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The development of somatic embryos of sago palm (Metroxylon sagu Rottb.) on solid media *) Perkembangan embrio somatik tanaman sagu (Metroxylon sagu Rottb.) pada medium padat Imron RIYADI; J.S. TAHARDI TAHARDI; . SUMARYONO
E-Journal Menara Perkebunan Vol 73, No 2: Desember 2005
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

SummarySago palm (Metroxylon sagu Rottb.) isusually propagated vegetatively by suckers.However, the limited availability of uniformsuckers is a major obstacle in the establishmentof cultivated sago plantations. Tissue culture hasthe potential for large-scale mass clonalpropagation of superior genotypes of sago palm.In vitro culture of sago palm has been establishedthrough somatic embryogenesis. Embryogeniccallus derived from shoot apical tissue of youngsuckers was cultured on a modified Murashigeand Skoog (MMS) medium containing 30 g/Lsucrose, 2 g/L Gelrite, 1 g/L activated charcoal,5.0 mg/L 2,4-D, and 0.1 mg/L kinetin to inducesomatic embryos. Callus clumps formed somaticembryos within four weeks. In the subsequentculture, approximately 0.3 g initial globularcallus grown on MMS medium containing 1.0mg/L kinetin, 0.01 mg/L ABA and 0.1 mg/L GA 3produced 140 to 200 somatic embryos at differentdevelopmental stages four weeks later. All stagesof developing embryos with different sizesand colors were present at any one time ofculture. Secondary (repetitive) somatic embryo-genesis was also found in the culture.Transferring of the mature stage of somaticembryos to solid media with half-strength macro salts and with sucrose at concentration of 20 or 30 g/L without growth regulators led to the development of normal plantlets.RingkasanTanaman sagu (Metroxylon sagu Rottb.)biasanya diperbanyak secara vegetatif dengantunas anakan. Namun, terbatasnya ketersediaantunas anakan yang seragam merupakanhambatan utama dalam pembukaan perkebunansagu. Teknologi kultur jaringan mempunyaipotensi untuk perbanyakan klonal tanaman saguunggul dalam skala besar. Kultur in vitrotanaman sagu telah dikembangkan melaluiembriogenesis somatik. Kalus embriogenik yangberasal dari eksplan pucuk tunas anakandikulturkan pada medium modifikasi Murashigedan Skoog (MMS) dengan sukrosa 30 g/L,Gelrite 2 g/L, arang aktif 1 g/L, 2,4-D 5 mg/Ldan kinetin 0,1 mg/L untuk menginduksi embriosomatik. Kalus membentuk embrio somatikdalam waktu empat minggu. Dalam kulturberikutnya, dari kurang-lebih 0,3 g embrio faseglobuler yang dikulturkan pada medium MMSdengan kinetin 1,0 mg/L, ABA 0,01 mg/L danGA 3 0,1 mg/L menghasilkan 140 sampai 200embrio somatik dengan fase perkembangan yangberbeda-beda. Embrio somatik dalam semuafase perkembangan dengan ukuran dan warnayang berbeda-beda ditemukan setiap saat dalamkultur. Di samping itu, embriogenesis somatiksekunder (berulang) juga terjadi dalam kultursagu. Embrio somatik fase dewasa biladipindah ke medium padat dengan garam makrosetengah konsentrasi dan sukrosa padakonsentrasi 20 atau 30 g/L tanpa zat pengaturtumbuh akan menjadi planlet normal.

