The determination of transcript accumulation values significantly affects gene expression in oil palm. Various genes are involved in pathogen infection, including probable 2‐oxoglutarate‐dependent dioxygenase At5g05600 (EgUnk3), zinc finger protein 2‐like (EgZFP2), and inositol polyphosphate multikinase beta‐like (EgIPK2b). Gene expression is typically measured using relative quantitative methods to calculate differences in quantitative values in the expression levels of targeted genes compared to a reference gene. However, the effectiveness of these methods in assessing the expression of EgUnk3, EgZFP2, and EgIPK2b, which are involved in Ganoderma boninense infection in oil palm seedlings, requires evaluation. This study aimed to establish an effective and straightforward method for analyzing the expression of EgUnk1, EgZFP2, and EgIPK2b genes in oil palm seedlings infected with G. boninense, utilizing Ct value correction through regression coefficients on the 2‐ΔΔCt and E‐ΔΔCt approaches. A correlation regression revealed values of 0.28, ‐0.32, and 0.29 for delta Ct of EgUnk1, EgZFP2, and EgIPK2b, respectively. However, a negative correlation in the Ct mean was corrected by linear regression for the targeted genes: ‐0.55, ‐0.81, and ‐0.29 for EgUnk1, EgZFP2, and EgIPK2b, respectively. The amplification factor (E) and efficiency value (R) using the EgActin gene were 1.95 and 94.92%, respectively. Normalization of log10 on the fold change value 2‐ΔΔCt and 1.95‐ΔΔCt approaches using the regression coefficient yielded consistent results for the EgUnk1, EgZFP2, and EgIPK2b genes. Overall, EgUnk3 and EgIPK2b genes exhibited downregulated expression in susceptible oil palm seedlings (‐0.60 for 2(‐ΔΔCt) and ‐0.58 for 1.95(‐ΔΔCt)), whereas EgIPK2b gene showed up‐regulated and the highest value in inoculated resistant seedlings (1.39 for 2(‐ΔΔCt) and 1.34 for 1.95(‐ΔΔCt)). Basal stem rot disease (BSR) in oil palm decreased EgUnk1 and EgIPK2b expression in susceptible seedlings but increased EgZFP2 gene expression in resistant ones. The results of this research provide valuable corrections to Ct values obtained directly from RT‐qPCR machines using simple linear regression. Consequently, the Ct values of target genes and reference genes exhibit smaller bias values, rendering gene expression levels more reliable.

Background The development of basal stem rot (BSR) disease in oil palm is associated with lignin during vegetative growth and salicylic acid (SA) biosynthesis. The increase in the lignin content, SA accumulation, growth, and root biomass could indicate the resistance of oil palm seedlings to BSR disease. Therefore, although there are many studies on the interactions between the Ganoderma boninense and oil palm, research on evaluation of physiological processes, biochemistry, and molecules occurring during early internal symptoms of BSR in roots of oil palm ( Elaeis guineensis Jacq.) are essential. Results Ganoderma boninense inoculation indicated that C01, C02, and C05 seedlings were susceptible, while the other three seedlings, C03, C07, and C08, were resistant based on Ganoderma Disease Index (GDI). Infection by G. boninense in the most susceptible seedlings C05 reduced fresh weight of roots (FW) by 9.0%, and lignin content by 10.9%. The most resistant seedlings C08 were reduced by only 8.4%, and 0.2% regarding their fresh weight and lignin content, respectively. BSR disease induced SA accumulation in the most susceptible C08 and decreased peroxidase (PRX) enzyme (EC 1.11.1.7) activities in root tissues of oil palm seedlings except C07 and C08 where PRX activities remained high in the 4 months after planting. Infection with G. boninense also increased glutathione S-transferase U19-like (EgGSTU19) gene expression in the root tissues of susceptible seedlings, while laccase-24 (EgLCC24) gene expression was associated with resistance against BSR disease. Based on the relative expression of twelve genes, two genes are categorized as receptors (EgWAKL5, EgMIK1), two genes as biosynthesis signal transduction compound (EgOPR5, EgACO1), five genes as defense responses (EgROMT, EgSOT12, EgLCC24, EgGLT3, EgGSTU19), and one gene as trans-resveratrol di-O-methyltransferase-like (EgRNaseIII) predicted related to BSR infection. While two other genes remain unknown (EgUnk1, EgUnk2). Conclusions Ganoderma infection-induced SA accumulation and lignification in resistant accessions promote the seedlings root biomass. Oil palm seedlings have a synergistic physical, biochemical, and molecular defense mechanism to the BSR disease. The utilization of nucleotide-based molecular markers using EgLCC24 gene is able to detect resistant oil palm seedlings to G. boninense .

Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense. Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense. A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense.

The Ganoderma boninense is the pathogen causing basal stem rot (BSR) disease in oil palm, degrades oil palm root cell walls and modify plant metabolisms. Research groups have studied molecular mechanisms responses to G. boninense infections. A few have indicated the importance of studying specific gene expressions in relation to resistance mechanisms. This study aims to identify differentially expressed genes in oil palm roots showing susceptible or resistance responses and visualize their accumulated transcript in the heatmap form. The susceptible and resistant seedlings showing symptoms of BSR infection and healthy ones were sampled. Sixteen genes associated with G. boninense infection were identified and their expression were analyzed. Total RNAs were extracted from root samples and cDNA synthesized. Quantitative real time PCR were performed using cDNA, and differential gene expressions were evaluated. Two genes showed the highest transcript accumulation in susceptible and resistant seedlings treated with or without G. boninense . Ten out of the 16 genes belonged to Group I and the other six to Group. Seven out of the 16 genes were differentially expressed in the samples either with or without G. boninense inoculation. The seven genes may be associated with early infection responses to G. boninense.

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.

Dalam penelitian ini, analisis akumulasi transkrip menggunakan Reverse-Transcriptase Quantitative PCR (RT-qPCR) diikuti dengan analisis interaksi statistik telah dilakukan pada akar dari dua genotipe tanaman kelapa sawit (A09 dan 23) yang rentan dan toleran terhadap Ganoderma boninense . Tiga gen putatif ( Eg#001 , Eg#004 , dan Eg#007 ) dari kelapa sawit diprediksi dapat digunakan sebagai biomarker dalam seleksi varietas tanaman kelapa sawit terhadap Ganoderma . Optimasi analisis RT-qPCR dilakukan dengan melakukan serial dilusi cDNA (1/5, 1/10, 1/25 dan 1/50) untuk mendapatkan nilai amplification plot dan melting curve yang ideal. Hasil analisis memperlihatkan serial dilusi 1/10 atau setara konsentrasi cDNA 50 ng/µL mendapatkan nilai Ct pada ambang 22-24 serta nilai melting curve sebesar 83,06. Secara umum, akumulasi transkrip gen pada genotipe 23 lebih melimpah dibandingkan dengan genotipe A09. Gen Eg#007 secara statistik memiliki akumulasi transkrip yang berbeda nyata pada genotipe 23 yang lebih tinggi (5,21E+00) dibandingkan pada genotipe A09 (1,93E+00). Di sisi lain, hasil identifikasi lokus ID pada genom kelapa sawit memperlihatkan bahwa gen Eg#007 merupakan penyandi protein yang tergolong dalam famili sulfotransferase (SOT) yang terkait dengan sistem pertahanan tanaman. Gen tersebut berpotensi mengungkap mekanisme aksi-reaksi antara G. boninense dan akar kelapa sawit. Kata kunci: kelapa sawit, Ganoderma , profil akumulasi transkrip, RT-qPCR, biomarker.

TheIndonesian government program of achieving self sufficiency of soybean by 2020 requires technological innovations for the farmers. The use of plant biostimulant is an innovative strategy and proven previously to increase the productivity of several other food crops. The aim of this study was to analyze the effect of PPBBI bi ostimulant on the growth, productivity and quality of a Wilis variety of soybean under greenhouse conditions. PPBBI biostimulant at 10 ppm and 20 ppm, was applied using foliar spray method with 20 mL volume to each plant. The applications were carried out 1 time, 2 times and 3 times . Six biostimulant treatments showed significant positive effects on the vegetative growth rate , generative organ development , and yield . The b iostimulant accelera ted vegetative growth to enter the generative phase earlier compared to control plants. The period of generative organs maturity required for treated plants was 7 14 d shorter compared to control plants so that the harvest period was 21 d shorter. Weight per 100 seeds of the P2 3 (application 3 times at 20 ppm); P2 2 (application 2 times at 20 ppm); P2 1 (application 1 time at 20 ppm) and P1 3 (application 3 times at 10 ppm) treatments were 20.16 g; 17.65 g; 18.89 g and 16.89 g respectively with no significant difference w hile the control plants was only 11.60 g . Based on the results of all parameters e.g.average number of seeds average weight per seed, and potential for yield improvement, the treatment of P1 3 (application 3 times at 10 ppm) was the best tr eatment with potential yield increase by 59.06% and oil content by 11.37%.

