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Kultur akar rambut Cinchona ledgeriana dan C. succirubra dalam kultur in vitro Hairy root culture of Cinchona ledgeriana and C. succirubra by in vitro culture Nurita TORUAN-MATHIUS; . REFLINI; . NURHAIMI-HARIS; . JOKO-SANTOSO; A PRIANGANI-ROSWIEM
E-Journal Menara Perkebunan Vol 72, No 2: Desember 2004
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Summary Problems encountered in hairy root culture of C. ledgeriana and C. succirubra are low percentage of transformation of explants by Agrobacterium rhizogenes and slow growth of hairy root. The objective of this research was to evaluate the potential of several A. rhizogenes strains for initiation hairy roots of C. succirubra and C. ledgeriana, and to obtain the best medium for hairy root culture of Cinchona spesies. Axenic shoot and leaves explants of eight-month-old of C. ledgeriana and C. succirubra seedlings were inoculated with A. rhizogenes strain ATCC-15834, ATCC-8196, R-20001, 07-20001, A4, R-MAFFA, TISTR509, TISTR510 and LBA9457. Inoculated explants were cultured in solid MS medium with the addition of 100 mg/L amphicylin. Subculture of the hairy root was performed by transferred of root pieces into fresh liquid basal medium MS, B5, White and Heller. Hairy roots from the best of basal medium were subcultured on the same medium with the addition of 50 and 100 mg/L L-tryptophane, three or five times concentration of MS vitamins. The integration of T-DNA of A. rhizogenes in hairy root was confirmed with specific primer for TL and TR-DNA of plasmid by Polymerase Chain Reaction analysis. The results showed that only A. rhizogenes strain LBA 9457 were effective for transformation of explants from both Cinchona species. The fastest hairy roots growth were found in MS medium, while growth in others medium was poor. Hairy roots of C. ledgeriana has vigor and growth better than hairy roots of C. succirubra. MS with the addition of 50 mg/L L-tryptophane and three times the concen-trations of vitamin is the best medium for hairy root growth and vigor. Hairy roots of C. succirubra and C. ledgeriana used in this studies were confirmed that hairy roots contained TL and TR-DNA region of Ri plasmid with molecular weight 780 and 1600 bp. The results showed that strain of A. rhizogenes, plant species, source of explant and composition of medium affect the initiation, growth, development and vigor of hairy roots.Ringkasan Masalah dalam kultur akar rambut C. ledgeriana dan C. succirubra adalah rendahnya tingkat keberhasilan transformasi eksplan dengan Agrobacterium rhizogenesdan pertumbuhannya yang lambat. Penelitian ini bertujuan untuk mengevaluasi potensi dari beberapa galur A. Rhizogenes untuk inisiasi, mendapatkan komposisi medium terbaik untuk pertumbuhan akar rambut C. ledgeriana dan C. succirubra, serta konfirmasi terintegrasinya TR dan TL-DNA Ri plasmid ke dalam jaringan eksplan. Eksplan batang dan daun berasal dari kecambah aksenik C. ledgeriana dan C. succirubra berumur delapan bulan diinokulasi dengan A. rhizogenes galur 15834, 8196, R-20001, 07-20001, A4, R.MAFFA,TISTR 509, TISTR 510 dan LBA 9457. Eksplan yang sudah diinokulasi dikulturkan dalam medium MS padat. Subkultur dilakukan dengan cara mentransfer potongan ujung akar rambut ke dalam medium cair MS, B5, White dan Heller. Akar rambut dari medium kultur yang terbaik kemudian disubkultur ke dalam medium yang sama dengan penambahan 50 dan 100 mg/L L-triptofan dengan konsentrasi vitamin sebanyak tiga kali dan lima kali dari konsentrasi normal MS. Integrasi T-DNA dalam akar rambut dikonfirmasi meng-gunakan Polymerase Chain Reaction dengan primer spesifik untuk TL dan TR-DNA plasmid. Hasil yang diperoleh menunjukkan bahwa hanya A.rhizogenes galur LB9457 yang efektif menginfeksi eksplan baik batang maupun daun dari kedua spesies kina. Induksi, pertumbuhan dan vigor akar rambut yang terbaik diperoleh dari medium MS dengan penambahan 50 mg/L L-triptofan dan tiga kali konsentrasi vitamin. Hasil konfirmasi akar rambut baik dari batang maupun daun menggunakan PCR, menunjukkan bahwa TL dan TR-DNA dari Ri plasmid A. rhizogenes mampu menghasilkan pita-pita DNA dengan BM780 dan 1600 pb. Hasil yang diperoleh menunjukkan bahwa galur A. rhizogenes, spesies tanaman, sumber eksplan dan komposisi medium berpengaruh terhadap inisiasi, pertumbuhan, perkembangan dan vigor akar rambut.