Pengaruh kitosan, mikroba antagonis, dan bakteri endofit dalam menekan perkembangan penyakit bercak daun pada bibit kelapa kopyor (Effect of chitosan, antagonist and endophytic bacteria in suppressing the development of leaf spot disease in kopyor coconut seedlings) Deden Dewantara ERIS; Sri WAHYUNI; Imron RIYADI; Happy WIDIASTUTI; . SISWANTO
E-Journal Menara Perkebunan Vol 87, No 1 (2019): April, 2019
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Currently, tissue culture is one of the best techniques in propagating of kopyor coconut. The important stage in plant propagation through tissue culture is acclimatization. Upon entering the acclimatization stage, the problem that couldarise in kopyor coconut seedlings is leaf spot disease caused by the fungus Colletotrichumsp., Curvularia sp. and Pestalotiopsissp. Environmentally friendly leaf spot disease control techniques can be done through the use of chitosan, antagonists microbes and endophytic bacteria. This study aimed to examine the use of chitosan, microbial antagonists, endophytic bacteria and their combinations to control leaf spot kopyor coconut disease in four different disease severity categories, namely severe/heavy, moderate, mild, and healthy.Disease development was observed every three weeks, while the rate of disease infection, area under the disease development curve (AUDPC) and disease progression/delta were observed 15 weeks after treatment. The result showed that in heavy severity category, endophytic bacteria treatment was more effective to inhibit leaf spot disease showed by AUDPC value of 131.95 units that significantly different compared to others. In the moderate category, combination treatment was more effective to suppress leaf spot disease, showed by the lowest infection rate by 0.03 unit per week, and percentage disease value progression changes was 12.10%, with no significantly different AUDPC value to the other treatments. In mild and healthy severity category, there were no significanly different between the rate of infection and AUDPC in all treatments. While the percentage of change progression disease was significantly different between endophytic treatment and control. [Keywords: coconut kopyor seedling, leaf spot disease, antagonist microbes, endophyticbacteria, chitosan]Abstrak Saat ini, teknik kultur jaringan merupakan salah satu teknik terbaik untuk memproduksi bibit kelapa kopyor. Tahap penting dalam perbanyakan tanaman melalui kultur jaringan adalah aklimatisasi. Pada saat memasuki tahap aklimatisasi, masalah yang dapat muncul pada bibit kelapa kopyor adalah serangan penyakit bercak daun yang disebabkan oleh cendawan Colletotrichumsp., Curvulariasp. dan Pestalotiopsis sp.Teknik pengendalian penyakit bercak daun yang ramah lingkungan dapat melalui pemanfaatan kitosan, mikroba antagonis dan bakteri endofit. Penelitian ini bertujuan untuk menguji pengaruh kitosan, mikroba antagonis, bakteri endofit dan kombinasinya untuk mengendalikan penyakit bercak daun bibit kelapa kopyor pada empat kategori keparahan penyakit yang berbeda yaitu berat, sedang, ringan, dan sehat. Perkembangan penyakit diamati setiap tiga minggu selama 15 minggu sedangkan laju infeksi penyakit, luas area dibawah kurva perkembangan penyakit (AUDPC)dan persentase selisih/delta perkembangan penyakit dihitung pada minggu ke 15.Hasil penelitian menunjukkan bahwa pada bibit kelapa kopyor dengan kategori keparahan berat, perlakuan bakteri endofit lebih efektif menghambat bercak daun dengan menghasilkan nilai AUDPC terkecil yaitu sebesar 131,95 unit dan berbeda nyata dibandingkan dengan perlakuan lainnya sedangkan laju infeksi dan persentase delta perkembangan penyakit tidak berbeda nyata dibandingkan dengan perlakuan lainnya. Pada bibit kopyor kategori keparahan sedang, perlakuan kombinasi lebih efektif menekan bercak daun ditunjukkan dengan laju infeksi terendah sebesar 0,03 unit per minggu yang menghasilkan delta perkembangan penyakit terkecil yakni sebesar 12,1%, dengan nilai AUDPC tidak berbeda nyata dibandingkan dengan perlakuan lainnya. Pada bibit kopyor kategori keparahan ringan dan sehat, tidak terdapat perbedaan yang nyata untuk parameter laju infeksi dan nilai AUDPC pada semua perlakuan. Sedangkan nilai persentase delta perkembangan penyakit pada perlakuan endofit berbeda nyata dibandingkan dengankontrol. [Kata Kunci: bibit kelapa kopyor, penyakit bercak daun, kitosan, mikroba antagonis, bakteri endofit]