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]

The Indonesian government program of achieving self-sufficiency of soybean by 2020 requires technological innovations for the farmers. The use of plant biostimulant is an innovative strategy and proven previouslyto increase the productivity of several otherfood crops. The aim of this study was to analyze the effect of PPBBI biostimulant on the growth, productivity and quality of a Wilis variety of soybean under greenhouse conditions. PPBBI biostimulant at10 ppm and 20 ppm, was applied using foliar spray method with 20 mL volume to each plant. The applications were carried out 1 time, 2 times and 3 times. Six biostimulant treatments showed significant positive effects on the vegetative growth rate, generative organ development, and yield. The biostimulant accelerated vegetative growth to enter the generative phase earlier than that of in the control plants. The period of generative organs maturity required for treated plants was 7-14 d shorter than that of in the control plants so that the harvest period was 21 d shorter. Weight per 100 seeds of the P2-3 (application 3 times at 20 ppm); P2-2 (application 2 times at 20 ppm); P2-1 (application 1 time at 20 ppm) and P1-3 (application 3 times at 10 ppm) treatments were 20.16 g; 17.65 g; 18.89 g and 16.89 g respectively with no significant difference, while the control plants was only 11.60 g. Based on the results of all parameters e.g. average number of seeds, average weight per seed, and potential for yield improvement, the treatment of P1-3 (application 3 times at 10 ppm) was the best treatment with potential yield increase by 59.06% and oil content by 11.37%.[Key words: generative, organic biostimulant, productivity, vegetative] AbstrakProgram pemerintah Indonesia dalam pencapaian swasembada kedelai pada tahun 2020 membutuhkan dukungan inovasi teknologi yang aplikatif untuk para petani. Biostimulan tanaman merupakan salah satu teknologi yang strategis dan terbukti dapat meningkatkan produktivitas beberapa tanaman pangan. Tujuan penelitian ini adalah untuk melakukan analisis pengaruh aplikasi biostimulan PPBBI terhadap partum-buhan, produktivitas dan kualitas hasil panen kedelai varietas Wilis pada kondisi rumah kaca. Biostimulan PPBBI dengan variasi konsentrasi 10 ppm dan 20 ppm, diaplikasikan pada tanaman kedelai dengan metode penyemprotan lewat daundengan volume 20 mL per tanaman.Penyemprotan dilakukan sebanyak 1 kali; 2 kali dan 3 kali aplikasi. Enam perlakuan biostimulanyang diujikan menunjukkan pengaruh positif yang signifikan pada kecepatan pertumbuhan vegetatif, stimulasi perkembangan organ generatif dan peningkatan hasil panen. Biostimulan PPBBI mempercepat laju pertumbuhan vegetatif untuk memasuki fase generatif lebih awal dibandingkan tanaman kontrol. Masa perkembangan dan pemasakan organ generatif polong pada tanaman perlakuan menjadi lebih pendek 7-14 hari dibandingkan tanaman kontrol sehingga secara keseluruhan masa panen tanaman perlakuan lebih singkat 21 hari. Bobot per 100 biji tanaman kedelai perlakuan P2-3; P2-2; P2-1 dan P1-3 berturut-turut mencapai 20,16 g; 17,65 g; 18,89 g; dan 16,89 g dengan tidak adanya perbedaan signifikan, sedangkan tanaman kontrol hanya 11,60 g. Berdasarkan hasil analisis seluruh peubah yaitu rerata jumlah biji, rerata bobot per biji, dan potensi produksi, maka perlakuan P1-3 (aplikasi tiga kali dengan dosis 10 ppm) merupakan perlakuan terbaik dengan potensi kenaikan produksi mencapai 59,06% dan kadar lemak 13,7%. [Kata kunci: biostimulan organik, generatif, produktivitas, vegetatif]

