Bridg, Hannia: Micropropagation and Determination of the in vitro Stability of Annona cherimola Mill. and Annona muricata L.

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Chapter 4. Purpose of this Work

4.1 Objectives

The purpose of this work is to develop an in vitro vegetative multiplication system assuring the clonal propagation or true-to-type regenerants for Annona cherimola Mill. and Annona muricata L., subtropical and tropical semideciduous woody fruit species from selected growing plants in order to improve a new an alternative propagation method that assures the rescue of natural selections.

The micropropagation of A. cherimola and A. muricata is studied in regard to the physical and chemical factors, related to the cellular dedifferentiation and differentiation processes of the selected plant explant under aseptic conditions until the promotion of in vitro plant clone regenerants.

The adaptation and endogenous preconditioning of the mother plants, source of explants in the greenhouse, to promote the in vitro establishment, is reported.

To advance in the aseptic in vitro establishment of A. cherimola and A. muricata, the effect of some disinfectant solutions in combination with several tissue explants is studied and their effect evaluated on vegetative, semi-woody and woody branches.

To proceed with the micropropagation of A. cherimola and A. muricata, different nutrient media formulations supplemented with plant growth regulators are evaluated to define the initial culture conditions of a selected explant.

A. cherimola and A. muricata are phenol reactive plants, the effect of explant size, branch lignification grade, photo-reaction stress in combination with antiphenol solutions is evaluated in terms of explant quality during all the micropropagation steps.

To forward the A. cherimola and A. muricata newly formed in vitro shoots, different salt media formulations in combination with several plant growth regulators are tested and evaluated in terms of shoot organogenesis and shoot differentiation.

To proceed with A. cherimola and A. muricata in vitro propagation, the previously formed shoots are stimulated to increase, not only in number and size, but also in height thus, some plant growth regulators in combination with nutrient media are evaluated on the quality of the new in vitro formed shoots. The limiting problems such as shoot chlorosis or shoot hyperhydration are prevented in advance.

A. cherimola and A. muricata are not root prolific plants, thus nutritional, hormonal and physical factors in the micropropagated shoots are evaluated under in vitro and ex vitro conditions. To stimulate rhizogenesis and root differentiation auxines, light, mineral salt composition and substrate the principal factors considered.

Shoot hardening of A. cherimola and A. muricata regenerants and their gradual adaptation to the greenhouse conditions is described in terms of optimal conditions to improve the survival rate of regenerants under in vitro conditions.

In order to support the in vitro vegetative propagation of A. cherimola and A. muricata, the quality of the in vitro regenerants was screened by the molecular DNA technique of Random Amplified Polymorphic DNA (RAPD).


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The DNA pattern band between A. cherimola and A. muricata ex vitro source of material and A. cherimola and A. muricata in vitro regenerants was compared. The reported results are discussed in terms of genetic stability and somaclonal variation.

Furthermore karyotype chromosome number is being complementarily reported from the plants obtained by means of in vitro culture.

To my knowledge this research presents for the first time a clonal micropropagation protocol for Annona cherimola and Annona muricata tested by RAPD. In addition rhizogenesis and genetic stability of the in vitro regenerants is discussed.


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