Cytokinin homeostasis and expression of the genes involved in cell cycle control during kohlrabi (Brassica oleracea L. var. gongylodes) regeneration in vitro

Regeneracija in vitro gajenog biljnog tkiva kontrolisana je regulatorima rastenja,naročito citokininima i auksinima. Egzogeno primenjeni biljni regulatori rastenja moguinteragovati sa endogenim biljnim hormonima i uticati na njihovu biosintezu ilidistribuciju, odnosno menjati hormonalnu homeostazu koja utiče na razviće in vitro.Proces regeneracije biljaka in vitro podrazumeva intenzivne ćelijske deobe, a centralnuulogu u kontroli ćelijskog ciklusa i razvića organa imaju kompleksi ciklina i kinazazavisnih od ciklina na čiju sintezu, odnosno aktivnost, utiču pomenuti biljni hormoni. Utom smislu, ciljevi ovog rada bili su uspostavljanje efikasnih protokola za regeneracijubiljaka kelerabe (Brassica oleracea L. var. gongylodes) in vitro, sa posebnim osvrtomna identifikaciju i kvantifikaciju različitih grupa citokinina i indol-3-sirćetne kiseline(IAA) u eksplantatskim tkivima sa regenerisanim izdancima, kao i analiziranje obrazacai intenziteta ekspresije gena koji kodiraju cikline i kinaze zavisne od ciklina tokom denovo organogeneze izdanaka. Efikasan sistem za repetitivnu somatsku embriogenezu(SE) kod kelerabe, sorta Bečka Plava (BP), je uspostavljen iz nezrelih zigotskihembriona. Najveća frekvencija SE detektovana je kod nezrelih zigotskih embriona uranom kotiledonarnom stadijumu razvića gajenih na hranljivoj podlozi bez regulatorarastenja.Uspešna indukcija de novo organogeneze izdanaka kelerabe, sorte BP i Bečka Bela(BB), postignuta je kod odsečaka hipokotila i intaktnih klijanaca, na podlogama sapojedinačnim citokininima (CK): 6-benzilaminopurin (BAP), tidiazuron (TDZ), transzeatin(transZ) ili cis-zeatin (cisZ). Frekvencija regeneracije kao i vijabilnostregenerisanih izdanaka obe sorte je bila veća kod intaktnih klijanaca, dok su međuprimenjenim citokininima BAP i TDZ bili najefikasniji u indukciji de novoorganogeneze. Kvantifikacija endogenog sadržaja citokinina i IAA u odsečcimahipokotila sa regenerisanim izdancima (HRI) kao i u intaktnim klijancima saregenerisanim izdancima (KRI) je ukazala na to da uočene razlike u organogenomodgovoru ova dva tipa početnog biljnog materijala mogu biti povezane sa njihovimhormonskim statusom...vitro plant regeneration is under control of plant growth regulators (PGRs), especiallycytokinins and auxins. Exogenously applied PGRs can affect the homeostasis ofendogenous plant hormones by influencing their biosynthesis and distribution,subsequently altering in vitro development. Intensive cell divisions represent the basisof in vitro plant regeneration, and they are controlled by complexes of cyclins andcyclin-dependent kinases. The activity of these complexes during cell cycle and organdevelopment is affected by plant hormones. Respecting this, the aims of this study weredeveloping efficient protocols for in vitro regeneration of kohlrabi (Brassica oleraceaL. var. gongylodes) with an emphasis on identification and quantification of differentgroups of endogenous cytokinins and indole-3-acetic acid (IAA) in explant tissue withrespective regenerated shoots, as well as analyzing expression of genes encoding cyclinsand and cyclin-dependent kinases during de novo shoot organogenesis. Efficient systemfor repetitive somatic embryogenesis (SE) was developed for kohlrabi cv. ViennaPurple (VP) using immature zygotic embryos. The highest frequency of SE wasobtained from immature zygotic embryos at the early cotyledonary stage grown onPGR-free medium.Successful induction of de novo shoot organogenesis in kohlrabi cv VP and cv. ViennaWhite (VW) was achived using hypocotyl explants and intact seedlings, cultivated onmedia supplemented with single cytokinin (CK): 6-benzylaminopurine (BAP),thidiazuron (TDZ), trans-zeatin (transZ) or cis-zeatin (cisZ). Higher frequency of shootregeneration as well as viability of regenerated shoots was detected for intact seedlings,with BAP and TDZ being the most efficient in de novo organogenesis induction.Quantification of endogenous cytokinin and IAA contents in both hypocotyl explantsand seedlings with regenerated shoots (HRSs and SRSs, respectively) suggested that theobserved differences in organogenic response between these two types of starting plantmaterial were related to their cytokinin and IAA contents. The level of total CKs washigher in HRSs, while SRSs displayed a higher IAA/bioactive CKs ratio..