Développement de technologies de culture in vitro pour la création de diversité génétique.

In vitro culture technologies for the genetic diversification of temperate woody fruit species.

Context

The genetic base of the temperate fruit trees is restricted. To renew and diversify the varieties assortment as well as integrating new agronomic characters, new technologies accelerating the classical breeding processes are suited. Single in vitro culture techniques make it possible to modify genotype at high frequency, in relatively short periods of time and without necessarily implying major or irreversible changes in the production process and quality. When they are combined to molecular markers technique, they also constitute potential alternatives methods to the genetic transformation.
The genetic transformation also requires powerful and reliable systems of plant regeneration from the point of view of the stability of genes expression.
To date, the following orientations are developed:
- Use of the somaclonal variation to change the color of the fruits and the growth habit of the trees.
- A. rhizogenes-mediated transformation in order to create new Prunus rootstocks influencing the growth habit, flowering and cuttings rootability.
- Research on regeneration competence and linked genetic variation induced in vitro.
- Construction of stable chimaeras to improve the diseases tolerance and the control of the tree growth.
The techniques developed in the studies are
- adventitious budding,
- multi-apexing from shoot meristem
- somatic embryogenesis
- protoplast culture

Objectives

- Color of the pome fruits and tree habit by somaclonal variation.

Mutations concerning fruits color and growth habit of fruit trees appear naturally in the orchards in a random way.
Adventitious budding induced in vitro increases the frequency and widens the range of variations to other fruit characteristics such as the shape, the firmness or the time of maturation that prove to be stable.
The regenerants screened precociously in vitro are transferred in orchard by ex vitro grafting of microshoots. The first fruits are observed 4 to 5 years after the first in vitro handling. An extension of the study to the pear tree is envisaged with Doyenné du Comice variety.
Boskoop apple treated in the same way gave regenerants that are observed about the “spur” trait of the shoot growth.

- Expression of ‘rol’ genes from A. rhizogenes in Prunus rootstock.

The increase in the volume of the rooting system as well as stressing the side branching and the flowering naturally conferred on certain plants by A. rhizogenes is properties sought in fruit or in forest species.
The first experiments led to the regeneration of various clones with “Inmil “ (P. incisa x serrula) dwarfing rootstock for cherry tree, through the successive organogenic processes of rhyzogenes, followed by the somatic embryogenesis and finally adventitious budding. These trees present a survival problem in greenhouse growth conditions.
The experiments are repeated with P. avium after budding regeneration from transformed roots.

- Study of aneuploid apple trees

Aneuploïdy constitutes a source of genetic diversity for several fruit species. Trees issued from crossing Jonagold (3n) and Mc Intosh ' Wijcik' (2n) (columnar growth) apple varieties declines when growing on their own roots. A germination carried out in vitro and followed by ex vitro micrografting leads to fruit bearing and fertility of the plants.
AFLP and microsatellites molecular analyses on seedlings of an aneuploid line (H48) confirm that the F2 progeny come from natural hybridizations.
Some aneuploids reveal certain genetic variability during the process of adventitious budding. This is detected after flow cytometry analysis. They can be quantitatively very weak or of polyploid nature.
The observation of the behaviour continues within the orchard for single regenerants and genotypes from the progenies on the basis of columnar growth habit.

- Regeneration competence.

With the woody fruit species, the regeneration of whole plants constitutes the major factor limiting the application of biotechnologies. The techniques require a continuous optimization of cell competence to regenerate with the purpose of controlling the origins of buds or somatic embryos and the development of protoplasts culture.
Mutli-apexing practised on meristematic dome (0.1mm) implies a minimum of cells in the neo-formation of buds and decreases consequently the risk to regenerate chimaeras.
The unicellular origin of the regenerants constitutes however the ultimate objective. It can be reached by somatic embryogenesis and protoplasts culture. Somatic embryogenesis is limited to a few genotypes of Prunus species with which it is applied in routine. The genetic transmission underlined in ' Inmil' progeny must be the subject of a thorough study. In the same way, the means of extending the protoplasts culture succeeded with Prunus incisa x serrula (protoclones are flowering in the orchard) in other Prunus species must be required according to the advance of knowledge in this field.