Proceedings Volume 2010 (format .pdf) - SimpBTH

the same seedling exposed to both SD and LD and found high statisticalsignificance for both photoperiod and genotype at individual level.2. MATERIALS AND METHODSPlant materialSpinach seeds of cultivar Matador were washed with running water and a fewdrops of detergent, then immersed in 30% commercial bleach (4% NaClO) for30 min, and in 15% bleach for 15 min. The seeds were rinsed with sterile distilledwater, blotted dry on a piece of sterile filter-paper and planted in 90 mm Petridishes(20 seeds per dish) containing 25 ml of basal plant growth regulator(PGR)-free medium for germination. The cultures were exposed to a 16 h daylength at photon flux density of 55 µmol m -2 s -1 . Non-contaminated seedlings werecollected on new Petri-dishes (three seedlings per dish) containing the samemedium and grown for an additional 1-2 weeks, until the seedlings developed 4leaves and the root system was well developed.Basal mediumThe basal medium contained MS (Murashige and Skoog 1962) mineralsolution and 20 g/l sucrose, 100 mg/l myo-inositol, 2 mg/l thiamine, 2 mg/lpyridoxine, 5 mg/l nicotinic acid and 2 mg/l adenine. The media were gelled with0.7% (w/v) agar (Torlak, Belgrade, Serbia) and pH was adjusted to 5.6 beforesterilization, by using pH-meter. The media were sterilized by autoclaving at 114ºCfor 25 min.Induction of regeneration from root sections and culture conditionsProcedure for induction of regeneration was essentially as was described inKnoll et al. (1997), with some modifications. Seedling’s lateral roots were excisedand 1 cm long apical sections were cut off and placed on basal medium with20 µM α-naphthaleneacetic acid (NAA) and 5 µM gibberellic acid (GA 3 ). Forevery seedling, one half of root sections was exposed to short days (SD) whereasthe other half was exposed to long days (LD). Explants were subcultured on thesame medium at 4-week intervals.All cultures were maintained at 25 ± 2ºC, under cool white fluorescent tubeswith a photon flux density of 55 µmol m -2 s -1 and two photoperiods: SD (8hlight/16h dark period) and LD (16h light/8h dark period).Recordings and statistical analysisFor induction of regeneration, 30 randomly chosen seedlings were used. FourPetri-dishes with 5 root sections, were prepared for each of the two treatments (SDand LD photoperiods) for each seedling. One seedlings was referred as onegenotype throughout the text. The number of somatic embryos (SE) was recordedwith the aid of a stereomicroscope at 4-week intervals over 12-week period. SEwere harvested and removed at the end of each subculture.The effects of photoperiod and genotype on SE induction were evaluated usingstandard two-factor factorial Analysis of Variance (ANOVA). The difference56