Genome editing using CRISPR/Cas9 construct containing sgRNA is the newest technology used to assemble organisms with desired characters through knock out of the target gene. As part of the effort to develop oil palm planting material tolerant to Ganoderma, this research aimed to obtain a technique for introducing CRISPR/Cas9 modules containing sgRNA into oil palm tissue. Based on differential expression studies, two genes encoding isoflavone reductase (IFR) and methallothionine-like protein (MT) were selected as the target for edited genes. The CRISPR-IFR and CRISPR-MT modules each carrying a sequence of gene recognition (sgRNA) were constructed and transformed into oil palm calli using Agrobacterium tumefaciens. The transformed calli were cultured on a modified DF medium for callus propagation, and then sub-cultured into DF media for induction of somatic embryos. PCR analyses using specific primers flanking the DNA targeted region were carried out and the results showed that both constructs had been introduced into the cells. DNA sequence analysis indicated the presence of substitution of several bases in the target area of both genes. The transformed calli grew in a selection medium containing hygromycin. Development of somatic embryos was detected after 3-4 weeks culturing on modified DF media.

Tandan Kosong Kelapa Sawit (TKKS) merupakan salah satu limbah perkebunan yang jumlahnya sangat melimpah. Telah banyak penelitian dilakukan yang bertujuan untuk memanfaatkan limbah ini menjadi produk yang bernilai ekonomi tinggi dan salah satu adalah mengomposkan TKKS tersebut. Teknik pengomposanTKKS yang selama ini memerlukan waktu 2-4 bulan dan pengangkutan produk kompos yang dihasilkan memerlukan biaya yang mahal. Waktu pengomposan yang lama tidak dapat mengatasi permasalahan banyaknya limbah TKKS ini dihasilkan di pabrik (21-23% dari Tandan Buah Segar). Sehingga diperlukan teknik pengolahan limbah yang lebih cepat. Pada penelitian sebelumnya TKKS terbukti dapat dikonversi melalui proses pirolisis yang relatif lebih cepat menjadi arang hayati dan asap cair. Arang hayati memiliki banyak manfaat khususnya untuk meningkatkan produktivitas tanaman. Selain itu juga dapat memacu aktivitas mikroba tanah, terutama yang berasosiasi dengan akar tanaman dan mampu meningkatkan konservasi unsur hara mudah larut sehingga pencucian hara menjadi minimal. Asap cair selama ini banyak digunakan sebagai bahan pengawet makanan dan penghilang bau pada industri karet. Pada penelitian ini akan dilakukan karakterisasi arang hayati dan asap cair dari TKKS . Dari 6 kg TKKS dapat dihasilkan 1,9 kg arang hayati dan 3,6 L asap cair. Arang hayati TKKS memiliki kadar makronutrien : C 60%; N 1,07%; P 1,29%; K 13,37%; Mg 1,02%; Ca 1,71% dan mikronurien Fe 0,95%; B 31 ppm dan Zn 248 ppm, dengan pH 9. Asap cair yang dihasilkan memiliki pH 3,5 dan dapat dihilangkan kandungan tar-nya dengan pengendapan semalam. Kandungan asap cair ini. mengandung karbonil 2,984 %; turunan fenol 13,169 %; dan asam organik 74,268 %.

Plant biostimulants are applied to meet the challenge for sustainability of agriculture production. IRIBB has developed organic-based biostimulant formulas (OBFs) and tested in the field of several crops. OBF-1 improved the vegetative growth and productivity of some annual food crops. Compared with the control plants, the productivity of OBF-1 treated rice, maize, potato and shallot increased by 25%, 31%, 30% and 23%, respectively. In addition, OBF-1 treated crops indicated more tolerant to pests and diseases. OBFs applications on perennial estate crops demonstrated that OBF-2 and OBF-1 combination increased tea yield around 48% and sugarcane yield up to 50%. Whereas foliar sprays of OBF-3 on oil palm increased the CPO yield by up to 32%. The increased CPO yields were contributed to the increase of total FFB (fresh fruit bunches) weight and oil extraction rate in the fruit mesocarp. OBF-3 is a formula enriched with oil-biosynthetic activators. The farmers and planters should gain additional profits from the increased crop yields in response to the OBF application. How much profits gained were depended also on the price of the commodities. In general, the additional profits were much higher than the OBFs and labour costs.

