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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.
Stability of T-DNA Integration in Phalaenopsis “Sogo Vivien” Transgenic Orchid Carrying 35S::Gal4::AtRKD4::GR Endang Semiarti; Exsyupransia Mursyanti; Ahmad Suyoko; Faiza Senja Widya Perdana; Catharina Tri Widyastuti; Aditya Nur Subchan
Biology, Medicine, & Natural Product Chemistry Vol 7, No 1 (2018)
Publisher : Sunan Kalijaga State Islamic University & Society for Indonesian Biodiversity

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (843.81 KB) | DOI: 10.14421/biomedich.2018.71.5-13

Abstract

Orchid is an elegant ornamental plant and favoured by the society. Phalaenopsis "Sogo vivien" is a mini-sized orchid with an interesting white-striped purple petals. This study was aimed to analyze the stability of the integration of embryonic gene carrier T-DNA from Arabidobsis AtRKD4 into the P. "Sogo vivien" genome produced in 2016. The study was conducted in 3 stages: 1) Transgenic plant phenotype analysis (1 year old); 2) Examination of T-DNA integration in orchid genotypes using PCR. 3) Analysis of transgenic plant leaf explants’ ability to produce somatic embryo in vitro. In vitro cultures were performed on the base medium of New Phalaenopsis (NP), plus various concentrations of TDZ (0, 1, 2 mg.L-1) and IBA (0, 1, 2 mg.L-1) or without TDZ and IBA as controls. The transgenic Phalaenopsis ‘Sogo vivien’ were transferred to pot mediums via ex vitro with two treatments: the first leaves were cut as explants for in vitro culture, and the plants were transferred to the mixture of fern medium with shavings of bark. The integration of T-DNA in the genome was detected by DNA genome amplification from the second leaves using the AtRKD4 gene primers and the POH1 gene. The results showed that the highest number of somatic embryo (SE) propagules or protocorm like bodies (PLBs) amounted to 27 were derived from transgenic plant # 2 cultured on NP + 2 mg.L-1 TDZ +1 mg.L-1 IBA medium. The presence of AtRKD4 transgenes were detected with the amplification of 380 bp of the RKD4 gene from the genome of transgenic plant # 2 by using PCR. There were 2 out of 15 plants that positively carry the AtRKD4 gene and produce SE. Thus, the stability of the AtRKD4 carrier T-DNA integration in the genomes of transgenic plants was 13.3%.
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

Abstract

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.
Improvement of Genetic Transformation Efficiency in Vanda tricolor Orchid Using Acetosyringone Dwiyani, Rindang; Purwantoro, Azis; Indrianto, Ari; Semiarti, Endang
ANNALES BOGORIENSES Vol 14, No 2 (2010): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/53

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  improving   Vanda  orchid  through  genetic modification could be valuable for horticulture and, indirectly, also for conservation. In this research, a method of  Agrobacterium-mediated transformation of two  Vanda  tricolor  obtained  from  Salak Mount, West Java and merapi Mount, Yogyakarta in Indonesia protocorms was improved using acetosyringone (AS). Concentrations of 0 and 25 ppm AS were used in transformation of pG35S binary vector containing kanamycin resistance gene into V.  tricolor  protocorms.  The  result  showed  that  25  ppm  AS  was  required  on  innoculation  with Agrobacterium  solution, without AS on cocultivation. Five week s after  treatment  on the 300  ppm kanamicyncontaining medium, green protocorms were obtained, that  was  11.01% for  V.  tricolor  from Salak Mount with pre-culture treatment prior to innoculation, 9.39% for  V.  tricolor  from Merapi  Mount with pre-culture treatment prior to  innoculation,  and  1.37%  for  V.  tricolor  from  Merapi  Mount  without  pre-culture  treatment  prior  to innoculation. The  best  condition  to  set  high  efficiency  of  transformation  is  pre -culture  protocorms  prior inoculation, soaking protocorm on 25  ppm  AS for 30 minutes, then cocultivate its on AS-free  callus induction medium Key words: Vanda tricolor, Agrobacterium, orchid protocorms, acetosyringone
Karakterisasi Isolat Rhizoctonia sp. Patogenik dan Rhizoctonia Mikoriza Pada Tanaman Anggrek Tanah Spathoglottis plicata Soelistijono, Soelistijono; Priyatmojo, Achmadi; Semiarti, Endang; Sumardiyono, Christanti
Biota : Jurnal Ilmiah Ilmu-Ilmu Hayati Vol 16, No 2 (2011): June 2011
Publisher : Universitas Atma Jaya Yogyakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (300.536 KB) | DOI: 10.24002/biota.v16i2.121

