Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Abstract

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 #M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 #M benzylaminopurine, or first cultured for various periods at different levels of 2,4-D, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-D at a low concentration (1.4 #M) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-D concentration was raised to 14 #M. Prolonged culture on 14 #M 2,4-D resulted in less embryogenic callus formation.Abbreviations: BA -benzylaminopurine; 2,4-D-2,4-dichlorophenoxyacetic acid; NAA -naphthaleneacetic acid Embryogenic callus formation from leaf explants of cucumber has been reported by various authors (Malepszy and Nadolska-Orczyk 1983;Chee and Tricoli, 1988;Bergervoet et al. 1989;Debeaujon and Branchard, 1993;Lou and Kako, 1994). In our hands, the published procedures resulted in low frequencies of embryogenic callus formation. Here we report a method to improve the formation of embryogenic callus. Our research is based on the assumption that embryogenesis, just as other regeneration processes, consists of several phases each with its specific growth regulator requirements (Christianson, 1987).Seeds of Cucumis sativus L., cvs. Profito and Cordito, were kindly supplied by De Ruiter Seeds, Bleiswijk, The Netherlands. They were surface-sterilized for 45 min in 2% (w/v) sodium hypochlorite followed by three rinses in sterile distilled water, and cultured in glass jars at 25 °C and a 16-h photoperiod (35 #mol m -2 s -1). The medium contained full-strength MS macro-and micronutrients (Murashige and Skoog, 1962), McCown's woody plant vitamin mixture without glycine (Lloyd and McCown, 1981), 2% (w/v) sucrose and 0.6% (w/v) agar (Becton and Dickinson, granulated). The pH of the medium was adjusted to 5.8 before adding agar. Germination occurred after 3 days. Seedlings were raised in vitro until the appearance of the third normal leaf.We used the first and second normal leaves. From each leaf, five to seven explants were cut (5 x 5 mm, each explant contained a vein). The position of an explant in the leaf did not influence callus formation. The explants were placed with their abaxial side on the medium. They were cultured in the dark at 25 °C in 9-cm Petri dishes (10 explants per dish) on the same medium (25 ml) as above but with 3% (w/v) sucrose, 250 mg 1-1 bacteriological pepton (Oxoid), and various types and concentrations of auxin and cytokinin. The explants were subcultured at 3-week intervals. Each value in the graph is based on at least 25 explants. The significance of differences was determined with a X2-test.Explants of both cultivars, cultured continuously on standard medium with 4.5 #M 2,4-D and 4.4 #M BA (Bergervoet et al. 1989) f...

Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (C ucurbita pepo l.) and melon ( Cucumis melo l.)

“…It is possible that the presence of cytokinin increased the sensitivity of squash tissues to auxin, as has been previously reported by Aloni (1993). Kuijpers et al (1996) reported that leaf explants of cucumber required 14 µM 2,4-D to become competent to respond to the embryogenic stimulus but required less (4.5 µM) 2,4-D for embryo formation, as shown by a decrease in embryogenic callus formation in cultures inoculated for longer periods at a high auxin concentration. Similar effects of increased 2,4-D concentrations have been reported for cucumber (Cucumis melo) (Rajasekara et al 1983), melon (Cucurbita pepo) (Tabei et al 1991) and pepper (Kintzios et al 1996).…”

“…In this context it might be worthwhile to mention that in cucumber, leaf explant pretreatment at a high (14 µM) 2,4-D concentration for 10 days or 3 weeks resulted in a 40% increase in embryogenic callus formation compared to continuous culture on standard treatment (4.5 µM 2,4-D + 4.4 µM BA) (Kuijpers et al 1996). Moreover, explant pretreatment with high concentrations of growth regulators has been used successfully for the induction of roots of Pinus pinaster and P. contorta in vitro (Gronroos and von Arnold 1987;Rancillac et al 1982).…”

Observations on the combined effects of light, NAA and 2,4-D on somatic embryogenesis of cucumber (Cucumis sativus) hybrids

“…Once callus has been obtained, it can be subcultured and maintained in the dark (George 1993). Cade et al (1988); Kuijpers et al (1996) kept cucumber explants in the dark before transfer to the light.…”

Effect of liquid pulses with 6-benzyladenine on the induction of somatic embryogenesis from coffee (Coffea arabica L.) callus cultures

“…Explant pretreatment with high growth regulator concentrations has been used successfully for induction of somatic embryogenesis from other species such as cucumber (Kuijpers et al 1996;Kintzios et al 2002) and squash (Kintzios et al 2002). Our results indicate that initial pretreatment of coffee callus with BA could promote the initiation of somatic embryos without affecting the percentage of globular embryos which developed to the torpedoshape stage and eventually germinated.…”