Latex, a milky white liquid, is the main product from rubber tree (Hevea brasiliensis). Latex is the cytoplasm of complex cellular networks named laticifers in which it contains many different components, including important proteins. Various types of enzymes carrying functions associated with plant defense against pathogen and wounding have been detected in latex in which one of these enzymes is protease inhibitor (PI). Plant protease inhibitor has tremendous potential as an antifungal agent which can be developed as biofungicide. In this work, protease inhibitors from B-serum (lutoid) of rubber tree latex were isolated and purified using Ion Exchange Chromatography (IEC) technique. Of the total 70 fractions of proteins extracted from the columns, only 26 fractions showed measurable levels of protein. The concentration of obtained putative protease inhibitors (three fractions of IEC) ranged from 0.007 to 0.022 mL/g B-serum. Inhibitory activity against four protease enzymes (subtilisin A, trypsin, α-chymotrypsin, and papain) showed the characteristics of Hevea putative protease inhibitors from B-serum as serine and/or cysteine protease inhibitors with more than 15% inhibitory activity of target protease. Based on SDS-PAGE visualization, the molecular weight of dominant protein considered as Hevea putative protease inhibitors was 21.5 kDa. In vitro bioassay test of antifungal activity for Hevea putative protease inhibitors showed reduced mycelium growth of Ganoderma boninense, Sclerotium sp., and Rigidosporus lignosus.

Anthurium adreanum Lindl. cv. Nitta is an ornamental plant for cut flower industries. In vitro propagation enables a large scale production of high quality seedlings of A. adreanum quickly. The aim of this experiment was to overcome some problems in the in vitro propagation of A. adreanum, such as contamination, lack of plantlets vigor and low rate of survival in the acclimatization. In vitro propagation of A. adreanum was conducted via organogenesis from leaf explant. Explant sterilization using desogerm, antioxidant, alcohol, rifampicin, and NaOCl was the best because they reduced browning and contamination until 0 %. Organogenesis of A. adreanum was successfully initiated using MS medium added with 2,4-D and BAP at 1 mg L-1, which produced 74 shoots per explant. Shoots were best maturated in MS medium with a half strength of macro minerals added with 1 mg L-1 paclobutrazol. Survival rate increased by 7 and 14 days hardening treatment using double layer medium in the light culture room. Survival rate reached 89.3 % at 8 weeks after acclimatization.

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.

AbstractAcetyl-CoA Carboxylase (ACCase) is considered to beone of the key enzymes in palm oil biosynthesis. Availabilityof genes encoding this enzyme would give some advantagesin the molecular breeding of oil palm. Over expression ofthe genes in the oil palm mesocarp might increase the oilproduction in this tissue. On the other hand, downregulating of ACCase could divert the central metaboliteAcetyl-CoA to other product such as PHB (Polyhydroxy-butyrate), one of the known biodegradable plastic. Thispaper reported the work of cloning of the full length codingsequence of biotin carboxylase (BC), one subunit of theACCase. Based on the DNA sequence of the BC conservedregion that had cloned previously, primers pairs weredesigned to amplify 5’- and 3’- cDNA ends of BC usingRACE-PCR. The RACE products of 5’- and 3’- cDNA endsof BC were cloned into E.coli, and the DNAs weresequenced and analysed. The full cDNA of BC was obtainedby reisolation of the cloned 5’- and 3’- cDNA ends followedby digestion using KpnI, ligation into pGEM-T vector andcloning into E.coli. Colony PCR was carried out to confirmthat the target gene has been cloned. The recombinantplasmid containing full cDNA of BC was then isolated forDNA sequencing. The results showed that the 5’-BC (1367bp), 3’- BC (1032 bp), and the full length cDNA encodingBC (2182 bp) had been successfully cloned, and the DNAsequence had been confirmed as gene encoding ACCasesubunit biotin carboxylase.AbstrakAcetyl-CoA Carboxylase (ACCase) merupakan salahsatu enzim kunci dalam biosintesis minyak sawit. Keter-sediaan gen penyandi enzim ini sangat berguna dalampemuliaan kelapa sawit secara molekuler. Over-ekspresi genpenyandi ACCase pada mesokarp dapat meningkatkan pro-duksi minyak pada jaringan tersebut. Sebaliknya ekspresiACCase dapat ditekan melalui mekanisme down regulation sehingga metabolit central Acetyl-CoA dapat diarahkanuntuk menghasilkan produk lain seperti PHB (polyhydro-xybutyrate), salah satu jenis biodegradable plastik yangtelah banyak dikenal. Penelitian ini bertujuan untukmengklon cDNA lengkap penyandi ACCase subunit biotincarboxylase (BC) dari mesokarp kelapa sawit. Berdasarkansekuen DNA daerah konservatif BC yang telah diklon darimesokarp kelapa sawit pada penelitian sebelumnya, duapasang primer dirancang untuk mengamplifikasi daerahujung 5’- dan 3’- cDNA BC dengan RACE-PCR. Produk5’-RACE dan 3’-RACE diklon dan disekuen. cDNAlengkap penyandi BC diperoleh dengan jalan mengisolasikembali fragmen 5’- dan 3’- cDNA terklon, dilanjutkandengan digesti menggunakan enzim restriksi KpnI, ligasikedua fragmen ke vektor kloning pGEM-T, dan introduksike dalam E. coli. Setelah dilakukan PCR koloni untukmenguji keberhasilan kloning, plasmid rekombinan yangmengandung cDNA lengkap dari BC diisolasi untuk analisissekuen DNA. Dari penelitian ini fragmen cDNA 5’-BC(1367 pb) dan 3’- BC (1032 pb), serta cDNA lengkappenyandi BC berukuran 2182 pb telah diperoleh dan diklondalam E. coli. Analisis sekuen DNA mengkonfirmasi bahwacDNA terklon adalah benar gen penyandi ACCase subunitbiotin carboxylase.

