SOMATIC EMBRYOGENESIS AND REGENERATION OF PLANTS FROM PROTOPLAST CULTURES OF CELERY CELL-SUSPENSIONS (APIUM-GRAVEOLENS)

Protoplasts were isolated from cell suspensions of Apium graveolens L. variety rapaceum (celeriac) initiated and subcultured on Gamborg medium (B5) with 2,4-dichlorophenoxyacetic acid (2.26-mu-M) and 6-benzylaminopurine (1.1-mu-M). The microcalli fraction above 450-mu-m was taken from 6-days-old cell suspensions and put into an enzyme mixture of cellulase Onozuka R-10 (2%) and pectolyase Y-23 (0.1%). Protoplasts were cultured on Lazar et al. modified medium with 2,4-dichlorophenoxyacetic acid (2.26-mu-M), naphthaleneacetic acid (0.89-mu-M), and zeatin (0.5-mu-M) at a density of 3 x 10(5).mL-1. They were kept in darkness at alternating temperatures: 28 and 22-degrees-C during 16 and 8 h, respectively. The culture medium was partially renewed after 7, 14, and 21 days of culture. Cell division rate represented about 30-40% of viable protoplasts. Addition of 2,4-dichlorophenoxyacetic acid (2.26-mu-M) in the microcalli plating medium improved embryogenic differentiation and subsequent plantlet isolation. Calli from protoplasts showed a variable capacity for somatic embryogenesis on Murashige and Skoog medium containing kinetin (1.4-mu-M). Plantlet growth was observed more particularly on microcalli plating medium without growth regulators. After acclimatization, a great number of plants with abnormal morphology was noted from calli cultivated on medium with kinetin. No variation of the normal ploidy level (2n = 2x = 22) has been recorded among the examined plants.