Jurnal AgroBiogen
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Transformasi Gen Antisens ACC Oksidase pada Pepaya dengan Teknik Penembakan Partikel
Get PDFPapaya (Carica papaya L.) is a climacteric fruit that exhibit avery fast ripening rate. Ethylene controls the ripening eventin the papaya fruit. 1-aminocyclopropane-1-carbocxylic acid(ACC) oxidase gene encodes a specific enzyme for ethylenebiosynthesis. The gene had become a target for manipulationto make a gene construct of an antisense ACC oxidaseto regenerate transgenic papaya that has a characteristic ofdelayed ripening. The objective of the experiment is to engineer transgenic papaya that has a delayed ripening characteristic by transforming papaya with the antisense ACC oxidase gene through particle bombardment technique. The immature embryos of papaya variety Burungwere used for the explants. Antisense ACC oxidase and reporter (gus) genes were co-transformed to papaya calli. Four hundreds eighteen calli were bombarded by the antisense ACC oxidase gene. The transformation experiment resulted 25 putatives transgenic plants out of fifty plantsacclimatized in a greenhouse. Gus gene expression assay observed at 9 days after bombardment showed that the papaya explants bombarded twice at 9 cm shoot distance had 53.3% transformation rate of gus positive and 5.25 blue spots number in average. The results of PCR analysis showed that four out of 25 transgenic putative papaya plants (TR6, TR9, TR20, dan TR24), indicated a positive PCR of the antisense ACC oxidase gene with the amplified fragment DNA size of 800 base pair
Molecular Analysis and Effectiveness Assay of AV1 Gene in Transgenic Tobacco for Resistance to Begomovirus
Get PDFGenetic transformationof tobacco plant using AV1 gene was conducted atthe previously experiment and generated transgenic tobaccoplants positively carrying the selectable marker nptII gene.The objectives of this experiment were to (1) analyze thepresence of Begomovirus AV1 gene in T0 generation putativetransgenic tobacco plants using PCR technique with specificprimers and its correlation with resistance phenotype, (2)analyze the integration and copy number of the transgene inT0 generation putative transgenic tobacco plants and itscorrelation with resistance response, (3) screen the T0generation putative transgenic tobacco plants with the targetvirus infection and to detect the presence of the virus in thetransgenic plant tissue using universal primers. PCRdetection of AV1 gene in tobacco transgenic was conductedby using specific primer for Begomovirus AV1 gene.Meanwhile, Southern Blot analysis was conducted by usingthe AV1 gene probe. The effectiveness of AV1 gene intobacco transgenic was tested by inoculation of target virususing whiteflies vector. Result of the experiments showedthat there was a positive correlation between the presenceof the AV1 transgene in T0 generation putative transgenictobacco plants and the resistant phenotype. Transgenicplants with a single copy integration of the transgeneexhibited more resistant than the multiple copy one. andnon transgenic plant. The resistance as a result of AV1 geneexpression was indicated with no symptom in T0 generationtransgenic tobacco plants and the accumulation of the virusin the transgenic plants tissue. Northern and Westernhybridization analysis need to be perfomed for investigatingthe presence of mRNA or protein accumulation so that theresistance mechanism of the AV1 gene could be explainedmore detail
Penggunaan Aksis Jantung Pisang untuk Penyediaan Sumber Eksplan Bebas Bakteri
Get PDFThe sterile culture is very important in cryopreservation works. Bacterial-free explant sources are difficult to obtain during invitro culture of banana. Floral bud is expected as bacterial-free explant sources because the organ emerges above the groundand protected by bracts. The purposes of this study are to obtain optimal concentration of BA to regenerate male bud floralaxis explants of Barangan variety and to prove that cultures derived from these explants were free from bacterialcontamination. Two-millimeter pieces of male bud floral axis of Barangan variety were planted on MS medium containing of 1μM IAA, 200 g/l CH, and 2% sucrose. Experiment was arranged in Completely Randomized Design with the treatment of BA (5,10, 15, 20, and 25 μM) in 10 replications. Subculture was conducted by using MS media containing of 10 μM BA and 1 μM IAA.The variable observed were percentage of browning, number of nodules, number of shoots, number of normal shoots,number of abnormal shoots, and number of nonsurvived shoots. The screening towards bacterial contamination wasconducted by using medium containing of 10 g/l trypton, 10 g/l glucose, and 5 g/l yeast extract. The results showed that theexplants could regenerate into shoots. The 25 μM BA was the best treatment because it could produce the highest number oftotal and normal shoots, i.e. 9.2 shoots/explant and 6 shoots/explant, respectively. All of the shoots regenerated from male budfloral axis were 100% free from bacterial contamination, whereas all of the shoots regenerated from suckers werecontaminated by bacteria
Construction of Cry1Ac Plasmid Vector and Its Transformation into Agrobacterium tumefaciens