Pengaruh jumlah subkultur dan media sub-optimal terhadap pertumbuhan dan kemampuan regenerasi kalus tebu (Saccharum officinarum L.) (Effect of repeated subculture and suboptimum media on the growth of sugarcane calli (Saccharum officinarum L.)) Hayati MINARSIH; . Suharyo; Imron RIYADI; Diah RATNADEWI
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Sugarcane (Saccharum officinarum L.) is an important crop for sugar production. One attempt to increase sugarcane productivity is through micropropagation and quality improvement of sugarcane seedlings in vitro. This research aimed to study the effect of repeated subcultures on callus capacity for regeneration and plant survival in acclimatization phase, as well as the influence of suboptimum media on the recovery capability of sugarcane callus to proliferate in vitro. Fourth subcultured sugarcane callus derived from young leaves were used as material in this research. Basic medium of Murashige and Skoog (MS) added with 3 mg/L 2,4-D, 10% coconut water, and 3% sucrose was used for callus initiation. For callus regeneration, the MS medium was supplemented with 2 mg/L BAP, 0.2 mg/L IAA, 10% coconut water, and 3% sucrose. Study on the effect of subculture numbers consisted of three stages, i.e. initiation, regeneration, and acclimatization, while the study on resting phase or the use of sub-optimal media included six treatment media and two pathways. Results showed that the fifth subcultures produced embryoid callus (91%), the highest non mucilaginous callus (97%), and the highest abnormality rate (6%). Results from the suboptimum media treatment, showed that B pathway (4 week resting phase) was better than the A pathway (8 week resting phase), based on fresh weight and callus abnormality percentage. A and B pathways indicated that the growth of callus can be recovered when it was grown back to the normal media and 1.5D-MS treatment of the resting phase showed the best growth and appearance.

Identifikasi dan pencegahan kontaminasi pada kultur cair sistem perendaman sesaat Identification and prevention of contamination in liquid culture of temporary immersion system Masna Maya SINTA; Imron RIYADI; . SUMARYONO
E-Journal Menara Perkebunan Vol 82, No 2: Desember 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

AbstractLiquid culture is commonly used to scale up in vitro culture production as well as to optimize the developmental phase of plant in vitro culture. One of the liquid cultures that has been used widely is temporary immersion system (TIS). The main problem of liquid culture is contamination. The use of antibiotics sometimes controls the contaminants less effectively and hinders the growth of plant culture. The purpose of this research was to determine sources of contaminant on whole sequence of TIS to identify and to prevent the emergence of the contaminants. Sampling method was applied to each section and stage of TIS culture and the contaminants found were identified. The results revealed that compartment of TIS was the main source of contaminant (100%). Furthermore, from all components of TIS compartment, washer (a small ring seal connecting screen disc and basket) was the main source of TIS contaminant (41.2%). Four contaminants found were identified as Bacillus macerans, Bacillus megaterium, Bacillus sphaericus and Bacillus firmus. Two times sterilization of washer in an autoclave at temperature of 121 oC and air pressure of 1 kg/cm2 for 20 minutes before and after being installed reduced the contamination level on TIS culture significantly.AbstrakKultur cair umumnya digunakan untuk meningkatkan skala produksi dan mengoptimalkan fase perkembangan kultur in vitro tanaman. Salah satu jenis kultur cair yang banyak digunakan adalah sistem perendaman sesaat (SPS). Masalah utama dalam kultur cair adalah kontaminasi. Penggunaan antibiotika terkadang kurang efektif dalam me-ngendalikan kontaminan dan menghambat pertumbuhan kultur tanaman. Tujuan dari penelitian ini adalah untuk mengetahui sumber kontaminan pada seluruh rangkaian kultur SPS serta mengidentifikasi dan mencegah munculnya kontaminan tersebut. Metode yang digunakan adalah pengambilan contoh pada tiap bagian dan fase kultur SPS, serta kontaminan yang ditemukan kemudian diidentifikasi. Hasil penelitian memperlihatkan bahwa kompartemen SPS merupakan sumber utama kontaminan (100%). Selanjutnya, dari seluruh komponen kompartemen SPS, washer (cincin penutup yang menghubungkan penyaring dan keranjang) di dalam rangkaian SPS merupakan sumber utama kontaminan (41,2%). Empat kontaminan yang ditemukan diidentifikasi sebagai Bacillus macerans, Bacillus megaterium, Bacillus sphaericus dan Bacillus firmus. Sterilisasi cincin penutup sebanyak dua kali dalam autoklaf pada suhu 121 oC dan tekanan udara 1 kg/cm2selama 20 menit sebelum dan sesudah dirangkai secara nyata menurunkan tingkat konta-minasi pada kultur SPS.

