Imron Riyadi
Indonesian Research Institute for Biotechnology and Bioindustry

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Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium-mediated transformation Asmini BUDIANI; Imam Bagus NUGROHO; Hayati MINARSIH; Imron RIYADI
Menara Perkebunan Vol. 87 No. 2 (2019): 87 (2), 2019
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v87i2.336

Abstract

AbstrakCekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]Abstract      Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS (Δ1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]
Propagasi in vitro tanaman kurma (Phoenix dactylifera L.) pada bioreaktor dengan perendaman sesaat Rizka Tamania SAPTARI; Masna Maya SINTA; Imron RIYADI; . PRIYONO; . SUMARYONO
Menara Perkebunan Vol. 88 No. 2 (2020): 88 (2), 2020
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v88i2.394

Abstract

The cultivation of date palm in Indonesia has increased since the last decade. However, the superior date palm seedlings are still limited and most of them are imported from other countries. The mass supply of superior date palm seedlings can be provided by in vitro propagation in the bioreactor. Therefore, the research was conducted to develop a protocol of date palm in vitro propagation by using Temporary Immersion Bioreactor (TIB). The in vitro propagation was carried out through somatic embryogenesis technique using meristematic tissues isolated from offshoots of date palm female clone cv. Zambli as explants. The explants were sterilized and then cultured to produce embryogenic calli and somatic embryos. Afterwards, somatic embryos germination and plantlets formation were conducted in TIB with treatments of immersion period: 3, 10, and 30 minutes every 6 hours, with 8 replications, The results showed that the optimal somatic embryo germination in TIB was with the immersion period of 30 min every 6 h, resulting in the most formation of shoots and fresh biomass weight increment up to nearly threefold in 6 weeks. Thereafter, plantlets formation in TIB with immersion period of 10 min and 30 min every 6 h exhibited similar performances in producing more plantlets with higher total fresh weight and better vigor than those of 3 min every 6 h. However, there were more rooted plantlets in the TIB with immersion period of 10 min every 6 h. Based on the results, an in vitro propagation protocol via somatic embryogenesis in TIB has been successfully developed for mass propagation of date palm cv. Zambli, which produced plantlets with good vigor and rooting.
Determination of the optimum initial callus weight for the efficient propagation of sugarcane in temporary immersion bioreactor Rizka Tamania SAPTARI; Imron RIYADI; Masna Maya SINTA; M Eko Riyo Bayu PRASETYO; Sylvia LINDAWATI; Sumaryono SUMARYONO
Menara Perkebunan Vol. 90 No. 2 (2022): 90 (2), 2022
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v90i2.505

Abstract

AbstrakBioreaktor perendaman sesaat (BPS) telah digunakan secara luas untuk propagasi skala massal berbagai tanaman penting, termasuk tanaman tebu. BPS menyediakan sistem kultur semi-otomatis dan kondisi optimal bagi pertumbuhan tanaman. Beberapa faktor menentukan pertumbuhan tanaman pada BPS, salah satunya densitas dari eksplan. Oleh karena itu, penelitian dilakukan untuk menentukan bobot awal yang optimal untuk kalus tebu yang dikulturkan pada BPS, serta mengevaluasi pengaruh perbedaan bobot awal kalus tersebut terhadap proliferasi dan regenerasi kalus tebu. Kalus tebu diinduksi dari daun muda yang masih menggulung dari empat varietas tebu unggul Indonesia. Bobot awal kalus yang dikultur ke dalam bejana TIB yaitu 0,05 g; 0,1 g; 0,2 g; 0,5 g; dan 1,0 g untuk setiap bejana. Kalus kemudian melalui tahap proliferasi pada BPS sebanyak tiga siklus, kemudian kalus diregenerasi pada BPS dengan perlakuan auksin dan sitokinin. Hasil penelitian menunjukkan bahwa 0,2 g merupakan bobot awal kalus yang efisien untuk proliferasi kalus tebu pada TIB, dimana eksponensial multiplikasi kalus tercapai pada bobot awal tersebut, yaitu untuk masing-masing varietas 130,3 kali (PSKA 942), 136,8 kali (PS 094), 21,3 (PS 881), dan 12,9 kali (PS 091) setelah 12 minggu. Densitas kalus pada TIB berkorelasi negatif dengan karakteristik fisikokimia medium. Hal ini menggambarkan variasi intensitas pertumbuhan dan metabolisme kalus dengan adanya perbedaan densitas pada BPS. Penggunaan BAP 0,2 mg L-1 bersama kinetin 0,2 mg L-1 paling sesuai untuk memacu regenerasi kalus tebu dengan menghasilkan jumlah tunas terbanyak dalam waktu relatif lebih cepat (1 – 2 minggu lebih cepat) dibandingkan perlakuan lainnya dan dengan tingkat kejadian pencoklatan yang rendah.[Kata kunci: kultur in vitro, kultur cair, proliferasi]AbstractTemporary immersion bioreactor (TIB) has been utilized for the mass-scale propagation of many important plants, including sugarcane. TIB facilitates a semiautomated culture system and provides optimal conditions for plant growth. Several factors determine plant growth in the TIB, such as explant density. Therefore, an experiment was carried out to determine the optimal initial weight of sugarcane calli and to evaluate its effect on the proliferation and regeneration in TIB. Sugarcane calli were induced from spindle leaves isolated from four Indonesian prime sugarcane varieties. The initial weights of the calli cultured in the TIB flasks were 0.05 g, 0.1 g, 0.2 g, 0.5 g and 1.0 g per flask. The calli were proliferated through three cycles in TIB, and subsequently regenerated in TIB with auxin and cytokinin treatments. The results of the experiments showed that 0.2 g was the most efficient initial weight for sugarcane callus proliferation in the TIB, resulting in an exponential multiplication rate of 130.3-fold (PSKA 942), 136.8-fold (PS 094), 21.3-fold (PS 881), and 12.9-fold (PS 091) within 12 weeks. In the TIB, callus density showed a negative correlation with the physicochemical properties of the medium, demonstrating various growth intensities or metabolic activities of calli at different densities in the TIB. The use of 0.2 mg L-1 BAP along with 0.2 mg L-1 kinetin was suitable for promoting the regeneration of sugarcane calli and producing the highest number of shoots in a relatively short amount of time (1 – 2 weeks faster) with low incidences of browning.[Keywords: in vitro culture, liquid culture, proliferation]