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TRANSFORMASI GEN PEMBUNGAAN MELALUI AGROBACTERIUM TUMEFACIENS SECARA IN-VITRO PADA TANAMAN ANGGREK VANDA TRICOLOR DWIYANI, RINDANG; YUSWANTI, HESTIN; MERCURIANI, IXORA SARTIKA; SEMIARTI, ENDANG
Agrotrop: Journal on Agriculture Science Vol 6 No 1 (2016)
Publisher : Fakultas Pertanian Universitas Udayana

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Abstract

In Vitro Transformation of Flowering Gene through Agrobacterium tumefaciens on Vandatricolor Orchid. Research concerning of Agrobacterium-mediated transformation of Vanda tricolor orchid had been done during March to August 2014. Objectives of this research wasto investigate the effects of period of inoculation with Agrobacterium suspension and application of acetosyringone (AS) on the percentage of transforman candidates. The T-DNA harbored the PaFT gene under the control of promoter of Ubiquitin and the hpt gene, ahygromycine resistant gene as a selectable marker for transformant selection. This T-DNA was constructed in the PGAS vector and transferred to the plant genome mediated by A. tumefaciens. The period of inoculation with Agrobacterium and application ofAcetosyringone (AS) were trialed in this research. The result showed that a period of one hour inoculation with Agrobacterium suspension added with 25 ppm AS resulted in the highest number of transforman candidates. i.e. 7.04% compared to 4.10% (one hour inoculation without AS), 3.76% (two hours inoculation without AS), and 4.48% (two hours inoculation with 25 ppm AS).
INDUCTION AND GROWTH KINETICS CALLUS OF TOMATO (SOLANUM LYCOPERSICUM) Setiaji, Arkan; Annisa, RR Rifka; Rumiyati, Rumiyati; Semiarti, Endang
Biosaintifika: Journal of Biology & Biology Education Vol 12, No 1 (2020): April 2020 Article-in-Press
Publisher : Department of Biology, Faculty of Mathematics and Sciences, Semarang State University . Ro

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15294/biosaintifika.v12i1.21704

Abstract

Plant callus extracts are potential to be developed as ingredient in skincare products. Tomato callus is supposed to contain protein-derivatives and or other components such as secondary metabolites that play a role in skin regeneration. Therefore the production of calli is important to be studied for callus sustainable supply. This research aims to obtain optimum medium for callus induction and to analyze tomato callus development anatomically. In vitro culture response was assessed in tomato plant (Solanum lycopersicum L. ?Permata?) for optimum callus induction. Seeds were grown on ¼ MS medium for 10-15 days. Hypocotyl was excised and cultured on MS medium + 2 mg/l 2,4-D for 15 days as the explants for callus induction. Callus was transferred to MS medium with 8 variations of PGRs including the combination of BAP + NAA, and 2,4-D. Both fresh and dry weight was measured every 5 days over 60 days to establish the growth kinetics and growth efficiency of callus. Anatomic characters of calli were examined through paraffin-embedded method. The result showed of MS medium supplemented with 2.0 mg/l NAA and 0.2 mg/l BAP is optimum for tomato callus induction, based on highest number of the absolute growth rate on fresh weight (73.77% per day), dry weight (3.84% per day), and callus initiation time (5.56 days) achieved by the medium. Cells in the ground tissue of tomato hypocotyl are competent to be dedifferentiated into a callus. This research results were expected to find out suitable methods for tomato callus production in preparation for skincare uses.
THE INFLUENCE OF THIDIAZURON ON DIRECT SOMATIC EMBRYO FORMATION FROM VARIOUS TYPES OF EXPLANT IN PHALAENOPSIS AMABILIS (L.) BLUME ORCHID Mose, Windi; Indrianto, Ari; Purwantoro, Aziz; Semiarti, Endang
HAYATI Journal of Biosciences Vol. 24 No. 4 (2017): October 2017
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1348.365 KB) | DOI: 10.4308/hjb.24.4.201

Abstract

Phalaenopsis amabilis is an important national flower of Indonesia as a parent for orchidbreeding, so that needs a good strategy to produce high number of plants. The objective of this research is to analyze the use of thidiazuron (TDZ) for producing high number of plantlets, through directly induction of somatic embryos (SEs) from various explants. The method was used 20 each of protocorms, leaves, stems and roots as explants. The explants were dissected transversely, then put on various culture media: New Phalaenopsis (NP) and NP + (1, 2, 3) mgL?1 TDZ. Cultures were maintained at 25°C with continous white light. The formation of SEs was observed every week for 8 weeks. The results showed that SEs formation increased inline with the addition of TDZ concentration to the NP medium, for both velocity and amount of SEs formation. In NP0, SEs were formed at (26.07 ± 0.73) days after inoculation of protocorm, whereas on NP + (1, 2, and 3 mgL?1) TDZ, SEs were formed at (17.85 ± 0.67) days, (15 ± 0.64) days, and (11 ± 0.64) days, respectively. All types of explants formed SEs on NP + TDZ (1?3 mgL?1), whereas only 14 of 20 protocorms produced SEs (70%), and 8 of 20 stems formed SEs (40%) in NP0. In roots, SEs was formed on NP + 2 mgL?1 TDZ and NP + 3 mgL?1 TDZ. For stems, the highest amount of SEs (28.25 ± 1.07) was reached on NP + 3 mgL?1 TDZ, followed by protocorm (23.30 ± 1.13) SEs and roots (8.25 ± 0.68) SEs. In contrast, in NP0, the amount of SEs was very low (1.25 ± 0.46) from stem and (1.50 ± 0.65) from protocorms, there was no evidence of SEs formation in the leaves and roots.
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.
HOMOLOGI FUNGSI GEN KNAT1 ( Knotted 1– like Arabidopsis thaliana) PADA ANGGREK BULAN Phalaenopsis amabilis (L.) Bl. DENGAN MEDIATOR Agrobacterium tumefaciens Isminingsih, Sulastri; Semiarti, Endang; Purwantoro, Aziz
Jurnal Agroekoteknologi Vol 1, No 1 (2009)
Publisher : Jurusan Agroekoteknologi Fakultas Pertanian Untirta

