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Journal : Indonesian Journal of Biotechnology

Induction of Somatic Embryogenesis through Overexpression of ATRKD4 Genes in Phalaenopsis “Sogo Vivien” Mursyanti, Exsyupransia; Purwantoro, Aziz; Moeljopawiro, Sukarti; Semiarti, Endang
Indonesian Journal of Biotechnology Vol 20, No 1 (2015)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1208.768 KB) | DOI: 10.22146/ijbiotech.15276

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 toinduce somatic embryogenesis of P.”Sogo Vivien” through insertion of AtRKD4 gene into orchid. T-DNAcontaining 35S::GAL4::AtRKD4::GR was inserted into 16-22 days after sowing orchid protocorms mediated byAgrobacterium tumefaciens EHA 105. Activation of the AtRKD4 gene was induced by glucocorticoid inductionsystem, using 15μM Dexamethasone (Dex). The results showed that 34 out of 2,648 orchid embryos developedinto protocorms on hygromycin selection medium, whereas only 4 out of 2,897 non-transformant protocormsdeveloped from embryos. A 500 bp of HPT genes was amplified from transformant candidates using specificprimers for HPT (HygF1 and HygR1) and 380 bp was amplified using specific primers for AtRKD4 (AtRKD4F1 and AtRKD4 R1), indicated that transgenes have been integrated into orchid genomes. Finally, 17 plantletswere positively carrying AtRKD4 and HPT genes, the efficiency of transformation was 0.63 %. Somatic embryoswere also emerged from leaf explants of transformant on hormone-free NP medium and became normalplantlets. It is probably due to the high activity of AtRKD4 genes in orchids
Selection of Phalaenopsis amabilis L. Blume Orchid Resistance to Hygromycin Mercuriani, Ixora Sartika; Purwantoro, Aziz; Moeljopawiro, Sukarti; Jang, Seonghoe; Semiarti, Endang
Indonesian Journal of Biotechnology Vol 17, No 2 (2012)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (258.811 KB) | DOI: 10.22146/ijbiotech.16000

Abstract

Examination of Phalaenopsis amabilis orchid resistance to hygromycin antibiotic is an important step to doprior to Agrobacterium-mediated genetic transformation in this orchids using Hygromycin phosphotransferase(HPT) gene as a selection marker in the T-DNA that harboring a desired gene to be transfered. We exposedthe plant on hygromycin containing medium. The experiment was conducted using 6 weeks old P. amabilisprotocorms. These protocorms were subcultured onto NP medium supplemented with various concentrationof Hygromycin (0, 5, 10, 20, 1nd 40 mg/l). The number of survival protocorms were examined every week for4 weeks after subcultured (WAS). The resistancy of hygromycin was calculated as ratio of death protocormsper total protocorms). The result showed that 10 mg/l hygromycin with 1 weeks of application caused deathclose to LD 50. This data indicate that P. amabilis resistance to hygromycin treatment on the appropriateconcentration 10 mg/l, and this concentration can be used for other purposes in orchid system.
APPLICATION OF CRISPR/CAS9 GENOME EDITING SYSTEM FOR MOLECULAR BREEDING OF ORCHIDS Semiarti, Endang; Nopitasari, Sri; Setiawati, Yuli; Lawrie, Muhammad Dylan; Purwantoro, Aziz; Widada, Jaka; Yoshioka, Yasushi; Matsumoto, Shogo; Ninomiya, Kana; Asano, Yuuki
Indonesian Journal of Biotechnology Vol 25, No 1 (2020)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/ijbiotech.39485

Abstract

Orchid is an important ornamental plant in Indonesia, because of the beauty of flowers. Therefore many people hunted in their natural habitat in tropical forests, to be traded. So it takes the effort of multiplication of orchid plants in large quantities for conservation, also needs to increase the beauty of orchid for plant breeding. The purpose of this study was to find an effective method for improving the quality and quantity with biotechnology, through the method of genome editing system using CRISPR/Cas9. The plant material used was Phalaenopsis amabilis protocorms growth on NP medium+pepton (2 gr/L). Protocorm is immersed in the culture of Agrobacterium tumefaciens which Ti-plasmid has been filled with a T-DNA construct of a pRGEB32 vector harboring the target genes PDS. Detection for transformants was conducted by PCR using  HPT primers (545 bp), Cas9 primers (402 bp), PDS primers (280 bp) and trnL-F (1200 bp) as an internal control. The results showed that PDS transformants had been obtained from 0.96% in PDS3T2. Some transformant phenotypes showed a pale leaf color compared to non-transformant plants. This suggests that the target genes could be truncated by CRISPR/ Cas9 system and it can be applied for functional gene editing in orchids