Get PDFIntroducing cry genesinto rice genome is reported able to produce rice plantresistant to stemborer. DNA sequence encodes cry1Ac genehas been inserted into pGEM4Z, but this construct does nothave a selectable marker gene for selection of transformedplant cells. The research aims were to construct a plasmidvector expressing a cry1Ac gene that has a transformationselectable gene and to transform it into Agrobacteriumtumefaciens. Materials used were pAY560325 binary plasmidvector, pGEM4Z-cry1Ac vector, Escherichia coli strain DH5-αand A. tumefaciens strain LBA4404 competent cells. Themethods consisted of plasmid DNA digestion using HindIIIand EcoRI, electrophoresis, DNA (backbone and insert)dissection from the gel, purification, and ligation using T4DNA ligase. Transformation of ligated DNA into E. coli byheat shock followed by cell plating onto selection medium,colony cultured, DNA isolation, and identification usingrestriction enzymes. Reconfirmation was done by cuttingusing restriction enzyme and PCR using F3 and R3, cry1Acgene specific primers. Research result were DNA fragmentsof 3.8 kb ubiquitin::cry1Ac insert and pAY560325, thebackbone vector, that after ligated and transformed into E.coli produced colonies. One of ten colonies containingplasmid DNA was evidently confirmed and namedpAY560325-cry1Ac. Subsequently, it was transformed into A.tumefaciens by electrophoration method. Plasmid DNA wasisolated from Agrobacterium that after digested with HindIIIand EcoRI produced DNA fragments of 9.44 kb (pAY560325)and 3.814 kb (ubiquitin::cry1Ac). While by PCR, plasmidproduced DNA fragment of about 711 bp. Thus, cry1Acplasmid vector (pAY560325-cry1Ac) was successfullyconstructed and transformed into A. tumefaciens and isready to be transformed into rice genome
Pembentukan Pustaka Genom, Resekuensing, dan Identifikasi SNP Berdasarkan Sekuen Genom Total Genotipe Kedelai Indonesia
Get PDFResequencing of the soybean genome facilitates SNP marker discoveries useful for supporting the national soybean breedingprograms. The objectives of the present study were to construct soybean genomic libraries, to resequence the whole genome offive Indonesian soybean genotypes, and to identify SNPs based on the resequence data. The studies consisted of genomiclibrary construction and quality analysis, resequencing the whole-genome of five soybean genotypes, and genome-wide SNPidentification based on alignment of the resequence data with reference sequence, Williams 82. The five Indonesian soybeangenotypes were Tambora, Grobogan, B3293, Malabar, and Davros. The results showed that soybean genomic library wassuccessfully constructed having the size of 400 bp with library concentrations range from 21.2–64.5 ng/μl. Resequencing of thelibraries resulted in 50.1 x 109 bp total genomic sequence. The quality of genomic library and sequence data resulted from thisstudy was high as indicated by Q score of 88.6% with low sequencing error of only 0.97%. Bioinformatic analysis resulted in atotal of 2,597,286 SNPs, 257,598 insertions, and 202,157 deletions. Of the total SNPs identified, only 95,207 SNPs (2.15%) werelocated within exons. Among those, 49,926 SNPs caused missense mutation and 1,535 SNPs caused nonsense mutation. SNPsresulted from this study upon verification will be very useful for genome-wide SNP chip development of the soybean genome toaccelerate breeding program of the soybean
Mikropropagasi Tanaman Manggis (Garcinia mangostana)
Get PDFThe conventional propagation of mangosteen plant is still facing some problems, such as the limited fruiting season and number of seedling, and slow growth of seedling. In vitro culture is an alternative technique to solve the problems. An experiment was done to obtain a suitable micropropagation technique for mangosteen plant through in vitro culture with high level of shoot multiplication and root formation, as well as high level of acclimated shoot or planlet growth. The treatments for shoot induction and axillary bud multiplication of mangosteen were three levels of BA (1, 3, and 5 mg/l) on the MS basal medium. The treatments for root induction were combinations between two kinds of basal medium (MS and WPM), two formulations of the media (full strength and 1/4 strength), and two levels of IBA (5 and 10 mg/l). Root induction was also done ex situ by dipping the shoots in IBA solutions (100-200 ppm) for 1-2 hours, followed planting onto the best acclimation media. The acclimation was done using two different media (soil only and soil + compost) under two different environments (green house and incubation room + green house). Results of the experiment showed that the highest percentages of seed growth and number of shoots per seed was obtained on the basal medium containing 5 mg/l BA. The highest number of axillary bud multiplication was obtained on the medium with 3 mg/l BA. MS medium + 5 mg/l IBA promoted 75% rooting. The plant acclimatization on soil + compost in the green house with 75% shading promoted the fastest plant growth. During the acclimatization, up to 75% of the shoots treated with dipping in 100 ppm IBA solution for one hour grew well. After four months, the roots of the plant developed secondary and tertiary roots
Transformasi Genetik Tembakau dengan Gen Cold Shock Protein melalui Perantara Agrobacterium tumefaciens
