Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
The effects of explant source, medium composition and growth regulators were examined in order to optimize the induction and selection of fast growing callus lines of European yew (Taxus baccata L. Washingtonii). Callus cultures were induced from isolated mature zygotic embryos or from segments of juvenile branches. Following two months of growth on induction medium (MS + 3.0 mg/L NAA, 0.5 mg/L kinetin, 100 mg/L arginine, 2.5 % sucrose and 0.8 % agar), callus proliferation was induced in 86.4 % of embryo explants and 100 % of branch-cutting explants. The growth potential of established callus lines was found to vary in response to genetic potential and culture medium composition. The growth rate of stem-derived callus obtained on induction medium was superior to that obtained using all other tested media modifications (duplication time 9.6 days). However, the growth of embryo-derived callus lines was enhanced by increasing the iron content from 27.8 to 55.6 mg/L FeSO4 ∙7H2O in the maintaining MS medium (duplication time for line E2 was 8.5 days). In two out of three embryo-derived lines, tissue growth was further improved by transferring onto modified B5 medium (duplication time for lines E2 end E5 was 4 and 5.7 days, respectively). HPLC analysis confirmed the presence of the anticancer agent cephalomannine in calli grown on B5 medium and a taxane-like substance in calli grown on MS medium.Ispitan je utjecaj eksplantata i sastav hranidbene podloge na rast kalusnih linija europske tise. Kalusne kulture potaknute su na eksplantatima čitavih zrelih zigotnih embrija i odsječaka jednogodišnjih grančica odrasle biljke. Nakon dva mjeseca uzgoja na indukcijskoj podlozi (MS s dodatkom 3,0 mg/L NAA, 0,5 mg/L kinetina, 100 mg/L arginina, 25 g/L saharoze i 8 g/L agara) proliferacija kalusa postignuta je na 86,4 % embrijskih eksplantata i na 100 % eksplantata grančice. Rast ustaljenih kalusnih linija ovisio je o genetskom potencijalu i sastavu hranidbene podloge. Najbolji prirast svježe mase kalusa dobivenog iz tkiva grančice ostvaren je na indukcijskoj podlozi (vrijeme udvostručenja mase kalusa bilo je 9,6 dana). Kalus induciran iz embrija rastao je brže na podlozi MS s povećanim udjelom željeza (55,6 mg/L umjesto 27,8 mg/L FeSO4 ∙ 7H20) pa je masa tkiva linije E2 udvostručena za 8,5 dana. Prirast mase tkiva u dvije od tri embrijske kalusne linije poboljšan je supkultiviranjem na modificiranu podlogu B5 (tkivna masa linija E2 i E5 udvostručena je nakon 4, odnosno 5,7 dana). Analizom HPLC u kalusnim kulturama uzgajanim na podlozi B5 dokazana je prisutnost antikancerogene tvari cefalomanina, a u kalusnom tkivu raslom na podlozi MS prisutnost neodređene tvari slične taksanu