RingkasanPada kelapa sawit, mesokarp merupakan jaringan yang lebih dikhususkan untuk mensintesis minyak. Akumulasi minyak pada jaringan ini terjadi selama perkembangan buah. Beberapa enzim yang terlibat dalam biosintesis minyak tampaknya disintesis hanya pada periode tertentu dari biosintesis minyak. Sedangkan protein regulator diduga ada pada saat minyak mulai disintesis atau beberapa saat sebelumnya. Sebagai bagian dari usaha mengklon gen kunci untuk biosintesis minyak, penelitian ini bertujuan mengidentifikasi dan mengisolasi protein yang terekspresi secara diferensial sesuai perkembangan buah. Sebagai bahan penelitian digunakan jaringan mesokarp dari berbagai umur buah sawit. Untuk setiap fase perkembangan buah dilakukan analisis kandungan minyak dan protein total. Elektroforesis gel poliakrilamid-SDS (SDS – PAGE) dan elektroforesis dua dimensi (2-D) digunakan untuk mempelajari dan mendeteksi adanya pita protein spesifik yang terekspresi secara diferensial sejalan dengan peningkatan kandungan minyaknya. Hasil penelitian menunjukkan bahwa minyak mulai aktif disintesis pada saat buah berumur 17 minggu setelah antesis. Konsentrasi protein total tidak meningkat sejalan dengan peningkatan kandungan minyaknya. Dari hasil SDS-PAGE terdeteksi dua protein, yaitu protein dengan berat molekul (BM) 31,0 kDa dan 34,3 kDa yang meningkat ekspresinya pada awal dan menjelang periode aktif sintesis minyak. Analisis lebih lanjut dengan elektroforesis 2-D menunjukkan bahwa protein 31,0 kDa terdiri dari dua protein dengan pI 4,64 dan pI 4,95, sedangkan protein 34,3 kDa merupakan protein tunggal dengan pI 4,56. Sikuensing secara parsial kedua protein tersebut menunjukkan adanya dua polipeptida dari protein 31,0 kDa yang mempunyai homologi tinggi dengan subunit biotin karboksilase ht-ACCase, dan empat polipeptida yang mempunyai homologi dengan enoilACP reduktase. Sedangkan protein 34,3 kDa mempunyai homologi dengan gliseraldehida 3-fosfat dehidrogenase. SummaryIn oil palm, mesocarp is tissue specialized for oil synthesis. Accumulation of oil in this tissue occurs during fruit development. It is likely that some enzymes involved in oil biosynthesis are synthesized only in a certain period of oil biosynthesis, while regulatory proteins may present at the beginning or right before the period of active oil synthesis. As a part of research work on cloning of gene encoding key enzymes for oil biosynthesis in palm mesocarp, this research was aimed to identify and isolate proteins differentially expressed during fruit development. Mesocarps from different developmental stage of fruit were used for analysis of oil content and protein concentra-tion. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and two dimentional (2-D) electrophoresis were used to study and detect specific protein bands differentially expressed during fruit development. It was shown that oil synthesis was started at 17 weeks after anthesis (WAA). There was no correlation between concentrations of total protein with oil content during mesocarp development. From the SDS-PAGE, two protein of 31.0 kDa and 34.3 kDa were detected that their expression increased at the beginning and just before the period of active oil biosynthesis respectively. Further analysis with 2-D electrophoresis showed that 31.0 kDa-protein consist of two proteins, with pI 4,64 and pI 4,95, while 34.3 kDa protein is a single protein with pI 4,56. Partial amino acid sequencing data of the 31.0 kD protein showed that two polypeptides highly homologous with ht-ACCase biotin carboxylase subunit and four polypeptides homologuus with enoyl-ACP reductase, whereas 34.3 kD protein showed homology with glyceraldehyde-3 phosphate dehydrogenase.

