FAIR-CT96-5034 Fundamental and technological aspects of rooting in micropropagation: relationship with quality ex vitro

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Objectives:

This project studied auxin receptors. It aimed to investigate shoot growth limitation and rooting recalcitrance in the rac tobacco mutant by hyperauxiny causing a saturation of the auxin-binding sites.

Activities and results:

Previous preliminary hormonal characterisation was performed on rac mutant tobacco to better understand its recalcitrance to rooting in comparison with its wild-type homologue. This included ethylene production and accumulation, peroxidase and IAA-oxidase activities, auxin protection, polyamine metabolism. This hormonal characterisation was completed first by a study of the endogenous auxin contents of the whole shoots and the basal parts of the stems of the two genotypes of tobacco during the growth cycle. More free and conjugated auxins were measured in the rac shoots. A maximum of free auxins occurred in both types of shoots: it was visible at day 14 in whole shoots and earlier (day 7) in the basal parts of the wild-type stems. A similar picture was found with the results of the conjugated auxins. The cytokinin levels were determined at the end of the growth cycle in the whole shoots of the wild-type and the rac mutant. At this stage, the auxins to cytokinins ratio was the same for the two types of tobacco. In order to check the "auxin-resistance" of the rac mutant, the wild-type and the mutant shoots have been treated by increasing concentrations of IBA and NAA included in the culture media for three subcultures. The wild-type shoots responded to both auxins showing increased growth rates at concentrations below 10 microM and growth inhibition at higher concentrations. As expected, the reactions of rac shoots were very low or nonexistent.

Further characterisation of the phenolic pattern in the shoots of the two types of tobacco was conducted by studying the phenylammonia-lyase (PAL) activity on the one hand and the soluble phenolic contents on the other during the course of their multiplication cycle. The rac shoots contained more phenolic compounds, due to a higher PAL activity, with a maximum at day 14 in both tobacco strains. These phenolics were determined by HPLC: chlorogenic acid was the major phenolic found in both shoots and is known for its auxin protector role. The rac shoots contained more routine (a flavonoid) which has been shown to be associated with the absence of root formation. Two other phenolics (which had the spectrum of hydroxycinnamic esters) have been found in higher amounts in the rac mutant.

The assumption of the association of the peroxidase minima and putrescine maxima with rooting induction in shoots from both tobaccos was checked. Since the wild-type tobacco roots automatically (without auxin treatment) in the multiplication medium used, this was modified in such a way that even the wild-type shoots did not root, except after auxin treatment. In the non-rooting (without auxin) conditions, the peroxidase activity hardly changed in either type of tobacco. The auxin treatment induced a peroxidase decrease with a minimum occurring after three or four hours in the wild-type and the rac shoots respectively.

Keywords: Rooting, micropropagation

Contacts

Scientific Supervisor

• Thomas GASPAR / Universite de Liege Institut de Botanique / BELGIUM