Fiziološki i biohemijski aspekti regeneracije košutice (Fritillaria meleagris L.) in vitro
Physiological and biochemical aspects of regeneration snake's head fritillary (Fritillaria meleargis L.) in vitro
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We have investigated in vitro regeneration of snake’head fritillary (Fritillaria meleagris L.), a perennial bulbous geophyte. The induction of morphogenesis in vitro of snake’head fritillary was achieved in mature zygotic embryo culture, scale segment and leaf base culture of in vitro formed bulbs. Plant regeneration via somatic embryogenesis and organogenesis was obtained in mature zygotic embryo culture on a growth regulator-free medium or on medium supplemented with TDZ. The induction of morphogenesis in vitro was achieved in scale segment culture of the in vitro formed bulbs on media supplemented with 2,4-D or TDZ, grown either on light or in darkness, with more efficient regeneration on media supplementned with TDZ. Somatic embryogenesis was induced in leaf base culture of the in vitro formed plants on media with 2,4-D, KIN or 2,4-D and KIN. Anatomical studies revealed that the somatic embryogenesis was direct, with somatic embryos of multicellular origin formed from epidermal and subepidermal leaf base cells . The effect of low temperature (4 and 15 °C), higher concentration of sucrose in the nutritional media and GA3 pretreatment on growth, differentiation and dormancy breaking of the in vitro formed bulbs was investigated. It was shown that pre-cultivation of the in vitro regenerated bulbs at lower temperatures and higher concentration of sucrose in the nutrition media (4,5 %) have stimulatory effect on growth and multiplication of the bulbs. Also, GA3 pretretment followed by cultivation at low temperature (4 °C) had a stimulatory effect on multiplication and germination of the bulbs. Cultivation at low temperature breaks dormancy of the bulbs and causees changes in the sugar content (sucrose, glucose and fructose), photosinthetic pigments and poliols. Activity of antioxidative enzymes (SOD, CAT, GR i POX) during dormancy breaking was investigated. It was shown that these enyzmes are actively involved in dormancy breaking of the in vitro formed bulbs, and in the process of acclimatization of the bulbs to ex vitro conditions. Antioxidative enzymes were active during the induction of morphogenesis in vitro in bulb segment culture and their activity depended on the nutritional media and the pretreatment to which the bulbs were exposed. During morphogenesis in vitro in the scale segment culture of snake’s head fritillary, up to 6 esterase isoforms have been detected, depending on the pretreatment and media composition. The content of arabinogalactan proteins (AGPs) in explants during the induction of morphogenesis in vitro in leaf base and scale segment cultures at media supplemented either with 2,4-D and KIN or with TDZ was determined. Concentration of AGPs increased after seven days of cultivation of explants on media with growth regulators in the leaf base culture and 21 days in the scale segment culture. In the leaf base culture, concentration of AGPs in explants was higher on a medium with 2,4-D and KIN than on a medium with TDZ. The AGP profile obtained by crossed electroforesis reveiled the presence of one AGP type during induction of morphogenesis in vitro of F. meleagris.
Keywords:Fritillaria meleagris; Morphogenesis in vitro; Bulb dormancy; Organogenesis; Oxidative stress; Somatic embryogenesis; Antioxidative enzymes; Esterases; Arabinogalactan proteins
Belgrade: University of Belgrade, Faculty of Biology (2012): 1-176[ Google Scholar ]