Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1999-11-24
2004-12-14
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
Reexamination Certificate
active
06830928
ABSTRACT:
The present invention relates to new methods of plant micropropagation and germplasm storage.
Plants can be propagated through their two developmental life cycles: the sexual or the asexual (or vegetative). In the vegetative (asexual) cycle the unique characteristics of any individual plant selected for propagation are usually perpetuated. In most cases, each new plant produced by this method may be considered to be an extension of the somatic cell line of one individual. Methods available for propagating plants in vitro (micro-propagation) are largely an extension of those already developed for conventional propagation (macro-propagation).
The methods that are theoretically available for propagating plants in vitro are essentially based on the multiplication of shoots from axillary buds, on the formation of adventitious shoots and on the formation of somatic embryos. These processes are initiated either directly from the original explant or indirectly from unorganised cells such as in callus or cell suspension cultures.
In practice most micropropagated plants are produced at present by the multiplication of shoots from axillary buds. Nevertheless, somatic embryos, shoots and/or plantlets do not always originate in a single way. For example, in shoot cultures, besides axillary shoots, there are sometimes adventitious shoots formed directly on existing leave or stem explants and/or shoots arising indirectly from callus at the base of the explant.
Considering these observations, somaclonal variation is frequently observed among the regenerated plants even through multiplication of shoots from axillary buds which is considered as the most stable source for plant micropropagation. In such mixed cultures, shoots arising indirectly from callus are not desirable because of the somaclonal variation. This is particularly important when the method is applied in a large-scale programme (industrial scale). In order to reduce this genetic/epigenetic variation, standardization of the cellular origin for the regenerated new shoots is therefore essential.
A second limitation of the method of axillary shoot production is associated with the use of exogenous phytohormones for plant micro-propagation purposes. The growth and proliferation of axillary shoots in shoot cultures is usually promoted by incorporating growth regulators into the culture medium. Depending on the plant species, usually cytokinins and auxins in appropriate combinations and concentrations are used. Such treatments effectively remove the dominance of apical meristems so that axillary shoots are produced. These shoots are used as miniature cuttings for plant multiplication.
The cytokinin growth regulators added to shoot culture media to promote axillary shoot growth, usually inhibit root formation. Therefore, single shoots must be moved to a different medium for rooting in vitro (generally medium containing auxins) before being transferred as juvenile plantlets to the external environment. The axillary shoot production thus requires a sequence of steps.
Nowadays, there is an increased tendency to consider that exogenous applied synthetic cytokinin, which is generally too high, may cause many abnormal features (side effects) such as production of small shoots which typically fail to elongate, induction of unusual leave shapes and particularly the tendency of the regenerated shoots to become vitrified.
In addition, high concentrations of usual cytokinins generally inhibit or considerably delay root formation and also prevent root growth and the promoting effects of auxins on root initiation. In this respect. frequently more than one subculture to a cytokinin-free medium may be required until the level of cytokinin within the tissues has been sufficiently reduced followed by a transfer of the shoots to a rooting medium.
Considering these aspects it is furthermore impossible to propagate particular plant species which are sensitive to such hormonal treatments.
It is thus the object of the present invention to provide an alternative micropropagation method with which the above identified drawbacks of the known methods are obviated.
According to the invention it has surprisingly been found that leafy galls, which are produced on dicotyledonous plants by infection with particular micro-organisms or by treatment with substances produced by these micro-organisms or derived from the infected and/or fasciated tissue (so-called fasciation), and which consist of multiple meristematic centers and shoot primordia that are suppressed for elongation, are an excellent starting point for micropropagation. In a gall no apical dominance phenomena are evident and the formation of numerous primordia is derepressed by the infection process. The gall is in fact a large mass of shoot primordia that are covered with leaves and that are infected by the micro-organism. In leafy galls specific metabolites are present that are involved in the establishment and the maintenance of the leafy gall structure.
In monocotyledonous plants the structures that arise upon contact with or infection by fasciation-inducing micro-organisms or substances have a different morphology than the above described leafy galls that are formed in dicotyledonous plant material. Infection either results in deformation of bulbs and/or the formation of several elongated shoots. These monocot elongated shoot structures are useful in the method of the invention and will be further identified as “shoot outgrows”, whereas the dicot structures are called “leafy galls”.
Apart form the morphogenetic aspects, a gall represents a center of elongation inhibition where the bacterium suppresses the outgrowth of the primordia in the gall. This phenomenon is translated in the apparent lack of apical dominance that would be exerted by one of the primordia and would lead to the outgrowth of distinct shoot primordia from the gall tissues. As such, each gall can be considered as a highly condensed plant where the amplified production of primordia and meristems—apical as well as axillary—are confined to one compact region through the suppression of both the elongation processes and apical dominance.
Furthermore, it was found according to the invention that due to the fact that the galls consist of large numbers of shoot primordia confined to a compact and small structure, they are also a very suitable material for germplasm storage.
Starting from these two observations the present invention now provides for a method for the in vitro preparation of starting materials for micropropagation and/or storage of germplasm, comprising the steps of:
a) contacting plant material with a micro-organism that induces fasciation and/or one or more fasciation-inducing factors derived from the micro-organism, or derived from the infected and/or fasciated tissue;
b) developing leafy galls or shoot outgrows on the plant material; and
c) isolating the leafy galls or shoot outgrows as the starting materials.
This method of the invention may then be incorporated in a method of micropropagation or a method of germplasm storage, respectively. A method for the in vitro micropropagation of plants comprises the above described method for the preparation of starting material, further characterized by the steps of
d) eliminating or inactivating the micro-organism that induces fasciation and/or the one or more fasciation-related factors derived from the micro-organism or derived from the infected and/or fasciated tissue;
e) culturing the leafy gall or shoot outgrows in or on one or more suitable culture media to allow further shoot and root development for obtaining plantlets, and
f) transferring the plantlets thus obtained, optionally after acclimatisation, to conventional growing conditions to obtain regenerated plants.
A method for the preservation of plants or germplasm comprises the method for the preparation of starting material, further characterized by the step of storing the leafy galls or shoot outgrows under growth limiting conditions. The growth limiting conditions may comprise low temperatures of about 4° C. or cryo-cons
El Jaziri Mondher
Goethals Koenraad Hilaire Maria
Van Montagu Marc Charles Ernest
Grunberg Anne Marie
Knobbe Martens Olson & Bear LLP
Para Annette H
Vlaam Interuniversitair Instituut Voor Biotechnologie
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