Daya hidup planlet karet asal in vitro microcutting pada berbagai periode penutupan sungkup plastik dan komposisi media tumbuh Survival rate of in vitro microcutting-derived rubber plantlets on various plastic cover closed periods and medium compositions . SUMARYONO; Masna Maya SINTA; . NURHAIMI-HARIS
Menara Perkebunan Vol. 80 No. 1: 80 (1), 2012
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v80i1.46

AbstractIn vitro culture through microcutting technology can be used for clonal propagation of rubber (Hevea brasiliensis Muell. Arg.) rootstocks. Acclimatization of in vitro plantlets to ex vitro conditions is a major bottleneck in the micropropagation of many plants.This research was conducted to study the effect of plastic cover closed period and media composition on the survival rate of rubber plantlets. Plantlets derived from microcutting were planted on plastic pots containing a mixture of soil, cocopeat, dung manure, and sand or zeolite. The plantlets were then placed inside a closed transparent plastic cover that opened after 2, 3, 4 and 6 weeks. The cover was placed under tree canopy. The second experiment used the same media composition with or without cocopeat and with sand or zeolite. At 1.5 month after culture, observation was done on the number of survived plantlets, plantlet height and the percentage of rooted plantlets. The results show that the best coverclosed period was six weeks and the best growing medium was a mixture of soil, cocopeat, dung manure, and zeolite (6:2:1:1v/v). On the two combined treatments, the survival rate was 73.3% after 1.5 month of acclimatization. The use of zeolite and a higher soil percentage gave positive influences on rubber plantlet survival rate. The second experiment results confirmed that the use of zeolite was better than sand and the use of cocopeat was definitely needed. It can be concluded that the best of acclimatization of rubber plantlets from microcutting was on a medium mixture of soil, cocopeat, dung manure, and zeolite (6:2:1:1) and placed inside a closed plastic cover for six weeks before the cover was opened gradually. AbstrakKultur in vitro melalui teknologi microcutting dapat digunakan untuk perbanyakan klonal batang bawah tanaman karet (Hevea brasiliensis Muell. Arg.). Aklimatisasi planlet in vitro ke kondisi ex vitro merupakan hambatan utama pada mikropropagasi berbagai jenis tanaman. Penelitian ini dilakukan untuk mempelajari pengaruh lama penutupan sungkup plastik dan komposisi media tumbuh terhadap daya hidup planlet karet. Planlet karet asal microcutting ditanam pada pot plastik berisi media dengan berbagai campuran tanah, cocopeat, pupuk kandang, dan pasir atau zeolit. Planlet selanjutnya diletakkan di dalam sungkup plastik transparan tertutup rapat yang dibuka setelah 2, 3, 4 dan 6 minggu. Sungkup plastik diletakkan di bawah tajuk pepohonan. Percobaan kedua menggunakan komposisi media serupa dengan atau tanpa cocopeat dan dengan pasir atau zeolit. Pada umur 1,5 bulan, pengamatan dilakukan terhadap jumlah planlet yang hidup, tinggi planlet, dan persentase planlet yang berakar. Hasil penelitian menunjukkan bahwa lama penyungkupan terbaik adalah enam minggu dan media tumbuh terbaik adalah campuran tanah, cocopeat, pupuk kandang, dan zeolit (6:2:1:1 v/v). Pada kombinasi kedua perlakuan tersebut, daya hidup planlet karet mencapai 73,3% setelah 1,5 bulan aklimatisasi. Penggunaan zeolit dan persentase tanah yang lebih tinggi berpengaruh positif terhadap daya hidup planlet karet. Hasil percobaan kedua menegaskan bahwa penggunaan zeolit lebih baik daripada pasir dan penggunaan cocopeat mutlak diperlukan. Dapat disimpulkan bahwa aklimatisasi planlet karet asal microcutting terbaik dilakukan pada media campuran tanah, cocopeat, pupuk kandang, zeolit (6:2:1:1) dan diletakkan di dalam sungkup plastik tertutup selama enam minggu sebelum sungkup dibuka secara bertahap.

Pengaruh periode pra-kondisi dan penutupan sungkup terhadap daya hidup planlet karet (Hevea brasiliensis Muell. Arg) Effect of pre-condition period and vessel closure on the survival rate of rubber (Hevea brasiliensis Muell. Arg) plantlets Masna Maya SINTA; . NURHAIMI-HARIS; . SUMARYONO
Menara Perkebunan Vol. 81 No. 1: 81 (1), 2013
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v81i1.47

