Embryogenèse somatique et regénération de plantes à partir de protoplastes issus de suspensions cellulaires de céleri (Apium graveolens)

Abstract

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 μM) and 6-benzylaminopurine (1.1 μM). The microcalli fraction above 450 μm 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 μM), naphthaleneacetic acid (0.89 μM), and zeatin (0.5 μM) at a density of 3 × 105∙mL−1. They were kept in darkness at alternating temperatures: 28 and 22 °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 μM) 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 μM). 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. Key words: Apium graveolens, protoplast, somatic embryogenesis, somaclonal variation, flowering.