FR2589673A1 - Composition based on bacterial inoculum and application of this composition to the propagation by cuttings and to the marcotting of plants, in particular apple root stocks - Google Patents

Abstract

The present invention relates to a composition for the marcotting and the propagation by cuttings of plants, characterised in that it comprises a bacterial inoculum with a plasmid comprising at least one functional portion of the Ri T-DNA and a carrier which is acceptable for marcotting and propagation by cuttings.

Description

 The present invention relates to a composition for layering and cutting plants, and in particular rootstock apple trees such as apple trees M9 and M26, making it possible to obtain chimeric plants.

 Large-scale production of graft portfolios requires vegetative propagation, especially by cuttings or layering.

 In the cuttings, a fragment of the plant is placed in the ground so that it can emit roots.

 In layering, the formation of roots on the trunk, branches or shoots is encouraged, without detachment from the mother strain. These elements are then separated when the roots are developed; after planting, these marcots give birth to new plants.

 The techniques which allow the preparation of several subjects from a single mother strain are only feasible for a limited number of species, and again, in some species, only certain varieties can be layered or can only be layered with difficulty, even with the use of substances which improve layering.

 The apple trees of clones M9 and M26 are widely used as rootstocks.

 However, it is well known that certain rootstocks, the M9 rootstock in particular, take very little root. The M9 rootstock is very difficult to lay or cut and its layering yield is quite low, which partly affects its development in arboriculture, although it is, moreover, quite suitable for cultivation in plantation dense and has precious qualities.

 The present invention relates to new cuttings and layering compositions which ensure very rapid root development of the layers and make it possible, moreover, to increase the number of layers by mother strain.

More particularly, these are compositions for cutting and layering plants, characterized in that they comprise a bacterial inoculum with a plasmid comprising at least one functional part of the
Ri T-DNA and an acceptable support for cuttings and layering.

 By "functional part of the Ri T-DNA" is intended to denote the region of the plasmid Ri ensuring the induction of roots (Ri = foot inducing ").

 This Ri plasmid can act as a vector and optionally contain, in addition to the Ri T-DNA, other homologous or heterologous genes intended to confer a particular phenotype in particular on the roots of the plant.

 The bacterium from which the inoculum is produced is preferably Agrobacterium. In particular, the species Agrobacterium rhizogenes is used.

These compositions can be used for a large
number of plants and in particular for production
industrial rootstocks such as M9 apple trees
and M26.

They lead to the production of chimeric plants, that is to say plants for which one or more genetic characters have been modified. These modified genetic characters are, of course, a function of genes
introduced into the plasmid Ri. In particular, when this plasmid contains only the functional part of the Ri T-DNA, the root system of the treated plants is considerably developed.

 These compositions can be in various forms, the bacteria must be in viable form, that is to say for example in lyophilized form or having undergone a simple desiccation under vacuum, or in a preservation medium which can serve as a support. . The support can also be a solution for reconstituting lyophilized bacteria and / or comprise elements promoting the adhesion of the bacteria to the trunk during layering.

 A. rhizogenes bacteria are known and can be obtained from collections or isolated from nature by known methods.

 The use of the compositions according to the invention is possible both in vitro and in vitro: it is sufficient to inoculate it at the level of an incision of the main stem, or of the branches, according to known cutting and layering techniques.

 In the case of rootstock apple trees, inoculation is preferably done at intervals of the main stem spaced about 15 to 25 cm apart; in this way, numerous inoculations are possible on the main stem of a single apple tree and allow many subjects to be obtained.

The detailed mode of use of the inoculum according to the present invention appears on reading the following examples with reference to the appended figures in which
. FIG. 1 represents a chimeric plant obtained from a layer excised from an M9 apple tree treated with A. rhizogenes strain A4 in an air-conditioned room;
. Figure 2 shows the root system of the chimeric plant of Figure 1;
. FIG. 3 represents the results of an electrophoresis carried out on the extracts of chimeric apple trees M26 (A) and M9 (B)
Plant material
M9 apple trees are commercially available and are grown in pots in the greenhouse or outdoors.

 The M26 variety is propagated in vitro using known techniques.

Bacterial strains
The strains of A. rhizogenes A4 and 8196 are standard isolates.

EXAMPLE 1
INOCULATION IN A GREENHOUSE OR IN AN AIR-CONDITIONED CHAMBER
(MACRO-CUTTING)
Apple trees (variety M9), the height of which is approximately 1.5 m, having grown in pots, were inoculated on internodes with A. rhizogenes strain A4, on the main or secondary branches, preferably sexed and having about 30-40 cm in length, making a vertical incision with a scalpel which is coated with the inoculum.

This induction is carried out by scraping with a scalpel a culture of A. rhizogenes developed for 2 to 4 days at 280C in a standard medium in a Petri dish.