Acclimatization of plantlets is a critical stage in the micropropagation of many plants. An experiment was conducted to determine the effect of pre-condition period and vessel closure on the growth and survival rate of rubber (Hevea brasiliensis Muell.Arg.) plantlets derived from in vitro microcutting during acclimatization. Plantlets were planted in plastic pots containing mixed growing media after being conditioned in ex vitro environment for 0, 3 and 6 days. Five closure vessel treatments were closed pots placed in opened container, opened pots in closed glass container, closed pots in closed glass container, opened pots in closed plastic container, and closed pots in closed plastic container. Observation on leaf conditions, rooting frequency, and plant height were conducted at 1.5 months and on the percentage of survive plantlets at 1.5 and 3 months after acclimatization. The results showed that pre-condition was required to increase survival rate and growth of the plantlets. Pre-condition period of six days gave a higher survival rate than 0 and 3 days which reached 100% and 93% on opened pot in closed plastic container and closed pot in opened container, respectively after 1.5 months and was reduced to 80% after three months of acclimatization. The highest formation of new leaves and roots were also obtained on six days pre-condition period. Plantlets with pre-condition for six days and were planted on closed pots in an opened container had the best rooting frequency which was 90%. The result showed that the highest survival rate (80%) of rubber plantlets after three months was obtained when the plantlets were pre-conditioned in ex vitro conditions for six days before acclimatization and planted on opened pots in a closed plastic container or closed pots in an opened container.

Optimasi sterilisasi permukaan eksplan stek mikro tanaman karet Optimization of surface sterilization on rubber microcutting explan Irfan MARTIANSYAH; Deden Dewantara ERIS; . NURHAIMI-HARIS; Darmono TANIWIRYONO
Menara Perkebunan Vol. 81 No. 1: 81 (1), 2013
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v81i1.55

AbstractAn increasing number of explants is necessary toobtain plantlets in large quantities, for mass propagationof rubber plants. However, high level of contamination atthe primary culture stage is still a major constraint in invitro microcutting of rubber. The aim of this study was tooptimize surface sterilization procedures to reduce micro-bial contamination at the primary culture. Sterilizationexperiment was conducted in two step., The first step wasto determine the effect of washing the explants withrunning water prior to sterilization and then using Deso-germe, ethanol or H 2 O 2 , while the second step was toidentify the suitable sterilization process on reducing thelevel of contamination. The results showed that the surfacesterilization with only one type of sterilization agent couldnot reduce contamination level caused either by bacteriaor fungi, while sterilization with three types of sterilizingagents increased the number of dead explants. The besttreatment for surface sterilization was the directsterilization of explants using 70% ethanol for one minuteand 17.6% H 2 O 2 for 20 minutes without washing with tapwater (A-CD treatment). The percentage of viable andaseptic explantsof this treatment was 76.7%, which wassignificantly higher than those of other treatments. Thistreatment reduced contamination level to 21.7%.AbstrakPeningkatan jumlah eksplan sangat diperlukan untukmemperoleh planlet dalam jumlah besar pada perbanyakanmassal tanaman karet secara in vitro. Namun, tingginyatingkat kontaminasi pada tahap kultur primer masih me-rupakan kendala utama dalam kultur stek mikro tanamankaret. Tujuan penelitian adalah mengoptimasi prosedursterilisasi permukaan eksplan untuk mengurangi jumlaheksplan yang terkontaminasi mikroba pada tahap kulturprimer. Percobaan sterilisasi dilaksanakan dalam duatahap, tahap pertama untuk mengetahui pengaruh pen-cucian eksplan dengan air mengalir pada awal sterilisasiserta penggunaan Desogerme, etanol dan H 2 O 2 , sedang-kan tahap kedua untuk mendapatkan proses sterilisasi yangpaling sesuai dalam menurunkan tingkat kontaminasi.Hasil penelitian menunjukkan bahwa perlakuan sterilisasipermukaan yang menggunakan satu jenis bahan sterilantidak dapat mengurangi kontaminasi, baik oleh bakterimaupun cendawan. Perlakuan sterilisasi eksplan dengantiga jenis bahan sterilan meningkatkan kematian eksplan.Perlakuan sterilisasi permukaan terbaik adalah sterilisasilangsung eksplan menggunakan etanol 70% selama satu menit dan H 2 O 2 17,6% selama 20 menit, tanpa pencuciandengan air mengalir (perlakuan A-CD). Persentase eksplansteril yang hidup sebesar 76,7%, berbeda nyata dibanding-kan dengan perlakuan lainnya. Perlakuan tersebut dapatmengurangi kontaminasi menjadi sebesar 21,7%.

