 1986).
This transfer of genes can also be carried out by the utilization of
Agrobacterium rhizogenes
which induces roots from the transformed tissue instead of transgenic plants. Plants having an abnormal phenotype can be regenerated from these transformed roots (Chilton et al., 1982, David et al., 1984).
A particularly interesting character is resistance to viral diseases. Genetically transformed plants resistant to different viruses have been obtained (Powell Abel et al., 1986) (Cuozzo et al., 1988) (Tumer et al., 1987) (Hoekema et al., 1989) (Van Dun and Bol., 1988). These plants (tobacco, potato and tomato) express a gene coding for the capsid protein of the virus to which they are resistant. The mechanism of protection has still not been elucidated.
The standard method for protecting plants against viral diseases consists of inoculating plants with an attenuated strain of the virus in order to prevent infection by more virulent strains. This practice, called cross-protection, has enabled yield losses due to viral infections to be reduced (Broadbent 1976) (Fernow 1967) (Costa and Miller 1980).
A system for regeneration of plants from individual cells or explants, placed in culture, is essential for the application of the techniques of genetic engineering.
Such methods have been described recently for the regeneration of non-transformed Cucurbitaceae:
The regeneration of the cucumber (
Cucumis sativus
), after induction of adventitious shoot buds on calli derived from cotyledons, has been described (Msikita et al., 1988; Kim et al., 1988); Wehner and Locy (1981) had previously described the induction of buds on cotyledons. Cucumber plants could be regenerated by somatic embryogenesis. These somatic embryos developed either in cell suspensions derived from calli developed from leaf explants (Chee and Tricoli, 1988) or hypocotyls (Rajasekaran et al., 1983), or directly on cotyledonous (Cade et al., 1988) or leaf (Malepszy and Nadolska-Orczyk, 1983) calli. In all of the cases described above, the plant material is required to pass through a phase of callus formation and cellular dedifferentiation. A prolonged period spent in the phase of callus formation can induce undesirable somaclonal variations. In some cases, these variations can cause sterility in the regenerated plants.
In the case of the melon (
C. melo
), regeneration through organogenesis has already been described either directly on cotyledons placed in culture (Smith et al., 1988; Niedz et al., in press; Dirks and Van Buggenum, 1989), or through the intermediary of calli derived from cotyledons (Mackay et al., 1988; Moreno et al., 1985; Orts et al., 1987; Bouabdallah and Branchard, 1986), hypocotyls (Abak and Dumas de Vaulx, 1980; Kathal et al., 1986) or leaves (Kathal et al., 1988).
The production of melon plants derived from somatic embryos has also been reported (Oridate and Oosawa, 1986; Branchard and Chateau, 1988).
All of these techniques require the passage through a relatively long step of dedifferentiation and callus formation which will precede the differentiation of buds or embryos. Such buds can develop and give rise either to plants having an abnormal phenotype or to sterile plants (Bouabdallah and Branchard, 1986). Furthermore, the induction of embryos or buds derived from calli is weaker than the direct induction of buds on cotyledons.
A regeneration procedure has also been described in another species of Cucurbitaceae: the squash (
Cucurbita pepo
) (Jelaska, 1972, 1974). This author has obtained plants starting from somatic embryos derived from calli cultivated for several months.
As far as the regeneration of genetically transformed plants is concerned, EP-A-0262972 describes the transformation of
Cucumis sativus
(cucumber) by means of
Agrobacterium rhizogenes
followed by regeneration and EP-A-0265100 describes the transformation of
Cucumis sativus
by fusion of protoplasts, followed by regeneration.
De Both and Ben Tahar (1989) have reported the production of transformed calli of melon. These calli which develop in the presence of kanamycin and express the gene for &bgr;-glucuronidase could not develop transgenic plants, in spite of the use of an experimental protocol already used with success in the regeneration of non-transformed melons.
The production of transgenic plants in the species
Cucumis melo
has never been reported.
The fact that, at present, there does not exist a method of regeneration starting from transformed tissues in the species
Cucumis melo
prevents transgenic melon plants from being obtained which express agronomically profitable characters such as resistance to viral diseases.
One of the objectives of the invention is to realize the regeneration of transgenic plants belonging to the species
Cucumis melo.
Another objective of the invention is to define the culture media which are necessary at each step for the regeneration of transgenic plants belonging to the genus Cucumis.
This invention applies more particularly to the genetic transformation of cotyledons, hypocotyls, leaves of the species
Cucumis melo
through the use of
Agrobacterium tumefaciens
followed by the induction of buds and the production of genetically transformed plants. The genetic transformation of the different tissues described above, followed by organogenesis, enables the cultivated spec

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