Pertumbuhan dan perkembangan kalus embriogenik dan embrio somatik kelapa sawit (Elaeis guineensis Jacq.) pada sistem perendaman sesaat Growth and differentiation of embryogenic callus and somatic embryos of oil palm (Elaeis guineensis Jacq.) in a temporary immersion system . SUMARYONO; Imron RIYADI; Pauline D. KAS; Gale GINTING
E-Journal Menara Perkebunan Vol 75, No 1: Juni 2007
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

SummaryIn temporary immersion system (TIS),plant materials are exposed to the medium fora short time, therefore they are more exposedto the air and a lack of oxygen frequentlyexperienced by a liquid culture can be avoided.This experiment was conducted to determinethe procedure for callus proliferation up tosomatic embryo germination of oil palm(Elaeis guineensis Jacq.) in TIS culture.Embryogenic calli of oil palm clone MK 638from Marihat Research Institute were culturedon solid medium in the dark culture room andthen used as materials for TIS. Immersion timefor all cultures was three minutes every sixhours. Callus proliferation was conducted inDF liquid culture with 5 mg/L 2,4-D and0.1 mg/L kinetin with transfer interval of 4, 6and 8 weeks. The treatments for somaticembryo maturation were kinetin and ABA,whereas for somatic embryo germination wasIBA, kinetin and GA 3 . The results show thatthe best transfer interval for callus proli-feration was four weeks. In this treatment therelative growth rate of callus was0.38 g/g/week. Somatic embryo initiation fromthe callus was done in DF mediumsupplemented with 1 mg/L 2,4-D and 0.1 mg/Lkinetin. The percentage of somatic embryowas 80% based on biomass fresh weight afterthe fourth subculture. The addition of 0.5 mg/Lkinetin and 0.05 mg/L ABA improved somaticembryo maturation of oil palm; the averagenumber of somatic embryos at advanced stages(torpedo and cotyledonary) was 16.3 embryosper flask. The addition of 2 mg/L IBA and0.5 mg/L kinetin in DF medium with half-strength macro-salt enhanced significantly thegermi-nation of somatic embryos. GA 3 at0.1 mg/L increased the total number ofgerminants.RingkasanPada sistem perendaman sesaat (SPS),bahan tanam hanya terpapar sebentar dalammedium sehingga paparan dengan udara lebihlama dan kekurangan oksigen yang seringterjadi pada kultur cair dapat diatasi. Penelitianini bertujuan menetapkan prosedur untukperbanyakan kalus embriogenik sampai denganperkecambahan embrio somatik kelapa sawit(Elaeis guineensis Jacq.) dalam kultur SPS.Kalus embriogenik kelapa sawit klon MK 638yang diperoleh dari Balai Penelitian Marihatdiperbanyak pada medium padat di ruang gelapyang kemudian digunakan sebagai bahan untukkultur cair SPS. Lama perendaman semuakultur di SPS diatur tiga menit denganfrekuensi setiap enam jam. Perbanyakan kalusdalam medium cair DF dengan 2,4-D 5 mg/Ldan kinetin 0,1 mg/L dilaksanakan denganinterval subkultur 4, 6 dan 8 minggu.Perlakuan pematangan embrio somatik adalahkinetin dan ABA sedangkan perlakuan untukperkecambahan embrio somatik adalah IBA,kinetin dan GA 3 . Hasil penelitian menunjuk-kan bahwa untuk proliferasi kalus embriogenikkelapa sawit, interval subkultur terbaik adalahempat minggu. Pada perlakuan ini laju tumbuhrelatif kalus mencapai 0,38 g/g/minggu.Inisiasi embrio somatik dari kalus dilakukanpada medium DF ditambah 2,4-D 1 mg/L dankinetin 0,1 mg/L. Persentase embrio somatikmencapai 80% dari total bobot basah biomassasetelah subkultur keempat. Penambahan kinetin0,5 mg/L dan ABA 0,05 mg/L meningkatkanpematangan embrio somatik kelapa sawit; rata-rata jumlah embrio somatik fase lanjut (torpedodan kotiledon) adalah 16,3 embrio per bejana.Penambahan IBA 2 mg/L dan kinetin 0,5 mg/Lpada medium DF dengan setengah garammakro meningkatkan perkecambahan embriosomatik secara nyata. GA 3 0,1 mg/L mening-katkan jumlah kecambah yang terbentuk.