Pengaruh ventilasi terhadap morfologi, stomata dan kadar klorofil tunas karet yang diperbanyak melalui microcutting Influence of ventilation on morphology, stomata and chlorophyll content of Hevea shoots propagated through microcutting . NURHAIMI-HARIS; Nurul Siti AYUNINGTIAS; Irma Herawati SUPARTO
Menara Perkebunan Vol. 79 No. 2: 79 (2), 2011
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v79i2.60

AbstractIn plant tissue culture, the culture vessels are usuallycovered tightly with screw caps, aluminium foils, parafilm,or plastic wrap. This condition restricts the exchange ofgases in culture vessels and will affect negatively thegrowth of explants. The use of ventilated closure improvesthe air quality in culture vessels. Microboxes provided withdifferent types of filter (yellow filter with Kv value13.09 Gas Exchange (GE)/day, green filter with Kv value81.35 GE/day and without filter) on their closure wereexamined as culture vessels for growing rubber explants atmultiplication step. The purpose of this research was toobserve the plant condition in different types of microboxcorresponding to the morphology, stomata and chlorophyllcontent of the shoots. The results showed no significantdifference of shoot height on each microbox. The use ofventilated closure increased significantly new leafformation and decreased leaf fall. Normal size and color ofleaves were found on shoots grown in microbox with greenfilter. Chlorophyll analysis revealed no significantdifferences on three types of microbox, however visualobservation showed that leaves were greener on microboxwith green filter. The stomata condition of shoots onmicrobox with green filter were similar with those ofmother plants in green house, while different condition ofstomata were found on shoots grown in microbox withyellow filter or without filter. In normal environment suchas at the field and green house, most of stomata wereclosed, in microbox provided with filter on the closure,most of stomata were half open, while on microbox withoutfilter most of stomata were wide open.bstrakDalam kultur jaringan tanaman, tabung/botol kulturditutup rapat dengan penutup yang dilengkapi ulir,aluminium foil, parafilm atau plastik wrap. Kondisitersebut menghambat pertukaran udara dalam tabung kultursehingga sering memberikan pengaruh negatif terhadappertumbuhan eksplan. Penggunaan penutup berventilasidapat meningkatkan kualitas udara dalam lingkungantabung/botol kultur. Oleh karena itu microbox denganpenutup berfilter diuji sebagai wadah untuk menumbuhkaneksplan karet pada tahap multiplikasi, yaitu microboxberfilter kuning dengan nilai Kv sebesar 13,09 (GasExchange (GE)/hari dan berfilter hijau dengan nilai Kvsebesar 81,35 GE/hari, sedangkan sebagai kontrol adalahmicrobox tanpa filter (tertutup rapat). Penelitian bertujuanmengamati kondisi tunas di dalam microbox berfilter,meliputi morfologi, stomata dan kandungan klorofil. Hasilpenelitian menunjukkan bahwa tinggi tunas tidak berbedanyata pada masing-masing microbox. Jumlah daun barudan daun gugur berbeda nyata, dimana pembentukan daunbaru terbanyak terdapat pada tunas dalam microboxberfilter kuning maupun hijau. Ukuran dan warna daunterlihat normal pada tunas dalam microbox berfilter hijau.Analisis kandungan klorofil tidak menunjukkan perbedaannyata, namun pengamatan visual menunjukkan bahwa daunlebih hijau pada microbox dengan filter hijau. Kondisistomata daun dari tunas dalam microbox dengan penutupberfilter hijau menyerupai stomata tanaman induk yangterdapat di rumah kaca dan lapangan, sedangkan kondisistomata berbeda ditemukan pada tunas dalam microboxberfilter kuning atau tanpa filter. Pada lingkungan normalseperti lapangan dan rumah kaca, sebagian besar stomatamenutup, pada wadah dengan tutup berfilter stomata agakmembuka sedangkan pada microbox tanpa filter sebagianbesar stomata terbuka lebar.

Isolasi dan kloning fragmen cDNA dari tanaman karet dengan sifat resisten dan rentan terhadap Corynespora cassiicola menggunakan metode cDNA-AFLP Isolation and cloning of cDNA fragments from rubber plant with resistant and susceptible characters to Corynespora cassiicola using cDNA-AFLP method . NURHAIMI-HARIS; Antonius SUWANTO; Maggy T SUHARTONO; Hajrial ASWIDINNOOR
Menara Perkebunan Vol. 78 No. 1: 78 (1), 2010
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v78i1.78