RingkasanRekayasa genetika kopi arabika tahan penyakit cendawan dapat dilakukan dengan cara memasukkan gen kitinase (gen chi) ke dalam genom tanaman tersebut. Penelitian ini bertujuan untuk mengintroduksikan gen chi pada kopi arabika serta meregenerasi eksplan yang ditransformasi menjadi plantlet. Gen chi disubkloning dari pBS G11 ke dalam plasmid pCAMBIA2301. Melalui Agrobacterium tumefaciens, plasmid rekombinan pCAMBIA2301/35s-chi kemudian dimasukkan ke dalam eksplan daun dan embrio zigotik kopi arabika. Eksplan daun transforman ditumbuhkan pada media seleksi yang mengandung kanamisin untuk induksi kalus embriogenik . Beberapa kombinasi 2,4-D dan dicamba serta kinetin, BAP dan 2-iP diuji kemampuannya untuk menginduksi terbentuknya kalus embriogenik. Embrio zigotik transforman ditumbuhkan pada media MS modifikasi yang mengandung kanamisin. Hasil penelitian menunjukkan bahwa perbedaan tipe sitokinin dan kombinasinya dengan 2,4-D atau dicamba. menyebabkan terjadinya variasi persentase pembentukan kalus embriogenik tahan kanamisin. Penambahan 100 mg/L kanamisin dalam media seleksi cukup efektif untuk menghambat pertumbuhan eksplan daun nontransforman. Persentase tertinggi induksi kalus embriogenik pada eksplan daun non transforman maupun transforman diperoleh pada media yang mengandung 5 mM 2,4- D dengan 5 mM of kinetin atau 5 mg/L dicamba dengan 5 mM BAP. Sedangkan dalam media dengan penambahan 5 mM kinetin, 100 mg/L asam sitrat dan 100 ppm asam askorbat, jumlah eksplan yang membentuk kalus mencapai optimum pada konsentrasi 0 dan 1 ppm dicamba untuk eksplan transforman dan 10 mg/L dicamba untuk non transforman. Pada eksplan embrio zigotik transforman, peningkatan konsentrasi kanamisin dari 100 mg/L hingga 500 ppm menurunkan persentase pengecambahan embrio dari 80.5 % menjadi 49%, persentase perakaran, dari 34 % menjadi 16%, jumlah akar, panjang akar dan tinggi tunas dari 7 mm menjadi 4 mm. Pada semua perlakuan kanamisin, embrio zigotik non transforman tidak membentuk akar dan pada umur kultur yang sama tunas yang dihasilkan lebih pendek dibandingkan dengan embrio-zigotik transforman. Hasil tersebut membuktikan bahwa gen ketahanan terhadap kanamisin (NPTII) telah terinsersi dan terekspresi dengan baik pada plantlet kopi arabika yang berasal dari eksplan embrio-zigotik transforman. Karena gen chi dikonstruksi dalam satu vektor dengan NPTII, maka diharapkan gen tersebut juga telah terinsersi ke dalam genom tanaman kopi.SummaryGenetic engineering of arabica coffee resistant to fungal diseases might be done by introducing a chitinase-encoding gene (chi) into genome of this plant. This research was aimed to introduce chi construct into arabica coffee and regenerate plantlets from the transformed explants. The chi gene was previously subcloned from pBS G11 into pCAMBIA2301 plasmid. With Agrobacterium tumefaciens,the recombinant plasmid pCAMBIA2301/35s-chi was then introduced into leaf and zygotic embryos explants of arabica coffee. The transformed leaf explants were cultured on the selection media containing kanamycin in the presence of several combinations of 2,4-D and dicamba with kinetin, BAP and 2-iP to induce the formation of embryogenic callus. The transformed zygotic embryos were cultured on the media of modified MS containing kanamycin. The results showed that the several types of cytokinin used in combination with 2,4-D or dicamba caused the percentage of kanamycin resistant-embryogenic calli was varied. The addition of 100 mg/L kanamycin in the selection media was effective for inhibiting the growth of untransformed explants. Among the several combinations of auxin and cytokinin tested, the highest percentage of embryogenisis for untransformed and transformed leaf explants were achieved on the media containing 5 mM 2,4-D and 5 mM kinetin or 5 mg/L dicamba and 5 mM BAP. However in the presence of 5 mM kinetin together with antioxidants of 100 mg/L citric acid and 100 mg/L ascorbic acid, the explants calluses was optimum at 0 - 1 mg/L dicamba for transformed explants and 10 mg/L dicamba for untransformed explants. In the explants of transformed-zygotic embryos, increasing kanamycin from 100 mg/L up to 500 mg/L decreases the percentage of embryo germination from 80.5 % to 49%, rooted-shoots from 34 % to 16%, number of roots, root length and shoot length from 7 mm to 4 mm. At all the kanamycin treatments, root was not developed from the untransformed-zygotic embryos and the lenght of shoots were shorter compared to the transformed-zygotic embryos. This result demonstrates that the kanamycin-resistant gene (NPTII) has been inserted and well expressed in the plantlets of arabica coffee derived from transformed-zygotic embryos. Since the chi gene was constructed in one vector with NPTII, this gene might also been inserted in the genome of coffee.

