| Summary: | Phalaenopsis amabilis (L.) Blume is one of Indonesian natural orchid which
has an aesthetic flower so it has a high economic value. The low multiplication rate
and long periods of life cycle are the main obstacles to propagate this orchid
conventionally. To produce high rate of plant�s multiplication, the KNAT1 (Knotted
like Arabidopsis Thaliana) gene of Arabidopsis was inserted into orchid protocorms
(developping orchid embryo) under the control of 35S-strong promoter. KNAT1 gene
is reported as a gene that involved in the shoot formation, and it had been
successfully introduced into Phalaenopsis amabilis (L.) Blume genom. After seven
times regeneration, the confirmation of the transgene existence in the genom is
needed to ensure whether the secondary growth plant could stably maintain the
transgene in its genome or not.
The experiment was carried out in 3 steps: 1). Co-integration analysis of
35S::KNAT1 into P. amabilis genom. This step including selection of putative
transgenic onto NP0 (New Phalaenopsis) and shoot inducing medium (NP SIM) with
addition of of kanamycin. Furthermore, the resistance plants that grow on selection
medium then subjected for PCR analysis to ensure whether its harbor the transgene or
not. 2). Phenotypic analysis on the multiplication rate, morphological variation, and
venation pattern. 3) Protein profile analysis of transgenic plants.
The results showed that the survival rate of putative transgenic was 58,7 % on
NPO medium and 62.5 % on NP SIM medium. PCR analysis confirmed that 82.5%
transgenic growth on NP0 and 93,33% on NP SIM contained DNA fragment of
KNAT1 gene, NPTII gene and trnL-F intergenic spacer, indicating that they are
positive transgenic. The 35S::KNAT1 transgenes and phytohormone were
independently involved in multishoots formation of P. amabilis transgenic plants.
The morphological performance was classified into 6 criterias, namely: normal shape,
lobed leaves, rossete, elongated stem, cup shoot, and widened leaves. The normal
type was the most abundant type of variation (± 29 %) in both medium. Protein
profile shows that all transgenic plants produced 45,8 kDa protein and that was
equivalent with molecular weight of KNAT1 protein. All those data show that
35S::KNAT1 transgene were stably integrated into the transgenic plant genome.
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