Evaluation of resistance of double haploid population of crosses between IR64 and Oryza rufipogon against Bacterial Leaf Blight (BLB) at seedling stage was conducted during dry season 2005/2006 in the screen house, at Rice Centre Research at Sukamandi. Inoculum was prepared by isolating BLB infected leaf in laboratory using Wakimoto's media. Seeds were germinated in petri dish for 48 hours, and then were sown in the plastic boxes size of 40 cm x 30 cm, each family was planted in 10 cm long row. TN1, IRBB, Code, Angke, dan O. rufipogon were used as control. Leaf inoculation of isolates of Xanthomonas oryzae pv. oryzae (XOO) ras III, IV, and VIII with concentration of 10 8 cell/ml, were applied to the plants at 18-21 day old plants by cutting method. Fertilizer application as recommended. Pest and weed control were based on necessity. Observation of disease severity was carried out after a sensitive control, TN1, was a severely affected. Observation method based on SES IRRI (1996) which are 1 for plant showed 0-3% of leaf damage, 2(4-6%), 3(7-12%), 4(13-25%), 5(26-50%), 6(51-75%), 7(7-87%), 8(88-94%), and 9 for plant with 95-100% of leaf damage. Result showed that Bio50-ACBlas/BLB03, Bio59-AC-BLB05 and Bio67-AC-BLB05 lines were resistant to phato-type III, 11 lines showed moderate resistant to phato-type IV, and Bio46-AC-Blas/BLB03, Bio47-AC-BLB05, and Bio48-AC-BLB05 lines were resistant to phato-type VIII. Apart of those, there were 2 lines, Bio38-AC-BLB05, and Bio63-AC-Blas/BLB03 showed moderately resistance to three phatotypes tested.
WHO recommends artemisinin-based combination therapy for curing malaria which is still a health problem in Indonesia. Artemisia annua L. is the primary source for artemisinin comes from subtropical China and India. Some introduction materials are grown in Indonesia, but they produce low concentration of artemisinin. Artemisinin synthesis uses the isoprenoid pathway, in which farnesyl diphospate (FDP) serves as the main precursor. FDP is the main precursor for sterol synthesis as well. The gene controls sterol biosynthesis is Squalene synthase (SQS). So then SQS is considered a competitive gene for artemisinin biosynthesis. CRISPR/cas9 is the most advanced system for genome editing, and can be used to induce a targeted-mutation. The aim of this research was to elevate Artemisinin content through disruption of the biochemical pathway of sterol synthesis using CRISPR/Cas9. Materials and methods used were as follows. Artemisia CRISPR/Cas9 seeds were processed and grown in selection medium and then soil. Seventy-seven Artemisia kanamycin resistant lines were produced. Fifty-two (of 77) lines were confirmed to have T-DNA by PCR with SQS-Fn and SQS-Rn primers, produced about 710 bp DNA fragment. By using SQS-Fs and SQS-Rs primers, those produced shorter DNA fragments (about 470 bp). A high-resolution electrophoresis QIAxcel was applied to identify for any nucleotide difference occured in PCR product of lines. Results showed that 44 lines (84.61%) have varied sizes with one to three nucleotides differences compared to control (plasmid, 468 bp). By assuming this analysis was right, it may be said that mutations has occured in SQS gene of Artemisia lines due to the CRISPR/Cas9.
The sugar yield lost caused by borer is about 10% or equal to 1000 kg of sugar/ha. There is no resistance trait to stem or shoot borer available in sugarcane germplasm. Genetic engineering by expressing of the Cry1Ab-Cry1Ac gene fusion is efforts to develop resistant variety to borer. A synthetic gene which consists of Rubisco gene promoter, chloroplast specific transit peptide (CTP) and the Cry1Ab-Cry1Ac was designed and assembled for a total size of 4019bp. It was inserted into pU3775CE plasmid cloning, and then into pCAMBIA5300_Ubi-tNOS plasmid vector at HindIII and KpnI sites, producing of pCAMBIA5300_RbcS::Cry1Ab- Cry1Ac. This plasmid was transforming into Agrobacterium tumefaciens and then transformed into Bulu Lawang (BL) sugarcane calli. Research aims were to identify lines produced from plant transformation molecularly and to evaluate their resistances against shoot borer. Methods applied were a DNA isolation and PCR using KAPA 2G ready mix and CryIAc-316F and CryIAc-316R primers, and plant bioassay with larva instar 1. Research progresses were 30 lines had been identified and proved containing of the cryIAc gene. Preliminary results of bioassay showed that there was variation among 10 BL Cry lines on shoots (from healthy to wilt and dry) and also on shoot borer conditions.