AbstractLeaf fall disease caused by Corynespora cassiicola fungi is one of the most important diseases in rubber plant. Rubber clone AVROS 2037 is considered resistant to this pathogen while clone PPN 2444 is susceptible. These two rubberclones were used to identify genes or transcripts differentially expressed during interaction between rubber plants and the fungi, using cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) method. Induced genes/transcripts expression was examined to compare differencies between plants uninfected and infected with C. cassiicola at 24, 36, 48 and 72 hours after inoculation. cDNA-AFLP analysis was performed using restriction enzyme VspI and TaqI so the adapters and primers also have the recognition site of these enzymes. By using 29 specific primers, 35 out of approximately 1450 fragments were differentially expressed between AVROS and PPN 2444 clones. All of these fragments were cloned and sequenced. The homology-based grouping of these sequences resulted in 19 contigs and nine individual sequences. Among these, 10 contigs and five sequences have significant sequence homology with known genes in gene bank data base, such as Ran binding protein, protein transporter and transcriptional regulators of some organisms; arginase, GTP-binding protein, heat shock protein (HSP) and aconitase. Ran binding protein, GTPbinding protein and protein transporter were known as membrane proteins while arginase and HSP usually expressed as a response to wounding or toxin treatment. The present of arginase is usually related to the availability of nitric oxide (NO) in plant tissue. NO is well known as a signal molecule on plant defense response. AbstrakPenyakit gugur daun yang disebabkan oleh fungi Corynespora cassiicola merupakan salah satu penyakit penting tanaman karet. Klon karet AVROS 2037menunjukkan sifat resisten terhadap patogen tersebut sedangkan klon PPN 2444 merupakan klon yang rentan. Kedua klon karet tersebut digunakan untuk mengidentifikasi gen atau transkrip yang diekspresikan secara diferensial selama terjadi interaksi antara tanaman karet dengan C. cassiicola menggunakan teknik cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP). Ekspresi gen/transkrip dipelajari untuk membandingkan perbedaan antara tanaman yang tidak diinfeksi dengan yang diinfeksi patogen pada waktu 24, 36, 48 dan 72 jam setelah inokulasi. Analisis cDNA-AFLP dilaksanakan dengan menggunakan pasangan enzim restriksi VspI dan TaqI sehingga adapter dan primer memiliki sekuen pengenalan kedua enzim restriksi tersebut. Dengan menggunakan 29 pasang primer spesifik, sebanyak 35 dari sekitar 1450 fragmen memiliki ekspresi berbeda antara klon AVROS 2037 dan PPN 2444. Semua fragmen yang berbeda tersebut kemudian diklon pada vektor kloning dan disekuen. Hasil sekuensing dikelompokkan berdasarkan homologi sekuennya dan menghasilkan 19 contigs serta sembilan macam sekuen yang tidak mengelompok. Sebanyak 10 dari 19 contigs dan lima dari sembilan sekuen tersebut memiliki homologi dengan produk gen yang telah dikenal yang terdapat di pangkalan data GenBank, seperti putative Ran binding protein, protein transporter, regulator transkripsi, arginase, GTP-binding protein, heat shock protein (HSP) dan aconitase. Beberapa di antaranya seperti putative Ran binding protein, protein transporter dan GTP-binding protein dikenal sebagai protein membran, sedangkan arginase dan HSP merupakan protein atau enzim yang ekspresinya pada tanaman antara lain dipengaruhi oleh adanya pelukaan dan perlakuan toksin. Keberadaan arginase sering berhubungan dengan ketersediaan nitric oxide (NO) pada jaringan tanaman. NO dikenal sebagai salah satu sinyal molekul dalam mekanisme pertahanan tanaman.

Pengaruh bahan pra-sterilan, tutup tabung kultur, dan musim terhadap tingkat kontaminasi eksplan pada kultur microcutting karet Effect of pre-sterilization agent, culture tube closure, and season on the contamination level of rubber microcutting culture . NURHAIMI-HARIS; . SUMARYONO; M.P. CARRON CARRON
Menara Perkebunan Vol. 77 No. 2: 77 (2), 2009
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v77i2.96

AbstractMicrobial contamination is a major obstaclein clonal propagation of hevea (Heveabrasiliensis) through microcutting technology;therefore the ability to reduce contamination willdetermine the success of the application of thistechnology. The aim of experiments was toincrease healthy and survived plantlets by testingpre-sterilization agents for cleaning explantsduring pre-sterilization step, culture tubeclosures suitable for explants growth and anappropriate time for introducing explants at theprimary culture phase. The pre-sterilizationagents tested were aganol, alcohol anddesogerme, the culture tube closures used wereparafilm and cotton, and the time for culturingexplants were determined by using rubbergenotypes introduced during the year of 2006 and2007. The results show that desogermedecreased significantly the level of explantcontamination compared to aganol and alcohol,meanwhile the type of culture tube closure didnot affect the level of explant contamination. Thetype of culture tube closure influencedsignificantly the survival of explants where thenumber of survived explants in culture tubescovered with cotton was higher than that of withparafilm. Season also affected the contaminationfrequency of the explants. Higher number ofhealthy plantlets were obtained whenintroduction of the explants were conducted fromJune to October considered as dry season inBogor compared to introduction of the explantsduring rainy season from January to May.Different genotypes of rubber introduced at theprimary culture phase did not affect thepercentage of explant contamination.AbstrakKontaminasi oleh mikroba merupakanmasalah utama pada perbanyakan klonal tanamankaret (Hevea brasiliensis) melalui teknologimicrocutting sehingga kemampuan mengurangikontaminasi menentukan keberhasilan aplikasiteknologi tersebut. Penelitian ini bertujuanmempelajari pengaruh jenis bahan pra-sterilanyang efektif untuk pencucian eksplan tahap pra-sterilisasi, mempelajari pengaruh tutup tabungterhadap perkembangan eksplan serta meng-identifikasi waktu yang tepat untuk melaksanakanintroduksi eksplan pada tahap kultur primer(kultur awal) sehingga jumlah eksplan sehat dantumbuh dapat ditingkatkan. Bahan pra- sterilanyang diuji adalah aganol, alkohol dan desogerme,tutup tabung yang digunakan adalah parafilm dankapas, sedangkan identifikasi waktu kulturdilakukan melalui introduksi eksplan sepanjang tahun 2006 dan 2007 terhadap berbagai genotipetanaman karet yang tersedia. Hasil penelitianmenunjukkan bahwa desogerme menurunkansecara nyata tingkat kontaminasi eksplandibandingkan dengan aganol dan alkohol,sedangkan jenis tutup tabung tidak berpengaruhterhadap persentase kontaminasi. Jenis tutuptabung berpengaruh sangat nyata terhadappersentase eksplan yang hidup dan membentuktunas, di mana persentase eksplan membentuktunas pada tabung dengan tutup kapas lebih tinggidibandingkan dengan tutup parafilm. Musim jugasangat mempengaruhi tingkat kontaminasieksplan. Eksplan sehat jauh lebih banyakdiperoleh apabila penanaman eksplan dilakukanpada bulan Juni sampai Oktober, yang merupakanmusim kemarau di Bogor dibandingkan denganintroduksi eksplan pada bulan Januari sampaiMei, yang merupakan musim hujan. Jenisgenotipe yang ditanam pada tahap kultur primertidak berpengaruh terhadap persentasekontaminasi.