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Abstract

ABSTRACTTo understand the function of KNAT1 gene Arabidopsis in the forming and developing of Indonesian origin orchid shoot Phalaenopsis amabilis through transform p35S::KNAT1 and pGreen to protocorm like bodies (plb) of the orchid mediated by Agrobacterium tumafaciens LBA4404. The plb transformants were grown on New Phalaenopsis selection medium containing 5 mg/l BAP, 0.15 mg/l NAA, 15 mg.l Kanamycin and addition of 300 mg/l Cefotaxim to eliminate the overgrowth of Agrobacterium. The analysis of positive transformant use Polimerase Chain Reaction (PCR) with the specific oligonucleotide primers for KNAT1 gene: KNAT1F1R1 and universal primers for pGreen: 35SO and Tnos. The result shows that 3 shoots of 1850 transformants positively carry out the 35S::KNAT1 construct (frequency of transformation was 0.16 %) while 5 shoot of 1850 transformants also positively carry out the pGreen vector, with the frequency of transformation was 0.27 %. The phenotype analysis of 35S::KNAT1 transformants show multiplication on forming of the leaf from a plb to + 10 shoots and forming of the leaf shape which has terompet like shapes and rectangular shape.Key words: Arabidopsis, KNAT1 gene, Phalaenopsis amabilis, shoot
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

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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
Early detection of the orchid flowering gene PaFT1 in tobacco cells using a GFP reporter Wahyuningsih, Sri; Lawrie, Muhammad Dylan; Daryono, Budi Setiadi; Moeljopawiro, Sukarti; Jang, Soenghoe; Semiarti, Endang
Indonesian Journal of Biotechnology Vol 21, No 1 (2016)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1401.252 KB) | DOI: 10.22146/ijbiotech.26781

Abstract

Here we describe a novel method of using green fluorescence protein (GFP) as a reporter gene for early detection of an integrated T­DNA containing the orchid flowering gene, PaFT1 (Phalaenopsis aphrodite Flowering locus T1) in the tobacco genome. Functional assays that report the presence of exogenous DNA early in development are especially useful in plants where the desired phenotype is only apparent after long periods of vegetative growth. The objective of this study is to establish a method for detecting an inserted Phalaenopsis orchid flowering gene and examining its function in tobacco. The p35S::PaFT1­ 35S::GFP construct was introduced into Agrobacterium tumefaciens strain EHA101. Transformed tobacco leaves were cultured on MS medium with addition of 1 mgL-1 NAA+3 mgL-1 BAP+50 mgL-1 Kanamycin+300 mgL-1 timentin for selection. Results showed bright green GFP fluorescent signals in 11 out of 15 (73%) tobacco leaf cells at a 2­month time point after transformation. GFP and PaFT1 fragments were amplified in genomic PCR using GFP and PaFT1 specific primers. The accumulated PaFT1 transcripts were observed in 3 month­old transgenic tobacco plants containing p35S::PaFT1­35S::GFP. Green florescence was observed only in the transgenic plants at the 5 month­old stage but not in the wild type controls.
PENGARUH CAHAYA DAN TEMPERATUR TERHADAP PERTUMBUHAN TUNAS DAN PROFIL PROTEIN TANAMAN ANGGREK Phalaenopsis amabilis TRANSGENIK PEMBAWA GEN Ubipro::PaFT Putra, Rinaldi Rizal; Mercuriani, Ixora Sartika; Semiarti, Endang
Bioeksperimen: Jurnal Penelitian Biologi Vol 2, No 2: September 2016
Publisher : Universitas Muhammadiyah Surakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.23917/bioeksperimen.v2i2.2483

