Jurnal AgroBiogen
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Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato
Get PDFOverexpression of alanine aminotransferase (AlaAT) gene can improve nitrogen use efficiency (NUE) in plants. The previous isolated AlaAT genes cannot be freely applied to generate NUE plants due to IPR restriction. Therefore, isolation of the gene from targeted monocot and dicot plants is necessary. The objectives of this study were to isolate AlaAT genes from barley, foxtail millet, cucumber, and tomato and analyze their homology to other isolated AlaAT genes in sequence databases (gene bank). Total RNA was isolated from roots of barley, foxtail millet, cucumber, and tomato, and then converted into cDNA using reverse transcription method. The cDNA was then cloned into pGEM®-T Easy plasmid and the verified clones were sequenced. The amino acid sequences were analyzed for their homologies using Clustal Omega software and phylogenetic tree was constructed. The results showed that the amino acids of AlaAT gene from barley was different from AlaAT genes of tomato and cucumber with homology level less than 80%. Phylogenetic tree predicted that AlaAT genes clustered into three groups with AlaAT genes of foxtail millet and barley clustered in one group together with other monocots in group I. AlaAT genes derived from dicots clustered into two groups in which AlaAT gene of tomato clustered in group II, while that of cucumber was in group III. The identity differences of AlaAT gene of tomato and that of cucumber as well as the estimates of the same enzymatic functions can open up enormous opportunities in genetic engineering research for the development of NUE rice
Validating Plant Genes Involved in Pepper Yellow Leaf Curl Indonesia Virus Infection Using VIGS in Model Plant Nicotiana benthamiana
Get PDFPepper yellow leaf curl disease caused by Pepper yellow leaf curl Indonesia virus (PepYLCIV) has become a challenge to chili pepper cultivation. Development of resistant variety by utilizing recessive resistance gene is expected to control the disease in the field. This study aimed to validate three plant genes, namely deltaCOP, hsc70, and BAM1, in PepYLCIV infection by applying Virus-induced Gene Silencing (VIGS) in a model plant, wild type Nicotiana benthamiana. PepYLCIV and construct of Tobacco rattle virus (TRV) which induced silencing of each gene were co-inoculated into N. benthamiana plants through agroinfiltration. Gene expression and the relative amount of viral DNA were determined by quantitative reverse transcription PCR (qRT-PCR) and quantitative PCR (qPCR), respectively, at 15 days post inoculation. The results showed a decreased level of deltaCOP, hsc70, and BAM1 expressions to 66.4%, 53.0%, and 47.0%, respectively, compared to that in the control (100%). Silencing of the three genes decreased the accumulation of PepYLCIV to 0.1%, 18.4%, and 63.0%, respectively, compared to that in the control. deltaCOP and hsc70 genes were indicated to be involved in the viral infection and could be good candidate genes for obtaining chili pepper varieties resistant to PepYLCIV. This result affirmed that the reverse genetics technique could be an alternative approach for identifying plant genes involved in viral infection, including PepYLCIV. The use of an infectious clone in this study allows the virus inoculations could be carried out without rearing and maintaining its natural vector, hence reduces the risk of virus transmission to healthy plants
Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method
Get PDFCold shock protein (Csp) is an essential bacterial protein for increasing abiotic stress tolerance, especially cold stress. Several studies discovered that overexpression of the gene successfully improves the tolerances of several types of plant not only under cold stress, but also other abiotic stresses, e.g. hot and drought conditions. The objectives of this study were to construct a binary vector containing the LcCsp gene modified from Lactobacillus casei and transform the gene into Nipponbare rice genome. The native LcCsp gene sequence, however, has low GC content (46.7%) while rice as transformation target plant has 52% GC content. The native LcCsp gene sequence, therefore, was optimized to the level close to 52.7% similar to GC content of the rice genome. This LcCsp gene was synthesized by using DNA printing technology (gBlocks® Gene Fragments Entry, IDT). The synthetic LcCsp gene was successfully inserted into the pCAMBIA1300-int binary vector driven by Ubiquitin1 promoter and NOS terminator. The T-DNA cassette was successfully transformed into Nipponbare rice genome by Agrobacterium tumefaciens strain LBA4404 using immature embryo transformation protocol. A total of 51 T0 Nipponbare lines survived from hygromycin selections and 21 lines were successfully acclimatized. Molecular analysis of the candidate lines showed that all Nipponbare transgenic putative lines contain the LcCsp gene demonstrating high transformation efficiency of 11.8%. The rice lines resulted from this study should be further analyzed and might be useful for developing rice transgenic lines tolerance to heat, drought, or saline stress condition
Molecular and Phenotypic Analyses of Inpari HDB/K15 F2 Lines Containing sd1 Mutant Gene Resulted from Genome Editing Method
