Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-03-25
2003-11-18
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S288000, C800S293000, C800S294000
Reexamination Certificate
active
06649812
ABSTRACT:
The present invention relates to a method of production of transgenic plants from meristems, the said plants being wholly transformed in the T
0
generation. The invention also relates to the transformed explants obtained during the method.
Genetic engineering techniques are now commonly applied in the breeding of plant species. These techniques allow the introduction of new characters which are difficult or impossible to introduce by conventional techniques. In spite of the development of these techniques, there are however certain species which cannot be easily subjected to transformation or which cannot be regenerated from differentiated explants such as cotyledons or leaves. For these species (for example sunflower, cotton, pea, bean, soybean and the like), it has been demonstrated that these difficulties could, in part, be overcome using, as explant for the transformation, a meristematic explant, for example an apical meristem.
The use of meristems allows the regeneration of fertile plants without a callus formation stage. The technique of regeneration from meristems applies to a large number of different species and, within the same species, to a large number of genotypes. By virtue of this technique, it has been possible for numerous recalcitrant species to be transformed and regenerated (see for example: Bidney et al., Plant Molecular Biol. 18, 301-313, 1992; Bidney et al., Proc. 13e Int. Sunflower Conf. Vol II, Pisa Sept. 1992; Schrammeijer et al., Plant Cell Rep. 9: 55-60, 1990; Christou et al., The Plant Journal, 2(3), 283-290, 1992; Gambley et al., Plant Cell Rep. 12: 343-346, 1993; Russel et al., Plant Cell Rep. 12: 165-169, 1993).
This method has, however, some disadvantages. The transformation frequency is extremely low and the regenerated plants are almost exclusively chimeric, that is to say that some tissues are transformed while others are not. This characteristic results from the fact that the meristem produces the cellular material at the origin of all the tissues of the various plant organs (stem, roots, leaves). Now, regardless of the technique used, the transformation operation only leads to the transformation of a limited number of cells within the explant, these cells being distributed randomly. The meristem is never wholly transformed following such a manipulation. The plants regenerated from a meristem having undergone a transformation stage are therefore chimeric. Wholly transgenic plants are obtained only in the progeny and provided that the germinal line cells have been affected by the transformation. This type of process is described for example in: Bidney et al., Plant Molecular Biol. 18, 301-313, 1992; Schrammeijer et al., Plant Cell Rep. 9: 55-60, 1990.
The disadvantages presented by the production of chimeric plants are recognized in the art, but it has not been possible to offer any solution. A system of labelling which makes it possible to recognize, among the T
0
chimeric plants, those which had undergone transformation of the germinal line and which were therefore capable of transmitting the new character to their progeny, has been described (Christou et al., The Plant Journal, 2(3), 283-290, 1992). This method facilitates the production, in the next generation, of wholly transformed plants, but the plants produced in the T
0
generation are still chimeric.
Up until now, no method exists which makes it possible to produce in a systematic and predictable manner, transgenic plants, wholly transformed in the T
0
generation, from meristematic explants.
The present invention solves this technical problem. The invention is based on the development, by the inventors, of a method which makes it possible to enrich the meristems in transformed cells, in order to arrive at wholly transformed meristems, that is to say meristems all of whose cells are transformed. According to the invention, the regeneration of plants is then carried out exclusively from these wholly transformed explants, which will guarantee the transgenic character of the plants obtained. The inventors have also demonstrated that, under certain conditions, the new formation of newly formed leaf meristems and buds on the leaves of explants derived from meristems can be deduced. The existence of these newly formed leaf buds, containing newly formed leaf meristems, has never been described in the literature. The method of the invention also allows the production of these newly formed leaf meristems in a wholly transformed state. They constitute, in this case, an excellent cellular material for the regeneration of wholly transformed plants in T
0
.
In general terms, the invention therefore provides a method of production of wholly transformed meristems, and a method which makes it possible to regenerate exclusively from these explants wholly transgenic plants in T
0
.
More particularly, the invention relates to a method of production of transgenic plants, wholly transformed in the T
0
generation, comprising:
a) a stage for the genetic transformation of a meristematic explant, and
b) a stage for selective culture which allows the specific development, among all the transformed cells, of those which are at the origin of the secondary meristems and/or of those capable of giving rise to newly formed leaf meristems;
c) the regeneration, from the cellular material obtained during stage ii), of transgenic plants.
Within the context of the present invention, the term “meristematic explant” means an explant consisting essentially or exclusively of meristematic tissue or of tissue capable of becoming, during its development, meristematic. According to the invention, this term covers especially:
an apical meristem (also known in the art as “primary meristem”), particularly an apical stem meristem, that is to say at the origin of the stem;
a newly formed leaf bud;
part of a young leaf capable of giving rise to newly formed leaf buds.
The term “newly formed leaf bud” means a bud carried on the upper epidermis of a leaf. It contains a newly formed leaf meristem. Its development is induced “artificially” by a series of precise culture conditions.
The nature of these various explants will be described later.
The term “secondary meristem” means a meristem derived from a primary meristem. In particular, this term relates, within the context of the invention, to the axillary meristems situated at the axil of the leaves and responsible for the construction of the lateral branches of the plant.
The term “axillary bud” means a bud situated at the axial of a leaf, containing a secondary meristem.
The term “newly formed leaf meristem” means a meristem contained in a new formed leaf bud. The formation of these meristems is caused “artifically” by culture conditions which will be defined later.
At the cellular level, the secondary meristems and the newly formed leaf meristems have a structural organization and an action which are identical to those of the principal meristem, but are smaller in size. Moreover, the newly formed leaf meristems are smaller than the secondary meristems.
The studies leading to the development of the present invention are based on the following morphogenetic principles. The apical meristem contains, at the axil of the leaf Primordia, cells which, in a predetermined manner, will give rise, by cell multiplication, to secondary meristems. These “preprogammed” cells could be transformed during a genetic transformation procedure. The secondary meristems derived from the multiplication of these cells will be composed exclusively of transformed cells. The same is true of the cells which, under favourable culture conditions, will give rise to newly formed leaf meristems. Consequently, the plants regenerated from these transformed secondary meristems and from transformed newly formed leaf meristems will be wholly transgenic.
The inventors have developed a selective culture system which favours the development, among the transformed cells, of those which are at the origin of the secondary meristems and of the newly formed leaf meristems. The other cells will be eliminated.
Preferably, the s
Knittel Nathalie
Lenee Philippe
Biogemma
Crouch Deborah
Merchant & Gould P.C.
Woitach Joseph
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