Transgenic callus culture establishment, a tool for metabolic engineering of Rhodiola rosea L.

• Autorzy: Mirmazloum I. , Forgacs I. , Zok A. , Pedryc A. , Gyorgy Z.

Warianty tytułu

PL

Zakładanie hodowli kalusowej jako narzędzie w inżynierii metabolicznej Rhodiola rosea L.

• Języki publikacji: EN

Abstrakty

EN

Agrobacterium tumefaciens EHA101 (pTd33) strain carrying uidA (GUS) reporter gene was used in model experiments on roseroot callus transformation. The T-DNA of pTd33 binary vector plasmid harbors nptII gene conferring resistance to kanamycin, and a uidA reporter gene, encodes the ß-glucuronidase enzyme. Roseroot seeds were sterilized and germinated on half strength MS media of which 70% germinated without any pretreatment. Calli were obtained from leaf segments of the in vitro grown seedlings. Calli was grown on solid MS medium supplemented with 1 mg l⁻¹ NAA and 0.5 mg l⁻¹ BAP. Different types of calli were obtained of which the green and compact type was chosen for transformation experiments. After co-cultivation with agrobacteria, calli were transferred to the same medium supplemented with 20 mg l⁻¹ kanamycin, 200 mg l⁻¹ carbenicillin and 300 mg l⁻¹ claforan with antioxidants (Polyclar and DTE) for selection. GUS test using a titron buffer was applied for monitoring the transformation of the calli. DNAs of 20 individual samples was extracted and subjected for PCR analysis proved the stable transformation in all of the taken samples by amplifying the nptII gene fragment. The method introduced here can be a tool for inserting and over-expressing the genes encoding for hypothesized enzymes to be involved in the biosynthesis of pharmaceutically important bioactive molecules of roseroot and therefore facilitating the applications for callus culture of roseroot in different bioreactor systems for pharmaceutical productions.

PL

Szczep Agrobacterium tumefaciens EHA101 (pTd33) przenoszący gen reporterowy uidA (GUS) został użyty w modelowych doświadczeniach nad transformacją kalusa różeńca górskiego. T-DNA z plazmidy wektora binarnego pTd33 mieści w sobie gen nptII przekazujący odorność kanamycynie, natomiast gen reporterowy uidA koduje enzym ȕ-glukuronidazy. Nasiona korzenia różeńca gorskiego wysterylizowano i poddano kiełkowaniu na połowie zestawu pożywki MS. Spośród nich 70% wykiełkowało bez żadnego wcześniejszego zabiegu. Kalusy otrzymano z segmentów liści sadzonek wyrosłych in vitro. Kalusy wyhodowano na stałej pożywce MS z dodatkiem 1 mg l⁻¹ NAA oraz 0,5 mg l⁻¹ BAP. Uzyskano różne typy kalusa, z których typ zielony i zwarty zostaá wybrany do doświadczeĔ dotyczących transformacji. Po wspólnej hodowli z agrobakteriami kalusy byáy przeniesione to tej samej pożywki uzupełnionej 20 mg l⁻¹ kanamycyny, 200 mg l⁻¹ karbenicyliny oraz 300 mg l⁻¹ klaforanu z przeciwutleniaczami (Polyclar i DTE) do selekcji. Zastosowano test GUS przy użyciu bufora trytronowego do monitorowania kalusów. Wyodrębniono 20 indywidualnych próbek DNA i poddano je analizie PCR, która wykazała stabilną transformację we wszystkich próbkach poprzez amplifikowanie fragmentu genu nptII. Metoda przedstawiona tutaj może być narzędziem insercji i ekspresji kodowania genów do hipotetycznych enzymów, które mają brać udział w biosyntezie ważnych z punku widzenia farmaceutycznego molekuł różeca górskiego, co ułatwi zastosowanie hodowli kalusów różeńca górskiego w różnych systemach bioreaktorów w produkcji farmaceutycznej.

Słowa kluczowe

EN

transgenic culture; callus culture; establishment; metabolic engineering; Rhodiola rosea; roseroot

• Wydawca: -
• Czasopismo: Acta Scientiarum Polonorum. Hortorum Cultus
• Rocznik: 2014
• Tom: 13
• Numer: 4
• Opis fizyczny: p.92-106,fig.,ref.

Twórcy

autor

Mirmazloum I.

• Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O.Box 53, H-1518 Budapest, Hungary

autor

Forgacs I.

• Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O.Box 53, H-1518 Budapest, Hungary

autor

Zok A.

• Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O.Box 53, H-1518 Budapest, Hungary

autor

Pedryc A.

• Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O.Box 53, H-1518 Budapest, Hungary

autor

Gyorgy Z.

• Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O.Box 53, H-1518 Budapest, Hungary

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