We have optimized a procedure for genetic transformation of a major leafy vegetable crop,
Amaranthus tricolor L., using epicotyl explant co-cultivation with
Agrobacterium tumefaciens. Two disarmed
A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (
nptII) and the β-glucuronidase gene (
gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated
Agrobacterium culture corresponding to OD
600???0.6 and diluted to 10
9 cells ml
?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml
?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the
nptII and
gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T
0). Among T
1 seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (
χ 2) test. Southern hybridization of genomic DNA from kanamycin-resistant T
1 transgenic segregants to an
nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T
1 transgenic plants, thereby confirming stable expression of the
nptII gene.
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