THE EFFECT OF WATER PARAMETERS ON THE DEVELOPMENT OF HEVEA-BRASILIENSIS CALLI IN INVITRO CULTURE

The use of micropropagation methods in hevea culture, especially somatic embryogenesis, would improve intraclonal homogeneity as well as vigour and productivity of trees. However, difficulties encountered in the setting up of somatic embryogenesis with hevea led to the study of a number of parameters which are particularly important to the sucess of this technique. The quantification of water parameters of Hevea brasiliensis calli and their environment during the somatic embryogenesis process revealed stress at different levels, at the beginning of culture and during the days which followed related to the relative humidity of vessel atmosphere and the water potential of the culture medium. The delay in mineral absorption and callogenesis of the internal tegument of immature seeds was related to the adjustment period of the intern tegument water potential to that of the medium (table I). Tissue growth and embryogenic induction were distinctly improved by water conditioning of the initial explant (table II) (partial drying under air flow for 5-10 min). A close relationship was demonstrated between the evolution of water potentials of the medium and callus depending on the relative humidity level in the culture vessel. Keeping the relative humidity close to saturation stimulated an increase in callus water content and callus relative water content associated with higher embryogenic calli frequency (fig 2). On culture, only the embryogenic calli retained a high water potential (-0.9 MPa) identical to that of the medium (fig 3). Renewing the medium enhanced embryogenic induction (table III). The medium water potential appeared as an important culture parameter: at a high level (-0.7 MPa) it strongly stimulated embryogenic calli initiation (fig 4). These effects are related to a clear water status, with better tissue hydration (high water potential and high relative water content), which thus appears necessary for somatic embryogenesis. Similar results were obtained with an early addition of abscissic acid whose optimal concentration (10(-7) M) stimulated embryogenic calli formation and the acquisition of this specific water status. Analysis of nitrogen, potassium and phosphorus revealed stimulation of mineral uptake from the medium by the embryogenic calli. This was probably enhanced by optimal physiological conditions (fig 5). The present study demonstrates that hevea callus is a system which is very sensitive to environmental changes: callus browning and inhibition of the embryogenic process provide evidence of this. Hydric regulation of the callus appeared to be very limited. To avoid stress it is therefore necessary to work in optimal conditions for water avaliability in the vessel atmosphere and culture medium.