Mikropropagasi planlet tebu menggunakan sistem perendaman sesaat (SPS) Micropropagation of sugarcane plantlets using temporary immersion system (TIS) Hayati MINARSIH; Imron RIYADI; . SUMARYONO; Asmini BUDIANI
E-Journal Menara Perkebunan Vol 81, No 1: Juni 2013
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

bstractTo achieve Indonesian sugar self-sufficiency in2014, the national production needs to be escalatedthrough land extensification that requires a largenumbers of cane planting materials. This can be achievedby mass propagation of sugarcane through in vitroculture. Solid medium is commonly used for callusproliferation in sugarcane tissue culture. However, solidmedium is considered inefficient in terms of plantletproduction level, labour and space. The use of liquidmedium may solve the problem by allowing automationto increase plantlet production scale and uniformity.Temporary immersion system (TIS) is based on a shortperiodic immersion of explants in a liquid medium for aspecific frequency and duration. Research on in vitromass propagation of sugarcane using TIS was conductedat the Indonesian Biotechnology Research Institute forEstate Crops. Callus initiated from immature unfoldedleaves of PSJT 941 and PS 881 was cultured on liquidMS medium in TIS with different frequencies (12 and24 h) and durations (1 and 3 min) of immersion. Eachtreatment was replicated three times. The callus biomassof two elite cane varieties (PSJT 941 and PS 881)cultured in TIS for six weeks was higher (2 – 4 times fold)than that of on solid medium. The PSJT 941 varietyreached the highest calli biomass with immersion forthree min every 24 h. However, PS 881 variety reachedits highest biomass with immersion for one minute every24 h. The propagation of sugarcane using TIS culturewas proven to produce higher calli biomass up to fourfolds and to form more numbers and uniform shootscompared to the solid medium culture. The callus wassuccesfully regenerated to shoots and plantlets.AbstrakUntuk mencapai swasembada gula, perlu dilakukanpeningkatan produksi gula nasional melalui perluasanareal pertanaman tebu sehingga diperlukan bibit dalamjumlah besar. Hal tersebut dapat diatasi antara laindengan perbanyakan tebu melalui kultur in vitro. Peng-gunaan medium padat pada perbanyakan kalus tebumelalui kultur in vitro merupakan teknik yang umumdigunakan saat ini. Akan tetapi penggunaan mediumpadat dianggap kurang efisien dalam hal jumlah planletyang diproduksi, tenaga kerja dan ruang digunakan.Penggunaan medium cair dapat mengatasi kelemahantersebut dengan dimungkinkannya otomatisasi sehinggadapat meningkatkan skala produksi secara massal dankeseragaman planlet. Sistem perendaman sesaat (SPS)merupakan teknik kultur in vitro dalam medium cairmenggunakan bejana bersekat dimana kontak antaraeksplan dan medium terjadi hanya secara sesaat danperiodik. Penelitian perbanyakan massal bibit tebumelalui SPS dilakukan di Balai Penelitian BioteknologiPerkebunan Indonesia. Kalus diinisiasi dari daun meng-gulung varietas PSJT 941 dan PS 881 yang ditumbuhkanpada media MS cair dalam kultur SPS dengan frekuensiyang berbeda (12 dan 24 jam) dan lama perendaman (1dan 3 menit). Setiap perlakuan diulang tiga kali. Bobotbasah (biomassa) kalus dari dua varietas tebu (PSJT 941dan PS 881) yang ditumbuhkan dengan metode SPSsetelah enam minggu menunjukkan pening-katan yanglebih tinggi yaitu antara 2 - 4 kali lipat dibandingkandengan kontrol (media padat). Peningkatan biomassatertinggi pada varietas PSJT 941 diperoleh pada per-lakuan SPS dengan interval perendaman 24 jam dan lamaperendaman tiga menit. Sedangkan pada PS 881,peningkatan tertinggi biomassa diperoleh pada intervalperendaman 24 jam dan lama perendaman satu menit.Perbanyakan dengan metode SPS terbukti dapat mening-katkan biomassa kalus lebih dari empat kali lipat danpembentukan tunas yang lebih seragam dibandingkandengan pada media padat. Kalus yang dihasilkan dapatdiregenerasikan menjadi tunas dan planlet.