Summary Leaf rust disease caused by Hemileia vastatrix is considered to be one of the most important diseases on arabica coffee plantation. In order to understand the mechanism underlying resistance of arabica coffee against leaf rust disease, this research was aimed to study expression of β-1,3 glucanase (GLU) and chitinase (CHI) genes in the arabica coffee S1934 and BLP10 that have been reported respectively as a resistant and susceptible varieties to H. vastatrix. The two varieties were essayed against H. vastatrix, and an RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) using total RNAs from the S1934 and BLP10, both inoculated with H. vastatrix and uninnoculated was carried out for studying the expression of GLU and CHI. Two primer pairs were designed to amplify the conserved region of GLU and CHI. Amplification products were sequenced and the nucleotide sequences were subjected to BlastX analysis. The result of bioassay confirmed that arabica coffee S1934 was resistant to H. vastatrix, while BLP10 was susceptible. β-1,3 glucanase was expressed in all of the four samples, the inoculated and uninnoculated S1934, and BLP10 in different degree. S1934 expressed higher GLU compared to BLP10. In the inoculated S1934 the expression of this gene was higher compared to that of the uninoculated one. Expression of CHI was detected only in the S1934, both inoculated and uninoculated. Sequence analysis confirmed that the RT-PCR products were exon regions of genes encoding β-1,3 glucanase dan chitinase respectively. Both of the cDNA fragment have been cloned in E.coli. Ringkasan Karat daun yang disebabkan oleh jamur Hemileia vastatrix merupakan salah satu penyakit penting pada perkebunan kopi arabika. Untuk memahami mekanisme ketahanan kopi arabika terhadap karat daun, penelitian ini bertujuan untuk mempelajari ekspresi gen β-1,3 glukanase dan kitinase pada varietas kopi arabika S1934 yang dilaporkan tahan karat daun dan varietas BLP10 yang termasuk rentan karat daun. Untuk itu kedua varietas diuji kembali ketahanannya terhadap H. vastatrix melalui bioesai dan dilakukan RT-PCR menggunakan RNA total dari S1934 dan BLP10, baik yang diinokulasi dengan H. vastatrix maupun yang tidak diinokulasi, untuk mempelajari ekspresi gen GLU dan CHI. Dua pasang primer spesifik dirancang untuk mengamplifikasi daerah konservatif kedua gen tersebut. Hasil amplifikasi disekuen dan dianalisis menggunakan program BlastX. Hasil bioesai mengkonfirmasi bahwa S1934 tahan terhadap H. vastatrix, sedangkan BLP10 rentan. β-1,3 glukanase diekspresikan pada kedua varietas, baik yang diinokulasi maupun yang tidak diinokulasi, namun dengan tingkat ekspresi yang sedikit berbeda. Varietas S1934 mengekspresikan β-1,3 glukanase lebih tinggi dibandingkan dengan BLP10. Ekspresi gen tersebut pada S1934 yang diinokulasi lebih tinggi dibandingkan dengan yang tidak diinokulasi. Sedangkan kitinase hanya diekspresikan pada varietas S1934. Hasil sekuensing dan analisis DNA mengkonfirmasi bahwa sekuen hasil RT-PCR merupakan bagian ekson dari gen penyandi β-1,3 glukanase dan kitinase. Kedua fragmen tersebut telah diklon pada E. coli.