Testing the phytotoxic level of kanamycin showed
that there was a total inhibition of shoot initiation from
leaf explants at 50 mg/l kanamycin and tissues were
necrotic within 4 weeks. When selection medium contained
40 mg/l kanamycin, only few chlorotic shoots
were regenerated. Our study indicated the kanamycinresistant
level of haploid plants was the same as its diploid
control. To avoid false positives (escapes) to the
most extent, we used selection medium containing
50 mg/l kanamycin. At last, five independently kanamycin-
resistant lines were obtained. All of them grew
normally even when kanamycin content was up to
80 mg/l. Furthermore, PCR assay indicated that these
lines were all positive for the expected band, while
wild-type lines were not (Fig. 4). These results indicated
that the
betA
gene was transformed into these five
kanamycin-resistant lines, which showed a transformation
frequency more than 6%. The further biological
analysis of
betA
will be performed in future. Counting
the number of chromosomes in transgenic lines showed
that these lines remained haploid.