<p>Bulu Lawang (BL) is a sugarcane variety preferred by farmers in Indonesia due to its high yield, but this cultivar is susceptible to shoot and stem borer insect pests. Genetic engineering using cry1Ab and cry1Ac fusion gene is an effort to generate BL varieties resistant to the insect pests. This study aimed to 1) transform T-DNA containing cry1Ab-cry1Ac fusion gene into sugarcane genome by using Agrobacterium tumefaciens method, 2) obtain selection media composition of callus transformants, and 3) obtain transformation efficiency comparison of A. tumefaciens strains EHA105 and GV3101. The research was conducted at the Laboratory of Cell and Tissue Biology, the Laboratory of Molecular Biology, and the greenhouse of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor from March to August 2019. Research activities consisted of four parts namely 1) callus induction and Agrobacterium culture preparation containing pCambia5300-cry1Ab-cry1Ac ///// pCambia-5300_OaRbcS-prom-cTP-cry1Ab-cry1Ac plasmid, 2) callus incubation in cocultivation and resting media, 3) selection and differentiation of shoots on regeneration media, and 4) molecular analysis using PCR method. Results showed that the composition of media, both for selection and regeneration processes of putative plant transformants, was the key to the success of this experiment. A. tumefaciens strain EHA105 resulted in higher transformation efficiency (11.1%) compared to that of strain GV3101 (9.0%). Molecular analysis showed that cryIAb-cryIAc fusion gene was successfully inserted into the sugarcane genome suggesting that the transgenic plant containing cry1Ab-cry1Ac fusion gene was obtained. The putative transgenic plants need further assay through bioassay tests to verify its resistance phenotype to the insect pests.</p>
ABSTRAKStep I: the pBY520 and pRP9 were cut with BamHI and HindIII, and electrophorated with 1% agarose gel. DNA fragments of HVA1 and pRP9 were purified, ligated with T4 DNA ligase, and transformed into Escherichia coli DH5-α by heat shock. E. coli were grown onto solid medium (+ kanamycin 100 mg/l). A new plasmid DNA was isolated from single colony culture of the bacteria, confirmed, and named pRP9_HVA1.Step II: DNA of pRP9_HVA1 and pAY560326 were cut with XhoI dan SpeI enzymes, purified, and ligated. The next procedure was similar to step I, and the resulted plasmid was confirmed by PCR and digestion with XhoI dan SpeI enzymes, and named pAY_HVA1.Step III: pAY_HVA1 was first transformed into Agrobacterium EHA-105 and then into rive varieties Ciherang and Inpari 6 using the early infection of scutellum transformation method. Nine transgenic rice lines that positively contain HVA1 were obtained.
An enhancer trap system is an insertional mutagenesis based upon gene expression, instead of gene knock-out, so its insertion in genome is expected not linked to any dramatic changes in plant phenotypes. Gene knock-out, leading to lossof- function (LoF) mutation, is a dominant approach for rice functional genomic studies. The objective of this study was to find out whether Transcriptional Activator-Facilitated Enhancer Trap (TAFET) T-DNA insertion inducing mutant phenotypes in rice TAFET population. Materials used in this experiment were T1 generation of 270 rice TAFET lines. Eight plants of each were grown in the greenhouse and observed for any mutant phenotypes. Phenotypic, histochemical, Southern<br />blot analyses were carried out to define a mutant of pSKC66.1- 8e. Result showed that about 10% of the 270 lines produced chlorophyll-deficient leaves, ranged from yellowish green (viridis), white stripe green zebra-like stripe) to completely white (albino). Albino plants died after two weeks, whilst white stripe or viridis mutants became normal in the next generation<br />(T2). Another mutant was pSKC66.1-8e line which had floral dramatic phenotype change with various spikelet shapes and number of organs, and had a single twisted culm. The flower of mutant also had gus gene expression. Plants with wild type did not express gus gene and had six or more straight culms. Molecular, histochemical and phenotypic analyses of this particular line for three generations indicated that mutant phenotype was not due to the T-DNA insertion. Since there was approved that Tos17 is activated during tissue culture and induced mutant phenotype, this line might relate to Tos17 insertion, but it needs further investigation to gain such conclusion.
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