These incisions were covered with strips of PVC film (Scel-o-frais
Adventitious roots were formed after 3 or 4 weeks, at which point the PVC film was removed and replaced by wet peat which was held in place by another PVC strip. This was in turn replaced by a strained plastic pot to facilitate placement and containing wet peat.

 A few weeks later, when abundant roots invaded the substrate, the plastic pot was removed by cutting the stem flush with the bottom of the pot.

The excised marcots were put in pots and kept in a growth chamber for 5 weeks, at a humidity of 80%, under the conditions summarized in the following table:
PRE- DAY PRE- NIGHT
DAY DAY NIGHT NIGHT
Hours 3 10 3 8
Temperature (C) 20 25 20 17
Intensity
bright (lux) 4,750 9,500 4,750 0
We obtain in this way a multitude of layers which take root and develop.

 As can be seen in FIG. 1, no modification appears at the level of the aerial part of the chimeric plant obtained. The root system, on the other hand, has developed considerably due to the integration of the plasmid Ri into its genome (Figure 2).

EXAMPLE 2
IN VITRO INOCULATION (MICRO-CUTTING) These apple shoots (variety M26) are propagated in vitro vegetatively under conditions such that the roots do not form but that multiple shoots are produced.

 After 1 month, when the tips of the shoots are in a rapid growth phase, the base of the stem is inoculated with a paste of bacteria removed by scraping from the surface of a culture of A. rhizogenes (strain 8196) on solid medium .

 The inoculated shoots are transferred to a medium without growth substance and cultivated for 16 hours of daytime during, at 23 ° C., 1,800 lux.

 After 5 days, the cultures are transferred to 370C to eliminate the bacteria.

 The roots develop in less than 2 weeks and the seedlings are transferred to pots in the greenhouse or outside.

EXAMPLE 3
31) The effectiveness of adventitious root formation on young apple trees (clone M9) in the greenhouse varies according to the season of the year.

 In summer, 100% of inoculations produced roots. 80% of these yield viable plants after excision of the layers and transfer to pots. These young plants have transformed root systems, as demonstrated by the production of mannopine and agropine and the presence of the T-DNA of the plasmid Ri, and retain the apparent characteristics of the growth of the plant which was inoculated at the origin.

 The morphology of the root systems is modified; for example, these roots are very branched and often show plageotropic behavior.

 32) The apple trees produced in vitro (clone M26) were similar to those produced in the greenhouse, with abundant roots, and sometimes altered geotropic behavior.

 These plants have been compared to control plants for a period of 3 years and in no way appear to be disadvantaged by their transformed root systems.

 The compositions according to the invention thus allow a massive production of plants by vegetative reproduction, in particular a massive production of rootstocks.

 33) High voltage electrophoresis (silver nitrate staining) was performed to demonstrate the production of mannopine and / or agropine in the root system of transformed plants, and the absence of mannopine and / or agropine in the aerial part of said plants.

 The results of this electrophoresis are visible in FIG. 3: FIG. 3A relates to the tests carried out in vitro with the variety M26; FIG. 3B relates to the tests carried out in the greenhouse with the variety M9.

In both cases, "std" represents the controls in mannopine and agropine extracted from calluses induced by
A. tumefaciens strain A66.

 For FIG. 3A, column 1 represents the results obtained from an extract of leaves on a chimeric plant (variety M26) treated in vitro by microbouturage with A. rhizogenes strain 8196; columns 2 to 6 represent the results obtained from root extracts on several chimeric plants treated in the same way; these clearly reveal the presence of mannopine in the root extracts and the absence of mannopine in the leaf extracts.

 For FIG. 3B, columns 1 and 2 reveal the presence of agropine and mannopine in the roots formed by macro-cuttings on M9 apple trees using A. rhizogenes strain A4.

Claims (7)

 1) Composition for layering and cutting plants, characterized in that it comprises a bacterial inoculum with a plasmid comprising at least one functional part of the Ri T-DNA and an acceptable support for layering and cutting.  2) Composition according to claim 1, characterized in that the bacterium used is Agrobacteriurn, and in particular A. rhizoenes.  3) Composition according to one of claims 1 and 2, characterized in that the inoculated is in viable form.  4) Composition according to one of claims 1 to 3, characterized in that the inoculum is in a lyophilized form or in a form having undergone a simple desiccation under vacuum.  5) Composition according to one of claims 1 to 4, characterized in that the support comprises a medium for preserving the inoculum.  6) Application of the composition according to one of claims 1 to 5 in layering and cutting of plants, in vivo and in vitro, characterized in that it is inoculated at the incisions of the main or secondary stems of the plants .  7) Application of the composition according to one of claims 1 to 5 to layering and to cuttings in the apple tree of line M9, characterized in that the inoculation according to claim 6 is made at intervals of about 15 to 25 cm.