Physiological and biochemical aspects of regeneration snake's head fritillary (Fritillaria meleargis L.) in vitro

Proučavana je in vitro regeneracijia košutice (Fritillaria meleagris), višegodišnje lukovičaste geofite. Indukcija morfogeneze in vitro košutice postignuta je u kulturi zrelih zigotskih embriona, segmenata lukovica kao i bazalnih delova listova in vitro formiranih lukovica. U kulturi zrelih zigotskih embriona regeneracija biljaka je postignuta procesom somatske embriogeneze i organogeneze u isto vreme i na istom eksplalntatu na hranljivoj podlozi bez regulatora rastenja ili hranljivoj podlozi obogaćenoj sa TDZ. U kulturi segmenata in vitro formiranih lukovica indukcija morfogeneze postignuta je na hranljivoj podlozi sa 2,4-D ili TDZ na svetlosti ili u mraku. Regeneracija biljaka je bila postignuta na obe hranljive podloge putem somatske embriogeneze i organogeneze, s tim da je regeneracija putem organogeneze bila uspešnija na hranljivoj podlozi obogaćenoj sa TDZ. U kulturi bazalnih delova listova indukovana je somatska embriogeneza na hranljivim podlogama sa 2,4-D, KIN ili 2,4-D i KIN. Anatomska istraživanja su pokazala da je somatska embriogeneza direktna, a somatski embrioni imaju višećelijsko poreklo i nastaju od epidermalnih i subepidermalnih slojeva ćelija bazalnih delova lista. Ispitan je uticaj niskih temperatura (15 i 4 °C), povećane koncentracije saharoze u hranljivoj podlozi kao i predtretman sa GA3 na rastenje, razviće i prevazilaženje dormancije in vitro formiranih lukovica košutice. Pokazano je da predhodno gajenje lukovica na sniženim temperaturama i hranljivoj podlozi sa 4,5 % saharoze pozitivno utiče na rastenje i umnožavanje lukovica. Pored toga, predtretman rastvorom GA3 pre izlaganja lukovica niskoj temperaturi (4 °C) dovodi do stimulacije umnožavanja i klijanja lukovica. Izlaganjem in vitro formiranih lukovica košutice niskoj temperaturi dolazi do promena u sadržaju šećera (saharoze, glukoze i fruktoze), fotosintetičkih pigmenata i poliola. Ispitana je aktivnost antioksidativnh enzima (SOD, CAT, GR i POX) tokom prevazilaženja dormancije lukovica gajenjem na niskoj temperaturi. Analize su pokazale da enzimi antioksidativnog stresa aktivno učestvuju u procesima prekidanja dormancije in vitro formiranih lukovica košutice, kao i aklimatizacije lukovica na ex vitro uslove. Enzimi antioksidativne zaštite su aktivni i tokom indukcije morfogeneze košutice u kulturi segmenata lukovica, a njihova aktivnost zavisi od sastava hranljive podloge i predtretmana kome su lukovice prethodno izložene. Zimogramskom detekcijom esteraza tokom indukcije morfogeneze in vitro u kulturi segmenata lukovica utvrđeno je prisustvo 6 izoformi i njihova aktivnost kao i zastupljenost pojedinih izoformi zavise takođe od predtretmana na kojima su lukovice predhodno gajene kao i od regulatora rastenja u hranljivoj podlozi. Praćena je promena sadržaja arabinogalaktanskih proteina (AGP) u eksplantatima tokom indukcije morfogeneze in vitro u kulturi bazalnih delova listova i segmenata lukovica na dve hranljive podloge obogaćene sa 2,4-D i KIN ili TDZ. Koncentracija AGP u eksplantatima se povećava već posle 7 dana gajenja bazalnih delova listova odnosno posle 21 dana gajenja segmenata lukovica na hranljivim podlogama sa regulatorima rastenja. Koncentracija AGP je veća u bazalnim segmentima lista gajenim na hranljivoj podlozi sa 2,4-D i KIN nego na hranljivoj podlozi sa TDZ. Analizom profila AGP dobijenog ukrštenom elektroforezom pokazano je prisustvo samo jednog tipa AGP tokom indukcije morfogeneze in vitro.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