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The purpose of this study was to establish an efficient and
stable gene transfer system for Phalaenopsis. Two methods of
transformation were tested: pollen tube pathway and particle
bombardment. To establish the transformation system via pollen
tube pathway, the development of ovary and megaspore mother
cell in Phalaenopsis is microscopically examined. Pollens
germinated readily 3 days after pollination. Megaspore mother
cells undergo meiotic division at about 50 days after
pollination. Ten days later, megaspore gives rise to the one-
nucleatea embryo sac. The zygote was observed at 70 days after
pollination. Fertilization presumably occurs at this time. The
pRT99gus plasmid containing GUS and NPTⅡ genes was injected
into Phalaenopsis ovary at different times after pollination.
Transformed Seedlings evidenced by polymerase chain reaction
using primers specific for GUS gene were found when the time
for DNA injection was between 25 and 40 days after pollination.
An intrinsic GUS-like enzyme activity was found when the
seedlings were 30-day-old. Stable integration was demonstrated
by Southern blot analysis of genomic DNA isolated from pooled
seedlings. An alternative transformation protocol using
germinated seedlings as target tissues was developed by using
PDS1000/He particle gun with a gas pressure of 650 psi. The
ratio in a population of seedlings expressing GUS activity when
assayed 3 days after bombardment was as high as 30﹪. Southern
blot analysis of genomic DNA isolated from seedlings 30 days
after bombardment showed positive signals. This study
demonstrated that transgenic Phalaenopsis plants could be
produced using particle bombardment.
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