USE OF CALCIUM TO OPTIMIZE LONG-TERM PROLIFERATION OF FRIABLE EMBRYOGENIC CALLUSES AND PLANT-REGENERATION IN HEVEA-BRASILIENSIS (MULL-ARG)

In Hevea brasiliensis (Mull. Arg.), increasing the calcium content of the friable callus maintenance medium from 3 to 9 mM stimulated regene ration potential through somatic embryogenesis. This stimulation could be attributed to the homogeneous cytological structure of calluses, w hich were formed of undifferentiated cells capable of somatic embryoge nesis in optimal culture conditions. The very marked increase in the a ctive cell population was sufficient to cause a decrease and a stabili zation of water and osmotic potentials of the calluses, whereas their water content increased. The regeneration capacity of calluses culture d on a medium with additional CaCl2 was greater in terms of both quant ity (number of somatic embryos produced was increased 2-fold) and qual ity (germination efficiency trebled). High CaCl2 concentrations (9 mM CaCl2) in the embryogenesis induction medium favoured somatic embryo d evelopment when calluses were maintained 2 months on the same medium. In this case, addition of benzylaminopurine (BAP) and 3,4-dichlorophen oxyacetic acid (3,4-D) increased the number of embryos produced (243 e mbryos g(-1) FW callus) and their germination capacity (27%). These cu lture conditions were used to determine the optimum embryogenesis indu ction period. The length of the period affected both the intensity of embryogenesis (maximum 56-77 d) and somatic embryo quality (maximum 49 -70 d). The best results were obtained with a 70 d embryogenesis induc tion period, within which 355 embryos g(-1) FW callus were obtained, w ith 35% germination.