Pengaruh elisitasi terhadap pertumbuhan dan produksi alkaloida kinolin dari akar rambut tanaman kina (Cinchona succirubra Pavon ex Klotzsch) Effect of elicitation on growth and alkaloid quinoline production in hairy root of cinchona plant (Cinchona succirubra Pavon ex Klotzsch) Nurita TORUAN-MATHIUS; . NURHAIMI-HARIS; Joko SANTOSO; . ADE-HERI
Menara Perkebunan Vol. 74 No. 1: 74 (1), 2006
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v74i1.117

Summary Manipulation to increase alkaloid producti-vity in hairy root can be done by elicitation with the addition of certain enzymes. The objective of this research is to find out the effect of elicitation by cellulase and pectyoliase enzymes on the productivity of Cinchona succirubra hairy root culture. Hairy root was cultured on ½ MS macro nutrient with the addition of 40 g/L sucrose and 7 g/L agar. Elicitation was done by the addition of cellulase and pectiolyase at the concentrations of 0; 0.05; 0.10; 0.50; 1.00; 3.00; 5.00; 10.00; 15.00; 20.00 and 25.00 mg/L, respectively.The effect of elicitation was analyzed by fresh weight of hairy root, and alkaloid content in four-month-old culture.The result showed that the best growth was obtained by the addition of 15 mg/L cellulase, with fresh weight as much as 920 mg. The addition of 10 mg/L pectyoliase increased hairy root fresh weight as much as 880 mg. The highest quinoline alkaloid production was obtained by the addition of 25 mg/L celluase (Quinine 580, quinidine 492, cinchonine 234, dihydroxyn-chonine 195 and cinchonidine 165 µg/g fresh weight) and 1 mg/L pectyoliase (Quinine 2363, quinidine 238, cinchonine 104, dihydroxyn-chonidine 138 and cinchonidine 1558 µg/g fresh weight).Ringkasan Manipulasi untuk meningkatkan produk-tivitas akar rambut dapat dilakukan di antaranya dengan elisitasi melalui penambahan enzim tertentu ke dalam medium. Tujuan penelitian ini adalah untuk menetapkan pengaruh elisitasi dengan penambahan selulase dan pektioliase terhadap produktivitas akar rambut tanaman Cinchona succirubra. Akar rambut tanaman Cinchona succirubradikulturkan dalam medium MS ½ hara makro dengan penambahan sukrose 40 g/L dan agar 7 g/L. Elisitasi dilakukan dengan penambahan selulase dan pektioliase masing-masing pada konsentrasi 0; 0,05; 0,10; 0,50; 1,00; 3,00; 5,00; 10,00; 15,00; 20,00 dan 25,00 mg/L. Peubah yang diukur adalah bobot basah akar rambut dan kandungan alkaloida kinolin pada kultur berumur empat bulan. Hasil yang diperoleh menunjukkan bahwa pertumbuh-an terbaik terjadi pada penambahan konsentrasi selulase 15 mg/L dengan bobot basah 920 mg dan pektioliase 10 mg/L dengan bobot basah 880 mg. Produksi alkaloida kinolin tertinggi diperoleh pada konsentrasi selulase 25 mg/L (kinin 580, kinidin 492, sinkonin 234, dihidrok-sinkonin 195 dan sinkonidin 165 µg/g bobot basah) dan pektioliase 1 mg/L (kinin 2363, kinidin 238, sinkonin 104, dihidroksinkonin 138 dan sinkonidin 1558 µg/g bobot basah).

