ISSN:
1573-5044
Schlagwort(e):
agar/Gelrite combination
;
aminoglycosides
;
β-glucuronidase
;
Malus × domestica Borkh
;
Marshall McIntosh
;
neomycin phosphotransferase
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
Notizen:
Abstract The goal of this research was to develop an efficient transformation system for 'Marshall McIntosh' apple. To determine the optimum combination of agar and Gelrite gelling agents in the media to maximize regeneration and minimize hyperhydicity (vitrification), the following combinations of agar (A)+Gelrite (G) in g l-1 were tested: 7.0 A+0 G; 5.2 A+0.6 G; 3.5 A+2.5 G; 1.7 A+1.8 G; and 0 A+2.5 G. Both 5.2 A+0.6 G and 3.5 A+1.2 G provided greatest regeneration of healthy non-hyperhydric shoots. To determine the optimal concentration of aminoglycoside for the selection and regeneration of transgenic 'Marshall McIntosh' on agar-Gelrite-based media, kanamycin was tested at 0, 10, 25, 50, 75 and 100 mg l-1, and paromomycin was tested at 0, 50, 100, 150, 200 and 250 mg l-1. Kanamycin was more effective than paromomycin in the initial selection of transgenics. For selection of transformants of 'Marshall McIntosh', the use of kanamycin at 25 mg l-1on 5.2 A+0.6 G solidified medium is suggested. By optimizing the medium and selection conditions, a protocol was developed that resulted in four transgenic lines as confirmed by a GUS assay, NPT II ELISA, PCR, and Southern analysis. In repeated experiments with this protocol, transformation efficiencies of 3.1 and 2.6% were obtained.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1023/A:1026414031217
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