Pertumbuhan biak kalus dan suspensi sel tanaman kina (Cinchona ledgeriana Moens) Growth of callus and cell suspension cultures of cinchona (Cinchona ledgeriana Moens) . SUMARYONO; Imron RIYADI
E-Journal Menara Perkebunan Vol 73, No 1: Juni 2005
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

SummaryIn vitro technology of plants can be used topropagate plants and to produce secondarymetabolites with a short and continuousproduction cycle. Callus cultures of cinchona(Cinchona ledgeriana Moens) on solid media andcell cultures in liquid media have beenestablished. Callus could be easily initiated fromvarious explants of cinchona clone CB5, GA22and QRC312. The best callus initiation andproliferation were obtained on a Woody Plant(WP) solid medium supplemented with 15 µMpicloram,0.5 µM BAP and 1 µM phloroglucinol.In this medium the fresh weight of callusincreased by 12 to 14-fold within 5 to 6 weeks.Callus that constantly grew fast was selected as amaterial source for cell suspension cultures. InWP liquid medium with the same composition,the cells remained to grow fast where cell volumeafter sedimentation (CVS) increased by almost4-fold in two weeks. However, repeated sub-cultures decreased cell growth rate. The cellsuspension culture was then scaled-up in a 5-Lbioreactor. The culture medium was the same asin Erlenmeyer flasks. Cells in a bioreactor grewvery slowly, the cell biomass fresh weight andpacked cell volume (PCV) increased by 34% and50% respectively after 21 days of culture,although most of the cells remained viable.RingkasanTeknologi in vitro tanaman dapat digunakanuntuk memperbanyak tanaman dan memproduksisenyawa sekunder dengan siklus sangat singkatdan berkelanjutan. Biak kalus tanaman kina(Cinchona ledgeriana Moens) pada mediumpadat dan biak sel di medium cair telahdikembangkan. Kalus dengan mudah dapatdiinduksi dari berbagai jenis eksplan tanamankina klon CB5, GA22 dan QRC312. Inisiasi danproliferasi kalus terbaik diperoleh pada mediaWoody Plant (WP) padat dengan pikloram 15µM, BAP 0,5 µM dan floroglusinol 1 µM. Padamedium ini bobot basah kalus meningkat 12-14kali lipat dalam waktu 5-6 minggu. Kalus yangtetap tumbuh cepat dipilih sebagai sumber bahanuntuk biak suspensi sel. Dalam medium cair WPdengan komposisi yang sama, sel tetap tumbuhdengan pesat, volume sel setelah pengendapan(CVS) meningkat hampir empat kali lipat dalamwaktu dua minggu. Namun subkultur berulangmenurunkan laju pertumbuhan sel. Skala biaksuspensi sel kemudian diperbesar dalam bio-reaktor kapasitas 5 L. Medium kultur yangdigunakan sama dengan medium pada labuErlenmeyer. Pertumbuhan sel dalam bioreaktorsangat lambat, bobot basah sel dan packed cellvolume (PCV) hanya bertambah berturut-turutsebesar 34% dan 50% setelah 21 hari dalamkultur, walaupun sebagian besar sel tetap viabel.

Ekspresi dan kloning gen penyandi ADP-Glucose Phyrophosphorylase dari tanaman sagu (Metroxylon sagu Rottb.) Expression and cloning of gene encoding ADP-Glucose Phyrophosphorylase from sago palm (Metroxylon sagu Rottb.) Asmini BUDIANI1; Riza Arief PUTRANTO; Hayati MINARSIH; Imron RIYADI; . SUMARYONO; Barahima ABBAS
Menara Perkebunan Vol. 83 No. 2: 83 (2), 2015
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v83i2.4