Kultur akar rambut Cinchona ledgeriana dan C. succirubra dalam kultur in vitro Hairy root culture of Cinchona ledgeriana and C. succirubra by in vitro culture Nurita TORUAN-MATHIUS; . REFLINI; . NURHAIMI-HARIS; . JOKO-SANTOSO; A PRIANGANI-ROSWIEM
Menara Perkebunan Vol. 72 No. 2: 72 (2), 2004
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v72i2.123

Summary Problems encountered in hairy root culture of C. ledgeriana and C. succirubra are low percentage of transformation of explants by Agrobacterium rhizogenes and slow growth of hairy root. The objective of this research was to evaluate the potential of several A. rhizogenes strains for initiation hairy roots of C. succirubra and C. ledgeriana, and to obtain the best medium for hairy root culture of Cinchona spesies. Axenic shoot and leaves explants of eight-month-old of C. ledgeriana and C. succirubra seedlings were inoculated with A. rhizogenes strain ATCC-15834, ATCC-8196, R-20001, 07-20001, A4, R-MAFFA, TISTR509, TISTR510 and LBA9457. Inoculated explants were cultured in solid MS medium with the addition of 100 mg/L amphicylin. Subculture of the hairy root was performed by transferred of root pieces into fresh liquid basal medium MS, B5, White and Heller. Hairy roots from the best of basal medium were subcultured on the same medium with the addition of 50 and 100 mg/L L-tryptophane, three or five times concentration of MS vitamins. The integration of T-DNA of A. rhizogenes in hairy root was confirmed with specific primer for TL and TR-DNA of plasmid by Polymerase Chain Reaction analysis. The results showed that only A. rhizogenes strain LBA 9457 were effective for transformation of explants from both Cinchona species. The fastest hairy roots growth were found in MS medium, while growth in others medium was poor. Hairy roots of C. ledgeriana has vigor and growth better than hairy roots of C. succirubra. MS with the addition of 50 mg/L L-tryptophane and three times the concen-trations of vitamin is the best medium for hairy root growth and vigor. Hairy roots of C. succirubra and C. ledgeriana used in this studies were confirmed that hairy roots contained TL and TR-DNA region of Ri plasmid with molecular weight 780 and 1600 bp. The results showed that strain of A. rhizogenes, plant species, source of explant and composition of medium affect the initiation, growth, development and vigor of hairy roots.Ringkasan Masalah dalam kultur akar rambut C. ledgeriana dan C. succirubra adalah rendahnya tingkat keberhasilan transformasi eksplan dengan Agrobacterium rhizogenesdan pertumbuhannya yang lambat. Penelitian ini bertujuan untuk mengevaluasi potensi dari beberapa galur A. Rhizogenes untuk inisiasi, mendapatkan komposisi medium terbaik untuk pertumbuhan akar rambut C. ledgeriana dan C. succirubra, serta konfirmasi terintegrasinya TR dan TL-DNA Ri plasmid ke dalam jaringan eksplan. Eksplan batang dan daun berasal dari kecambah aksenik C. ledgeriana dan C. succirubra berumur delapan bulan diinokulasi dengan A. rhizogenes galur 15834, 8196, R-20001, 07-20001, A4, R.MAFFA,TISTR 509, TISTR 510 dan LBA 9457. Eksplan yang sudah diinokulasi dikulturkan dalam medium MS padat. Subkultur dilakukan dengan cara mentransfer potongan ujung akar rambut ke dalam medium cair MS, B5, White dan Heller. Akar rambut dari medium kultur yang terbaik kemudian disubkultur ke dalam medium yang sama dengan penambahan 50 dan 100 mg/L L-triptofan dengan konsentrasi vitamin sebanyak tiga kali dan lima kali dari konsentrasi normal MS. Integrasi T-DNA dalam akar rambut dikonfirmasi meng-gunakan Polymerase Chain Reaction dengan primer spesifik untuk TL dan TR-DNA plasmid. Hasil yang diperoleh menunjukkan bahwa hanya A.rhizogenes galur LB9457 yang efektif menginfeksi eksplan baik batang maupun daun dari kedua spesies kina. Induksi, pertumbuhan dan vigor akar rambut yang terbaik diperoleh dari medium MS dengan penambahan 50 mg/L L-triptofan dan tiga kali konsentrasi vitamin. Hasil konfirmasi akar rambut baik dari batang maupun daun menggunakan PCR, menunjukkan bahwa TL dan TR-DNA dari Ri plasmid A. rhizogenes mampu menghasilkan pita-pita DNA dengan BM780 dan 1600 pb. Hasil yang diperoleh menunjukkan bahwa galur A. rhizogenes, spesies tanaman, sumber eksplan dan komposisi medium berpengaruh terhadap inisiasi, pertumbuhan, perkembangan dan vigor akar rambut.

