Induction of Somatic Embryogenesis through Overexpression of ATRKD4 Genes in Phalaenopsis “Sogo Vivien”
https://doi.org/10.22146/ijbiotech.15276 Exsyupransia Mursyanti(1), Aziz Purwantoro(2), Sukarti Moeljopawiro(3), Endang Semiarti(4*)
(1) Biotechnology Study Program, Graduate School, Universitas Gadjah Mada, Yogyakarta, Indonesia Faculty of Technobiology, Universitas Atma Jaya Yogyakarta, Yogyakarta, Indonesia
(2) Biotechnology Study Program, Graduate School, Universitas Gadjah Mada, Yogyakarta, Indonesia Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia
(3) Biotechnology Study Program, Graduate School, Universitas Gadjah Mada, Yogyakarta, Indonesia Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia
(4) Biotechnology Study Program, Graduate School, Universitas Gadjah Mada, Yogyakarta, Indonesia Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia
(*) Corresponding Author
Abstract
Phalaenopsis “Sogo Vivien “is a mini orchid hybrid with beautiful flowers and numerous inflorescences.
Mass propagation of this orchid is needed to meet the market demand. Objective of this research was to
induce somatic embryogenesis of P.”Sogo Vivien” through insertion of AtRKD4 gene into orchid. T-DNA
containing 35S::GAL4::AtRKD4::GR was inserted into 16-22 days after sowing orchid protocorms mediated by
Agrobacterium tumefaciens EHA 105. Activation of the AtRKD4 gene was induced by glucocorticoid induction
system, using 15μM Dexamethasone (Dex). The results showed that 34 out of 2,648 orchid embryos developed
into protocorms on hygromycin selection medium, whereas only 4 out of 2,897 non-transformant protocorms
developed from embryos. A 500 bp of HPT genes was amplified from transformant candidates using specific
primers for HPT (HygF1 and HygR1) and 380 bp was amplified using specific primers for AtRKD4 (AtRKD4
F1 and AtRKD4 R1), indicated that transgenes have been integrated into orchid genomes. Finally, 17 plantlets
were positively carrying AtRKD4 and HPT genes, the efficiency of transformation was 0.63 %. Somatic embryos
were also emerged from leaf explants of transformant on hormone-free NP medium and became normal
plantlets. It is probably due to the high activity of AtRKD4 genes in orchids
Mass propagation of this orchid is needed to meet the market demand. Objective of this research was to
induce somatic embryogenesis of P.”Sogo Vivien” through insertion of AtRKD4 gene into orchid. T-DNA
containing 35S::GAL4::AtRKD4::GR was inserted into 16-22 days after sowing orchid protocorms mediated by
Agrobacterium tumefaciens EHA 105. Activation of the AtRKD4 gene was induced by glucocorticoid induction
system, using 15μM Dexamethasone (Dex). The results showed that 34 out of 2,648 orchid embryos developed
into protocorms on hygromycin selection medium, whereas only 4 out of 2,897 non-transformant protocorms
developed from embryos. A 500 bp of HPT genes was amplified from transformant candidates using specific
primers for HPT (HygF1 and HygR1) and 380 bp was amplified using specific primers for AtRKD4 (AtRKD4
F1 and AtRKD4 R1), indicated that transgenes have been integrated into orchid genomes. Finally, 17 plantlets
were positively carrying AtRKD4 and HPT genes, the efficiency of transformation was 0.63 %. Somatic embryos
were also emerged from leaf explants of transformant on hormone-free NP medium and became normal
plantlets. It is probably due to the high activity of AtRKD4 genes in orchids
Keywords
AtRKD4; glucocorticoid; Phalaenopsis “Sogo Vivien”; orchid hybrid; somatic embryo
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PDF DOI: https://doi.org/10.22146/ijbiotech.15276
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