Influence of growth regulators on callogenesis and somatic embryo development in date palm (Phoenix dactylifera L.) Sahelian cultivars

Abstract

This study provides a physiological analysis of somatic embryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80%) and Amsekhsi (76%) appeared highly callogenic, whereas Tijib (10%) and Amaside (2%) produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2 mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5 mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings.

Figure 1

Callogenesis and development of date palm somatic embryos. (a) compact nodular primary callus obtained from foliar explants after 2 months on 2,4-D (2 mg l⁻¹) containing medium; (b) friable secondary callus obtained 1 month after chopping of the primary callus; (c) suspension culture; (d) somatic embryos of stage I, (e) stage, II and III (f), respectively, after 2, 4, and 6 weeks of culture on PGR-free medium; (g) germinated somatic embryo. NC: Nodular callus, FC: Friable callus, MC: microcallus, SE: somatic embryo.

Figure 2

Effect of plant growth regulators (PGRs) in mg/L on the induction of callogenesis after 60 days of culture for cultivars Ahmar (a), Amsekshi (b), Tijib (c) and Amaside (d). Means were calculated from 48 explants per condition. Letters indicate significant differences according to Newman and Keuls test at the level of 5%.

Figure 3

Effect of 2,4-D (2 mg/L) without activated charcoal (AC) or at 50, 75, and 100 mg/L with AC (1000 mg/L) on growth rates of suspension cultures from two different Ahmar cultivars lines. Averages values were calculated from 5 repetitions per condition of medium; bars indicate confidence interval at 95% threshold.

Figure 4

Flow cytometry analysis of nuclear DNA from leaves of rice (Nippon bar) seedlings (green), date palm cv. Ahmar seedlings (violet), and date palm cv. Ahmar somatic embryo-derived plantlets (red, black, and blue).