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Although a transient transformation of T. borchii mycelium has already been obtained [ 35], stable transformants cannot be exploited to provide a direct functional evidence to support this hypothesis.
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Among transformants, diploid plants cannot be distinguished from haploid plants using morphological traits alone [ 37].
Frozen stocks of single transformants and pools of transformants were prepared by standard methods.
These transformants were named transformant cphA6803# 1and transformant cphA6803 2.
The plasmid from one transformant remains to be identified.
Transformants were selected on NACm30 and several transformants were subcultured into FTG broth.
Transformants were selected on −HIS media and then correct transformants were screened for by PCR.
60 putative transformants were obtained on kanamycin containing plates, and 6 real transformants were obtained from these 60 putative transformants by spraying with glyphosate.
Genomic PCR on independent transformants was performed, and 15 independent lines of T0 tobacco transformants were obtained.
The plates with potential transformants were incubated at 30°C for at least 3 days until transformants appeared.
Leu+ transformants were replica-plated onto 5FOA plates to select for correct replacement transformants.
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