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Penelitian ini bertujuan untuk mencari kondisi yang tepat dalam percepatan pembungaan tanaman Phalaenopsis amabilis transgenik yang telah disisipi gen penentu waktu pembungaan Ubipro::PaFT. Metode penelitian ini menggunakan tanaman transgenik pembawa gen Ubipro::PaFT umur 18 bulan setelah penanaman. Tanaman ditumbuhkan pada inkubator dengan pencahayaan menggunakan lampu LED putih dan kombinasi LED putih biru, dengan fotoperiodisitas 8 jam terang 16 jam gelap, suhu 25ºC pada fase terang dan 20ºC pada fase gelap selama 20 minggu. Setelah 20 minggu pertumbuhan tanaman, dilakukan analisis profil protein dengan metode SDS-PAGE untuk mengetahui protein yang diproduksi pada setiap fase pertumbuhan yang diamati.Hasil penelitian menunjukkan kombinasi cahaya LED putih dan biru meningkatkan pembentukan daun sebesar 60%, panjang daun 70,58%, tetapi belum diperoleh kemunculan infloresen. Analisis profil protein menunjukkan terbentuknya protein dengan ukuran 108,57; 71,30; 56,16; 40,85; 26,79; 13,27; dan 13,12 kilodalton pada tanaman transgenik, tetapi tidak terdeteksi protein dengan ukuran 19,65 kDa yang sesuai dengan berat molekul protein PaFT, sementara protein dengan ukuran sekitar 56,16 kDa sesuai dengan berat molekul protein POH1(Phalaenopsis Orchid Homeobox1). Hal ini menunjukkan bahwa gen vegetatif POH1 mampu menghambat aktivasi gen PaFT pada tanaman P. amabilis transgenik umur 20 minggu, sehingga tanaman masih dalam fase juvenil dan belum mampu diinduksi untuk berbunga.
Micropropagation of Mini Orchid Hybrid Phalaenopsis “Sogo Vivien” Mursyanti, Exsyupransia; Purwantoro, Aziz; Moeljopawiro, Sukarti; Semiarti, Endang
Journal of Tropical Biodiversity and Biotechnology Vol 1, No 1 (2016): June
Publisher : Faculty of Biology, Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (426.637 KB) | DOI: 10.22146/jtbb.12933

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Phalaenopsis “Sogo Vivien” is an orchid hybrid with mini size plant body, and exhibits numerous beautiful pink flowers, that is ideal as ornamental pot plant. Some plants of this orchid exhibit variegated leaves that improve the beauty of the plant, not only because of the flower but also as attracted leaves. This orchid has high economical value, but mass propagation of this orchid has not established yet. An effective method to propagate both the normal and variegated plants is worth to be generated. The objective of this research was to produce a large number of P. “Sogo Vivien” plants, including the variegated plants. The method used seeds from self pollinating variegated plant, and flower stalk nodes. The seeds were sown on three various medium: VW, NP and MS, and flower stalk nodes were planted on VW + BA 10 mg l-1 + active carbon. The results showed that the best medium for in vitro culture of P. “Sogo Vivien” was NP medium, in which all seeds could grew into plantlets. Most plantlets emerged from the seeds were non variegated, only one plantlet out of 1344 seeds was variegated (0.007%). Although all emerged plantlets from flower stalk exhibited variegated leaves. Particularly, the plantlets arised from the second and third basal nodes of flower stalk showed the highest growth rate than that from the other nodes. Histological analysis showed that at 11-13 days after shoot segment plantation on NP medium, the shape of apical cells in the nodes was changed, then followed by the change of cell shape in the basal part of the nodes, produced bipolar pattern, then gradually developed into shoot. These results suggest that mass propagation could be achieved using seed culture, but to get the variegated phenotypes, the second and third nodes of flower stalk from variegated plant were the best explants to be used.
KONSERVASI ANGGREK ALAM INDONESIA VANDA TRICOLOR LINDL. VARIETAS SUAVIS MELALUI KULTUR EMBRIO SECARA IN-VITRO Dwiyani, Rindang; Purwantoro, Azis; Indrianto, Ari; Semiarti, Endang
Bumi Lestari Journal of Environment Vol 12 No 1 (2012)
Publisher : Environmental Research Center (PPLH) of Udayana University

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Abstract

Vanda tricolor Lindl. var. suavis is an Indonesian wild orchid which is now extremely rare in nature due to its habitat destruction. Development of an appropriate method for propagation of this species through in vitro culture could be nessecary for conservation purposes. High phenolic content of plant tissue is a serious problem for V. tricolor research in the laboratory, which inhibits germination and growth of the embryo. To overcome this problem, seeds were sown in a medium with the addition of tomato extract as an antioxidant. The aim of this research is to find the most suitable concentration of tomato extract for germination and growth of the embryo of V. tricolor form Bali and Merapi in order to obtained healthy seedlings for conservation purposes. Orchid pods (7 months after polination) of V. tricolor Bali and Merapi were used as plant material. The treatment consisted of 5 concentrations of tomato extract which is 0, 50 100, 150, 200, 250 g L-1. Observation was done by counting the number of protocorms for each stage of growth at 4 weeks after seed sowing. The study concluded that V. tricolor Bali is more responsive to the tomato extract compared with Merapi. Concentration of 150 gL-1 tomato extract gave the highest percentage of protocorms for Bali form, whereas Merapi form did not give significant differences either with or without tomato extract added in the culture medium.