AbstractSago palm (Metroxylon sagu Rottb.) is a potential food and energy resources becouse it is the highest starch producing plant. Breeding of sago palm should be directed to produce elite genotype with superior characters such as high starch content, wider pith diameter, without spine and high starch quality. However, research on sago palm in Indonesia so far is limited espescially in the field of cultivation and breeding, and attempt to produce such elite would take long time. Availability of molecular marker for starch content would be beneficial to shorten the length period of breeding. ADP-Glucose Phyrophosphorylase is one of the important enzymes in starch biosynthesis. Therefore its gene is an interesting subject in order to develope molecular marker of high starch content. This research was aimed to study the expression of gene encoding AGP in the sago palm with high starch content versus low starch content, and to clone the full cds of the gene. RNA was isolated from leaf and pith of both palms. Exspression analysis and amplify-cation of full cds were conducted by Reverse Transcryptase-Polymerase Chain Reaction (RT-PCR) using specific primers. The results showed that sago palm with higher starch content expressed AGP higher than that of sago palm with lower starch content. Expression of AGP in the full developing leaf was higher than in the young leaf, and there was no expression detected in the pith. The full cds of AGP was successfully amplified and cloned. Even though the DNA sequence showed high homology with DNA sequence of the same gene that has been deposited in GenBank, there were differences in severall nucleotide including that in the active domain of the enzyme.AbstrakTanaman sagu merupakan sumber pangan dan energi yang sangat potensial untuk dikembangkan karena merupakan tanaman penghasil karbihidrat tertinggi. Pemuliaan tanaman sagu mestinya diarah-kan untuk menghasilkan bibit sagu yang selain memiliki rendemen pati tinggi, juga memiliki diameter empulur besar, tidak berduri dan memiliki cita rasa pati yang enak. Namun, sampai saat ini riset mengenai sagu di Indonesia masih sangat terbatas, sehingga pemuliaan sagu untuk menghasilkan bibit unggul demikian akan memerlukan waktu lama. Ketersediaan penanda rendemen pati akan sangat membantu mempercepat pemuliaan tanaman tersebut. ADP-Glucose Pyrophosphorylase adalah salah satu enzim yang berperan penting dalam biosintesis pati, sehingga gene penyandinya merupakan subjek yang menarik dalam pengembangan marka kandungan pati tinggi. Sebagai bagian dari upaya untuk mendapat-kan penanda rendemen pati tinggi pada tanaman sagu, penelitian ini bertujuan untuk mempelajari ekspresi gen penyandi AGP. RNA diisolasi dari daun tanaman sagu rendemen pati rendah dan tanaman sagu rendemen pati tinggi. Perbedaan tingkat ekspresi gen penyandi AGP dari tanaman sagu rendemen pati tinggi vs rendemen pati rendah, dianalisis dengan teknik Reverse-Transcryptase PCR menggunakan primer spesifik. Hasil penelitian menunjukkan bahwa tanaman sagu rendemen pati tinggi mengekspresikan AGP lebih tinggi dibandingkan dengan tanaman sagu rendemen pati rendah. Ekspresi gen tersebut pada daun tua (full developing leaf) lebih tinggi di-bandingkan dengan pada daun muda, dan pada empulur tidak dideteksi ekspresi gen tersebut. Daerah penyandi lengkap AGP subunit kecil telah diklon. Meskipun memiliki homologi yang tinggi dengan sekuen DNA gen yang sama yang telah dideposit pada GenBank, namun terdapat perbedaan beberapa nukleotida termasuk pada daerah domain aktif dari enzim tersebut.

Evaluasi varietas, sumber eksplan dan strain Agrobacterium terhadap keberhasilan transformasi tebu dengan gen P5CS Evaluation of varieties, explant sources, and Agrobacterium strains for successful sugarcane transformation using P5CS gene Hayati MINARSIH; Dwi SUBIYARTI; Imron RIYADI; Soekarno Mismana PUTRA; Laksmi AMBARSARI
Menara Perkebunan Vol. 83 No. 1: 83 (1), 2015
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v83i1.7

Abstract Genetic transformation can be used as an alter-native to develop sugarcane (Saccharum officinarum L.) tolerant to drought stress. P5CS gene has a role in biosynthesis of proline, an amino acid that accumulated under drought stress conditions. Transfer of a P5CS gene construct into plant cells in conjunction with regeneration of transgenic plantlets may develop sugarcane tolerant to drought stress. The aim of this research was to obtain an optimal transformation method which includes a suitable strain of Agrobacterium tumefaciens, and the best sugarcane explant and variety. The results showed that transfer of P5CS gene has been successfully carried out on sugarcane explants from solid media-derived calli, embryogenic calli and somatic embryos derived from temporary immersion system (TIS) culture. Whilst Agrobacterium strain LBA4404 was indicated as the most effective transformation vector. The regeneration of Kidang Kencana variety transformants from calli and somatic embryos was better than those of PS 881 and PS 891. The best performance of transformants based on the source of explants obtained from somatic embryos from TIS culture. Moreover, a succesfull Agrobacterium mediated transformation on sugarcane was indicated by transient expression of Gus gene and the ability of the transformants grew in a selection medium containing 50 ppm of kanamycin.Abstrak Transformasi genetik dapat digunakan sebagai upaya untuk merakit tebu (Saccharum officinarum L.) toleran terhadap cekaman kekeringan. Gen P5CS diketahui berperan dalam biosintesis prolin, yaitu asam amino yang umumnya terakumulasi ketika tanaman mengalami cekaman kekeringan. Transfor-masi gen P5CS dan regenerasi transgeniknya mungkin dapat menghasilkan tanaman tebu trans-genik yang toleran terhadap cekaman kekeringan. Tujuan penelitian ini adalah untuk mendapatkan metode transformasi yang optimum yang mencakup strain Agrobacterium tumefaciens yang sesuai, sumber eksplan dan varietas tebu terbaik sebagai target transformasi. Hasil penelitian menunjukkan bahwa transformasi gen P5CS telah berhasil dilakukan ke eksplan tebu baik yang berupa kalus asal media padat maupun kalus embriogenik dan embrio somatik asal kultur sistem perendaman sesaat (SPS). Sementara itu strain A. tumefaciens LBA4404 menunjukkan hasil yang paling efektif sebagai vektor transformasi. Pertumbuhan transforman baik pada kalus maupun embrio somatik pada varietas Kidang Kencana terlihat paling baik dibandingkan dengan varietas PS 881 dan PS 891. Sumber eksplan yang paling efektif adalah embrio somatik yang diperoleh dari kultur SPS. Keberhasilan transformasi tebu me-lalui Agrobacterium ditunjukkan oleh ekspresi transien dari gen GUS dan kemampuan dari trans-forman untuk tumbuh di media yang mengandung 50 ppm kanamisin.

