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.