Physiological and biochemical changes in cocoa seed (Theobroma cacao L.) caused by desiccation Perubahan fisiologi dan biokimia benih kakao (Theobroma cacao L.) akibat desikasi Nurita TORUAN-MATHIUS; . RACHMAWATI-HASID; . NURHAIMI-HARIS; Tolhas HUTABARAT
Menara Perkebunan Vol. 68 No. 1: 68(1), 2000
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v68i1.135

Ringkasan Benih kakao tergolong rekalsitran, benihnya sensitif terhadap desikasi dan apabila disimpan pada kondisi yang menyebabkan kehilangan air, benih akan kehilangan viabilitasnya. Viabilitas benih kakao hanya dapat dipertahankan beberapa hari saja dalam keadaan terbuka pada suhu kamar. Hal ini merupakan kendala dalam penyimpanan dan pengiriman benih kakao. Tujuan penelitian ini adalah untuk menetapkan pengaruh desikasi terhadap karakter fisiologis dan biokimia benih kakao. Benih ICS 60 (kakao lindak) dan DR2 (kakao mulia) diletakkan dalam cawan Petri kemudian disimpan pada suhu 25oC dan Rh 55-75% selama empat hari. Percobaan dilakukan dengan rancangan petak terpisah, petak utama adalah kandungan air awal dan kritikal. Sebagai anak petak adalah jenis kakao, masing-masing diulang empat kali. Peubah fisiologis yang diukur adalah viabilitas benih mencakup kandungan air benih, potensi tumbuh maksimum, daya berkecambah, kecepatan tumbuh, bobot kering kecambah normal, dan laju pertumbuhan kecambah normal. Di samping itu juga dilakukan pengamatan pola pita protein benih yang dianalisis dengan SDS-PAGE. Kandungan asam absisik (ABA) dan gula stahiosa, raftnosa, glukosa, fruktosa, arabinosa, silosa, serta sukrosa dalam benih yang ditetapkan dengan HPLC Integritas membran benih ditetapkan berdasarkan daya hantar listrik air perendaman benih yang diukur dengan konduktometer. Hasil yang diperoleh menunjukkan bahwa adanya interaksi yang nyata antara desikasi dengan seluruh tolok ukur fisiologis. Desikasi menyebabkan penurunan daya berkecambah, bobot kering dan laju pertumbuhan kecambah normal, potensi tumbuh maksimum dan kecepatan tumbuh. Sedang untuk, kandungan ABA, sukrosa, arabinosa dan rafinosa mengalami peningkatan. Di samping itu desikasi menyebabkan dibentuknya protein baru dengan BM 32,5; 47,0 dan 51,0 kDa (DR2); 47,0 dan 51,0 kD (ICS 60). Beberapa protein yang hilang oleh pengaruh desikasi yaitu dengan BM37, 0 (DR2), 19, 0 dan 37, 0 kD (ICS60). Benih ICS60 lebih tahan terhadap desikasi dibandingkan dengan benih DR2. Summary Seed of cocoa is recalcitrant and sensitive to desiccation. In open condition at room temperature, the viability of cocoa seed ultimately lost for several days. These characters are a problem for seed storage and delivery. The objectives of this study are to investigate the effect of desiccation on physiological and biochemical characters of cocoa seed. Seeds of ICS 60 (bulk cocoa) and DR2 (fine cocoa) were placed on Petri dishes and stored at 25oC, Rh 55-75% for four days (critical water content). The experiment was conducted with split plot analysis, (1) The main plot was the storage condition initial and critical seeds water content. (2) The sub plot was the variety of cocoa, with four replications of each treatment. The effect of desiccation on seeds viability was tested, based on seed water content, maximum growth potential, seed germination, germination rate, dry weight of normal seedling, and seedling growth rate. Besides, the changes of seed proteins band pattern were also analysed by SDSPAGE. Abscisic acid, stachyose, raffnose, fructose, arabinose, xyllose, and sucrose seed content were determined by HPLC. The integrity of seed membrane based on the leakage of electrolytes from seeds was measured with a CM 100 multicell conductivity meter. The results showed that there is an interaction with highly significant correlation between desiccation and all of the physiological and biochemical parameters. Desiccation caused the decrease of seed germination, dry weight and growth rate'of normal seedling, maximum growth potential, and germination rate and while the leakage of electrolytes, ABA, sucrose, arabinose and raffinose increased. Besides, desiccation was also caused the formation of new proteins with MW 32.5, 47,0 and 51,0 kDa (DR2); 47,0 and 51,0 kD ICS 60) . On the other hand, several protein were disappeared i.e. MW 37,0 (DR2), 19,0 and 37,0 kD (ICS60). Seeds of ICS 60 are more tolerant to desiccation than seeds of DR2.

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