Identifikasi dan pencegahan kontaminasi pada kultur cair sistem perendaman sesaat Identification and prevention of contamination in liquid culture of temporary immersion system Masna Maya SINTA; Imron RIYADI; . SUMARYONO
Menara Perkebunan Vol. 82 No. 2: 82 (2), 2014
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v82i2.21

AbstractLiquid culture is commonly used to scale up in vitro culture production as well as to optimize the developmental phase of plant in vitro culture. One of the liquid cultures that has been used widely is temporary immersion system (TIS). The main problem of liquid culture is contamination. The use of antibiotics sometimes controls the contaminants less effectively and hinders the growth of plant culture. The purpose of this research was to determine sources of contaminant on whole sequence of TIS to identify and to prevent the emergence of the contaminants. Sampling method was applied to each section and stage of TIS culture and the contaminants found were identified. The results revealed that compartment of TIS was the main source of contaminant (100%). Furthermore, from all components of TIS compartment, washer (a small ring seal connecting screen disc and basket) was the main source of TIS contaminant (41.2%). Four contaminants found were identified as Bacillus macerans, Bacillus megaterium, Bacillus sphaericus and Bacillus firmus. Two times sterilization of washer in an autoclave at temperature of 121 oC and air pressure of 1 kg/cm2 for 20 minutes before and after being installed reduced the contamination level on TIS culture significantly.AbstrakKultur cair umumnya digunakan untuk meningkatkan skala produksi dan mengoptimalkan fase perkembangan kultur in vitro tanaman. Salah satu jenis kultur cair yang banyak digunakan adalah sistem perendaman sesaat (SPS). Masalah utama dalam kultur cair adalah kontaminasi. Penggunaan antibiotika terkadang kurang efektif dalam me-ngendalikan kontaminan dan menghambat pertumbuhan kultur tanaman. Tujuan dari penelitian ini adalah untuk mengetahui sumber kontaminan pada seluruh rangkaian kultur SPS serta mengidentifikasi dan mencegah munculnya kontaminan tersebut. Metode yang digunakan adalah pengambilan contoh pada tiap bagian dan fase kultur SPS, serta kontaminan yang ditemukan kemudian diidentifikasi. Hasil penelitian memperlihatkan bahwa kompartemen SPS merupakan sumber utama kontaminan (100%). Selanjutnya, dari seluruh komponen kompartemen SPS, washer (cincin penutup yang menghubungkan penyaring dan keranjang) di dalam rangkaian SPS merupakan sumber utama kontaminan (41,2%). Empat kontaminan yang ditemukan diidentifikasi sebagai Bacillus macerans, Bacillus megaterium, Bacillus sphaericus dan Bacillus firmus. Sterilisasi cincin penutup sebanyak dua kali dalam autoklaf pada suhu 121 oC dan tekanan udara 1 kg/cm2selama 20 menit sebelum dan sesudah dirangkai secara nyata menurunkan tingkat konta-minasi pada kultur SPS.

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