Kultura korenova Gentiana punctata L. transformisanih pomoću Agrobacterium tumefaciens C58Cl(pArA4b)

Abstract

Gentiana punctata L. shoot cultures were transformed with Agrobacterium tumefaciens clone C58C1 harbouring A. rhizogenes A4 Ri plasmid (pRiA4b) to produce hairy roots capable of autonomous growth on growth regulator-free medium. Optimal medium comprised WPM macro-nutrient salts, MS micro-salts and iron, LS vitamins and 2% sucrose. Successful transformation was confirmed by PCR amplification of awe 1 and rol C sequences. Excised, transgenic root cultures characterized by intensive lateral root branching enabled high biomass production. Fast growth required use of explants with small initial weight and frequent subculturing. Cultures were maintained for 10 years (more than 100 subcultures) without changes in morphology or physiological responses.

Kulture izdanaka Gentiana punctata L. transformisane su transkonjugovanim sojem Agrobacterium tume-faclens klon C58C1 sa ubačenim A. rhizogenes A4 Ri plazmidom (pArA4b) koji produkuje korenove sposobne za autonoman rast na podlozi bez regulatora rastenja. Uspešna transformacija pokazana je PCR amplikacijom aih 1 i rol C sekvenci u uzorcima transformisanih korenova. Kulture transgenih korenova koje su se karakterisale intenzivnim bočnim grananjem, održavane su tokom 10 godina (90 i više subkultura) bez promena u morfologiji i fiziološkim svojstvima. Optimalni uslovi za rast ovih kultura bili su tečna podloga i 2% saharoza. Brz porast kultura zahtevao je male početne eksplantate i česta pasažiranja na svežu podlogu.