Get PDFColdshock protein (Csp) essential for organisms to survive inabiotic stress condition. CspB gene has been fused toubiquitin promoter in the T-DNA region of pCambia 1300int,and introduced into Agrobacterium tumefaciens LBA4404.This research had an objective to transform geneticallyNicotiana tabacum cv. Samsun by CspB gene under thecontrol of Ubiquitin promoter and NOS terminator mediatedby A. tumefaciens. Leaf discs were co-cultivated with A.tumefaciens LBA 4404. Based on the number of hygromycinresistantcalli, the efficiency of transformation was 57.5%. Inthe selective medium containing 50 μg/l hygromycin, theefficiency of regeneration of transgenic shoots was 82.6%.Based on PCR analysis using primers corresponding toubiquitin promoter and CspB gene, 18 putative tobaccotransgenic containing CspB gene under the control ofubiquitin promoter
Keragaman Genetik Kultivar Padi Beras Hitam Lokal Berdasarkan Penanda Mikrosatelit
Get PDFIndonesia has diverseaccessions of local black rice, which are important sourcesof germplasm. However, some of the local black ricecultivars have different names, leading a need to beidentified to determine their genetic diversity usingmolecular marker. This study aimed to identify geneticdiversity of eleven cultivars of local black rice, collection ofthe Assessment Institute for Agricultural Technology,Yogyakarta and compared them with two white ricevarieties using four microsatellite markers. Detection ofmicrosatellite alleles polymorphism was carried out byvisualization of PCR amplicons by electrophoresis onagarose gel. To estimate their genetic diversity, phylogenetictree and principal coordinate analysis were performed usingbinary data of SSR alleles. The results revealed that totalmarkers enabled to differentiate black rice cultivars asreflected by high value of polymorphic information content(PIC) mean (0.695). This value was consistent with the highgenetic diversity of black rice (genetic diversity index, h =0.283) in comparison with white rice cultivars (h = 0.020).The phylogenetic and main coordinate analyses suggestedthat black rice cultivars genetically differed from white rice.The local black rice cultivars were preferentially groupedbased on their genetic those were distributed in threecoordinates and did not represent their local geographicorigin. Genetic diversity analysis in this study will be usefulas an initial basis for proper identification and selection forappropriate parents to assist breeding program of black ricein Indonesia
Pemetaan, Karakterisasi, dan Pengembangan Primer-primer Lokus Pup1 (P uptake 1) pada Padi untuk Peningkatan Toleransi terhadap Defisiensi Fosfor
Get PDFPhosphorus (P) is the second most important nutrient forplants after nitrogen, but is available in very low amount. Pdeficiency in rice would reduce the number of tillers andgrain production. There are numerous publications onexploration of genes that are associated with P. Manyresearches on P that are directed to breeding program andinvolving many countries/institutions focus on Pup1research. Pup1 (P uptake 1) is associated with P uptake hasbeen well mapped on chromosome 12 at a distance of 15.31to 15.47 Mb and microsatellite markers between RM28073and RM28102 can be used as a selection tool in the MAB(Marker Assisted Backrossing) program. Indonesia is veryconcerned with this research because of P-deficientproblem. This review aims to provide current information ofresearch that explore the genes in Pup1 locus. This reviewoutlines the history of Pup1 mapping, to explain sequenceand expression analysis of Pup1, and to inform of Pup1specific primers. The latest information is expected to beuseful for rice breeders in Indonesia, especially for thosewho are interested to P deficiency research. Study of geneswithin Pup1 locus is still ongoing, and found that somegenes do not contribute directly to P uptake. This mayindicate that Pup1 locus use other mechanisms in the Puptake. This may indicate that some genes (dirigent-like,fatty acid α-dioxygenase, aspartic proteinases) play a role inthe increasing level of lignin in P deficient condition.Increasing level of lignin would increase the volume of rootsand thus increasing P uptake and resistance to biotic andabiotic stresses. Specific markers to detect the genes in thePup1 locus have been successfully developed, and can beused for breeding and exploration activities on Indonesianrice germplasm
Sebelas Tahun Perkembangan Jagung Bt dan Statusnya secara Global
Get PDFMajor insect pests of corn are the Asiancorn borer, the European corn borer, and the corn rootworm. The value of crop losses due to the insect pests inAmerica is 1.6 billion, Africa 0.6 billion. Prior to the use of Bt corn, farmersused a lot of insecticides to control the insect pests.Following introduction of the Bt corn in 1996, this crop hasbeen grown over 21 million hectares by millions of farmersfrom 13 countries in North America, Latin America, Asia,Africa and Europe. Globally, the farmers had been benefitedby grownt the Bt corn. The benefits varies, dependent oncountries and level of the corn borer infestations. In 2001,the US farmers gained 8 million from the Bt corn.Bt corn has not been grown commercially in Indonesia,although Bt corn MON810 has been declared as save torelease in the environment by the Indonesian BiosafetyCommittee. In 2001-2002, farmers in South Sulawesi withhad grown Bt cotton, this was the first time Bt crop in thecountry since the placement and implementation of thebiosafety regulation by the Indonesian Government in 1998