Explant Source and Tissue Culture Medium:
In our study, mature seeds of wheat genotypes (Table 1) were washed with tap water 10–15 min, then were surface sterilized with %70 ethanol for 5 min, washed several times with sterile distilled water in sterile laminar flow, after that seeds were incubated in shaker in a solution of 1% sodium hypochlorite involving a drop of Tween 20 (Sigma Aldrich) for 30 min, later seeds were rinsed with sterile distilled water three times and kept in sterile distilled water for overnight at 4°C.
Table 1
Wheat cultivars used in the present study, with their pedigrees and agricultural properties.
Genotypes
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Pedigrees
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Agricultural Properties
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cv. PALANDÖKEN 97
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AU//YT54*2/N10B/3/II8260/5/PNC/CM//NB6977/3/CC/INIA//BB/4/MXP//KR/FUNO
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Winter wheat; Recommended for arid regions
Resistant to cold, drought; Resistant to diseases of smut, bunt and rusts; Good tillering capacity; Medium late characteristic; Good response to fertilizers; Average yield 3500–4500 kg ha− 1
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cv. NENEHATUN
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ND//P101/BLUEBOY
SWM584 OP-1P-2P-OH
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Winter wheat; Recommended for arid regions; Resistant to cold, drought; Resistant to diseases of smut, bunt and rusts; Very good tillering capacity
Medium early characteristic; Good response to fertilizers; Average yield 3000–3500 kg ha− 1.
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cv. KIRİK
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Local Variety
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Alternative wheat; Recommended for arid regions in winter, irrigated regions in spring; Moderately resistant to cold, drought; Susceptible to diseases of smut, bunt and rusts; Good tillering capacity
Medium early characteristic; Good response to fertilizers; Average yield 1500–2000 kg ha− 1 in irrigated, 2000–2500 kg ha− 1 in arid conditions.
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Callus formation was performed in MS solution (Murashige and Skoog, 1962) supplemented with 2 mg L− 1 glycine, 100 mg L− 1 myo-inositol, 0,5 mg L− 1 nicotinic acid, 0,5 mg L− 1 pyridoxine HCl, 0.1 mg L− 1 of thiamine HCl vitamins, 1.95 g L− 1 of MES (2-(N-Morpholino) ethane sulfonic acid hydrate, 4- Morpholine ethane sulfonic acid), 50 mg L− 1 of ascorbic acid, 20 g L− 1 of sucrose, solidified with 7 g L− 1 of agar and pH was adjusted to 5.8 before autoclaving.
The explants obtained from calli of 20 mature embryos with endosperms were cultured in each petri dish as per replication (Filippov et al. 2006) containing callus media (Murashige and Skoog, 1962; Aydın et al. 2006) and each of 17 β-estradiol, estrogen, progesterone and testosterone hormones in three doses of 10− 4mM, 10− 5 mM, and 10− 6 mM for 21 days at 25 ± 1 in 16-hour light (62µmol m− 2 s− 1) / 8-hour dark photoperiod at 1500 lux illumination intensity. The experimental design was completely randomized design with four replications. Regular hygiene applications were carried out for sterilization of equipment (105 kPa pressure and 121°C) and plant growth solutions (0.22 µm porous cellulose nitrate filters). Hard-textured, dispersible, compact and cream-colored calli were considered embryogenic, while white and watery calli were considered non-embryogenic (Aydın 2010).
After three weeks of culture, callus formation rate (% CFR) was calculated as:
%CFR = (number of calli / number of cultured explants) x 100,
Embryogenic callus formation rates were calculated in two ways:
i) Embryogenic callus formation rate with respect to number of explants (%ECFE):
%ECFE= (number of embryogenic calli / number of explants) x 100
ii) Embryogenic callus formation rate with respect to number of calli (%ECFC):
%ECFC= (number of embryogenic calli / number of calli) x 100
For each sex hormone and its concentration, number of regenerated plants (NRP) were also counted. Statistical data were analyzed as factorial design of 3 (genotypes) x 4 (sex hormones) x 3 (concentrations) using analysis of variance (ANOVA).