Plant material and culture conditions
Adventitious buds of cv. Najda, obtained as described by Beauchesne et al. (1986) and different parts (distal leaf, proximal leaf, and root) of in vitro grown shoots obtained as described by Mazri and Meziani (2013) were used as explants. The adventitious buds were obtained after 12 months of culture (9-month initiation and 3-month multiplication) while the shoots were obtained after 15 months of culture (9-month initiation, 3-month multiplication and 3-month elongation-rooting). The explants were cut into 0.5 cm length segments (Fig. 1). Leaf segments were placed with the abaxial surface on the culture medium.
Fig. 1
Origin of the explants used to induce somatic embryogenesis. a Shoot obtained through in vitro organogenesis used as source of explant. b Root segments. c Proximal leaf segments. d Distal leaf segments. e Adventitious buds obtained through in vitro organogenesis ...
The mineral medium used was MS medium (Murashige and Skoog, 1962) supplemented with 30 g L−1sucrose (Sigma, Steinheim, Germany) and gelled with 6 g L−1 agar (Sigma, St. Louis, MO, USA). The pH was adjusted to 5.7 with NaOH or HCl (1 N) then the medium was distributed in 30 mL aliquots in 300 mL jars (6.5 cm in diameter and 12 cm in height). The culture medium was autoclaved for 25 min at 121 °C. The cultures were transferred onto fresh medium every month.
Callus induction
Eight media sequences (M1–M8) were tested to induce somatic embryogenesis (Table 1). The explants were cultured on MS medium supplemented with 1 g L−1 activated charcoal (AC) and various combinations of 45 µM auxin (2,4-dichlorophenoxyacetic acid (2,4-D); 1-naphthalene acetic acid (NAA); 2-naphthoxyacetic acid (NOA) or picloram) and 4.5 µM cytokinin (6-(Dimethylallylamino) purine (2iP) or kinetin). All PGRs were purchased from Sigma, Steinheim, Germany. The cultures were incubated for 4–6 months in darkness at 25 °C.
Table 1
Composition of media used for embryogenic calli induction and embryo regeneration. All media were supplemented with 30 g L−1sucrose, 1 g L−1 activated charcoal and 6 g L−1 agar
Somatic embryogenesis expression
Induced calli were transferred to the expression medium which consisted of MS medium basal formulation supplemented with 1 g L−1 AC and 10% PGRs concentration of the induction medium (Table 1). The cultures were maintained for one month under dark conditions at 25 °C
Somatic embryo maturation and germination
Embryogenic cultures were divided into 100 mg fresh weight (FW) calli then transferred to maturation medium, which consisted of PGR-free MS basal medium supplemented with 1 g L−1 AC, under dark conditions at 25 °C for 2 months. Tubular embryos were then transferred to PGR-free MS medium, with or without 1 g L−1 AC, at 25 °C under a 16 h light photoperiod for germination. Shoots from the germinated embryos were cultured for 3 months (25 °C, 16/8 photoperiod) on PGR-free MS medium supplemented with 1 g L−1 AC.
Plantlets transplantation
Plantlets with 3–4 leaves and elongated roots were removed from the culture medium; the root system was washed in running tap water then soaked for 15 min in a solution of 1 g L−1 Pelt 44 PM systemic fungicide (Bayer CropScience, Bayer Maghreb SA, Casablanca, Morocco). The plantlets were planted in a mixture of peat-gravel substrate (1:1, w/w) in plastic bags, then sprayed with 0.5 g L−1 Pelt 44 PM solution. Thirty days later, plantlets were transferred to open benches in the glasshouse with 27 °C and 70% relative humidity.
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