Abstract

Rhizoctonia mikoriza merupakan jamur yang mampu berasosiasi dengan anggrek tanah. Selain sebagai mikoriza, terdapat isolat Rhizoctonia sp. patogen dan penyebab penyakit busuk akar pada Spathoglottis plicata. Penelitian ini bertujuan mengetahui perbedaan antara Rhizoctonia sp. patogen dan Rhizoctonia mikoriza secara morfologi dan molekular menggunakan teknik RAPD. Hasil penelitian secara morfologi menunjukkan bahwa warna koloni, panjang dan jumlah inti sel isolat Rhizoctonia sp. patogen dan Rhizoctonia mikoriza pada S. plicata tidak berbeda, tetapi berbeda pada ketebalan sel dan pengelompokan isolat berdasarkan uji anastomosis hifa. Teknik molekuler RAPD menunjukkan bahwa setiap isolat Rhizoctonia sp. patogen dan Rhizoctonia mikoriza memiliki perbedaan pada struktur DNA.
Micropropagation of Dendrobium phalaenopsis Orchid Through Overexpression of Embryo Gene AtRKD4 Setiari, Nintya; Purwantoro, Aziz; Moeljopawiro, Sukarti; Semiarti, Endang
AGRIVITA, Journal of Agricultural Science Vol 40, No 2 (2018): JUNE
Publisher : Faculty of Agriculture University of Brawijaya in collaboration with PERAGI

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.17503/agrivita.v40i2.1690

Abstract

To increase the efficiency of crop production from Dendrobium phalaenopsis orchids, mass propagation has been performed by inducing somatic embryogenesis through Agrobacterium-mediated transformation of the Arabidopsis embryo gene AtRKD4 into orchid protocorm (developing orchid embryo). The three-week-old protocorms of D. phalaenopsis were genetically transformed with T-DNA carrying 35S :: GAL4 :: AtRKD4 :: GR through A. tumefaciens strain EHA 105. The cultures were maintained in VW medium with 10 mg L-1 Hygromycin. Due to the existence of glucocorticoid response element (GR) in the T-DNA construct, the transformed protocorms were transferred into VW medium with the addition of 15 μM Dexamethasone in 6 weeks after transformation to activate the transgene. A total of 12% protocorms has been confirmed for Hyg + by using PCR. The expression of embryo gene AtRKD4 was confirmed by cDNA analysis using AtRKD4 specific primers and Actin primers as a positive control experiment. The expression level of AtRKD4 in 2.5-month-old D. phalaenopsis transformant shoots was 7 times higher than non-transformant plants, and increased to 86 times higher in 8-months, that much higher than that of non-transformant. These results provide an improved method for genetic transformation of D. Phalaenopsis and will (eventually) increase production efficiency in the future.
Morphological And Biochemical Responses Of Saccharum Spontaneum L. Accessions To Drought Stress Munawarti, Aminatun; Taryono, Taryono; Semiarti, Endang; Sismindari, Sismindari
Journal of Tropical Life Science Vol 4, No 1 (2014)
Publisher : Journal of Tropical Life Science

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

Glagah (Saccharum spontaneum L.) has potential value as a crop species and may also be used in sugarcane breeding programs; however, this germplasm has not been extensively used in breeding programs, primarily in relation to improve drought tolerance. The objectives of this experiment were to evaluate the effect of drought stress initiated at vegetative growth stage on growth, leaf proline content and protein pattern of seven glagah accessions (BOT-53, BOT-54, BOT-60, BOT-77, BOT-78, BOT-84, and BOT-88). The plants were propagated from single node stalk segments in polybag in the field under non-stress condition for two months. The two month-old plants were then subjected to drought stress by withholding watering for eight weeks. Untreated control plants were watered every two days. Results indicated that drought stress reduced plant height, stalk diameter and green leaf number. On the other hand, there was a little difference between drought-stressed and control plants in terms of proline content. The protein pattern showed that drought stress caused a change in gene expression in the form of induction or repression of protein expression. A specific protein with a low range of molecular weight (Rf value about 0.647) showed constitutively expressed in accession BOT-53 but drought-inducible expressed in BOT-54. Keywords: Drought stress, glagah, proline, protein pattern, Saccharum spontaneum
Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids Endang Semiarti; Sri Nopitasari; Yuli Setiawati; Muhammad Dylan Lawrie; Aziz Purwantoro; Jaka Widada; Yasushi Yoshioka; Shogo Matsumoto; Kana Ninomiya; Yuuki Asano
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 due to their natural beauty of flowers. In the tropical forest, orchids are being acquired for trading and commercial market. Thus, the effort is required to proliferate orchid in large quantities for conservation and improve the floral variation for plant breeding. The purpose of this study is to develop a firmed methodology of molecular breeding of orchids using CRISPR/Cas9 KO system. The plant material used was Phalaenopsis amabilis protocorms growth on NP medium+pepton (2 g/L). Protocorm were submerged in the culture of Agrobacterium tumefaciens that Ti‐plasmid had been filled with a T‐DNA construct of a pRGEB32 vector harboring sgRNA with PDS3 sequence. Detection for transformants was confirmed 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 0.96% PDS transformants were obtained from PDS3T2 lines. Several transformant showed pale leaf color compared to non‐transformant plants. This study suggests that the target gene has successfully edited by CRISPR/Cas9 system and could be applied for that functional gene editing in orchids.
PENGARUH CAHAYA DAN TEMPERATUR TERHADAP PERTUMBUHAN TUNAS DAN PROFIL PROTEIN TANAMAN ANGGREK Phalaenopsis amabilis TRANSGENIK PEMBAWA GEN Ubipro::PaFT Rinaldi Rizal Putra; Ixora Sartika Mercuriani; Endang Semiarti
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

Abstract

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.
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
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.