Get PDFInpari HDB variety is resistant to bacterial leaf blight (BLB) disease, but due to its tall stature, the variety is susceptible to lodging. Inpari HDB with semidwarf stature, therefore, is of high interest for lodging resistant performance. sd1 gene encoding GA20ox-2 enzyme is one of the genes responsible for imparting semidwarf stature of rice. In previous study, sd1 mutant rice cv. Kitaake (K15) was developed by using CRISPR/Cas9 technology. The objective of this study was to analyze molecularly and phenotypically F2 lines containing sd1 mutant gene resulted from a cross between Inpari HDB and K15 to develop semidwarfInpari HDB rice variety. Thirty F2 Inpari HDB/K15 lines were analyzed at molecular level using DNA sequencing method together with phenotypic assessment of the lines to verify the integration of sd1 mutant gene. DNA sequencing analysis showed that 9 out of the 30 F2 Inpari HDB/K15 lines were sd1 mutants. The remaining F2 lines contained 17 heterozygotes and 4 nonmutants. All the nine mutant lines demonstrated shorter plant stature and showed more tiller number per plant compared to the nonmutant lines. The sd1 mutant gene in the F2 lines showed pleiotropic effects on panicle number andshowed no effects on other traits, such as flowering time, panicle length, filled and unfilled grain percentages. This study showed the introduction of sd1 mutant gene generated semidwarf Inpari HDB lines. The semidwarf Inpari HDB lines obtained from this research should be further evaluated to confirm their lodging resistant performances
Genetic Diversity Analysis and Development of DNA Fingerprints of 20 Indonesian Local Chili Pepper Varieties Based on SSR Markers
Get PDFChili pepper is one of the most valuable horticultural crops, widely cultivated in Indonesia. Analysis of its genetic diversity is needed to develop successful breeding programs of local varieties. Simple sequence repeat (SSR), a robust molecular marker used for genetic diversity analysis in plant species, offers potential, reliable DNA fingerprinting method to assess genetic variation and varietal identification of chili pepper. Fifteen SSR markers were used in this study to analyze the genetic diversity and develop profiling identification of DNA fingerprint of local chili pepper varieties. Twenty local and two improved varieties of three chili pepper species, consisting of 3, 1, and 18 varieties of Capsicum frutescens, C. chinense, and C. annuum, respectively, were assessed for their SSR polymorphism. A total of 87 alleles was obtained from the polymorphism analysis with high alleles variation (2–16 alleles) with average total allele of 5.8 and average polymorphism information content (PIC) of 0.59 (0.34–0.83). Clustering and Principle Coordinate Analyses (PCoA) classified the varieties into two groups with coefficient of similarity of 0.65 indicating their high genetic variability. Most local varieties belonged to the same cluster and separated from the two improved varieties. Based on PIC values and dendrogram with selected markers, five SSR markers, i.e. EPMS441, EPMS331, EPMS335, GPMS194, and CaSSRBio1.1, were identified as SSR marker set for DNA fingerprinting purposes. SSR marker set used in this study was successful in developing the varietal identity of local chili pepper varieties, as indicated by unique code of each variety
Effect of Introgression of Pup1 Locus on Rice Seedling under Phosphorus Deficiency
Get PDFThe lack of soil-phosphorus (P) element will result in plant growth retardation. Plants could survive in P deficiency stress by increasing the ability of P uptake or by increasing the efficiency in the P utilization. The aims of this study were to understand genetic composition of rice genotypes possessing Pup1 locus and to know root and leaf growth responses at different P availability condition. The three rice genotypes (IR74, IR74-Pup1, and Kasalath) were analyzed for their genetic composition using SNP markers. The phenotypic experiment was arranged using a Completely Randomized Design with four replications and performed hydroponically in nutrient solution with different availability of P. The result showed that IR74-Pup1 had 84.4% similarities to its parent (IR74) with 13.6% of donor segments, where the Pup1 locus located. The influence of Pup1 locusintrogression on total length, surface area, diameter, and volume of the root varied at each growth stage. IR74 and IR74-Pup1 had root and leaf growth restriction on low P, but Pup1 locus introgression showed better growth performance, both in normal P and in low P conditions. The introgression of Pup1 locus increases plant ability to reduce the impact of growth inhibitioncaused by P deficiency.
Genetic Diversity Analysis of 41 Chili Pepper Genotypes (Capsicum annuum L.) Based on SSR Markers
Get PDFChili (Capsicum annuum L.) is an important horticultural crop which possesses high genetic diversity. Their genetic diversity needs to be assessed through molecular marker-based analysis to facilitate chili breeding scheme. The objective of the study was to analyze the genetic diversity of 41 chili genotypes using Simple Sequence Repeat (SSR) markers. Eleven SSR markers were used to amplify all genotypes through PCR technique, visualized on Polyacrylamide Gel Electrophoresis (PAGE). The data were analyzed using NTSYSpc version 2.1 and PowerMarker version 3.25 softwares. Results showed that the average of SSR alleles was 7.9 with a range of 4-11 alleles per marker. All SSR markers showed PIC value >0.5, indicating their suitable use for chili genetic studies. Cluster analysis of 41 genotypes generated three main groups according to coefficient similarity of 77.4%. The genetic clusters appeared as reflection of the genetic background of each genotype. In addition, the SSR markers used can identify potential parent as indicated by the genetic distance between genotypes. The highest genetic diversity (0.698) was demonstrated by combinations of Yuni × Ayesha, Yuni × Ayesha 2, and Yuni × Namira. The estimated genetic diversity produced by SSR markers in this study should be useful as guidance for chili breeders to select suitable parents for new cultivar development for high productivity and resistance to diseases
Response of Anther Donor Genotypes (F1) from Indica x Indica Crosses to Rice Anther Culture
Get PDFAnther culture is one of the in vitro techniques that can be used to accelerate the obtainment of purelines in the form of dihaploid (DH) plants resulted from androgenesis. The objectives of this research were to study response of anther donor genotypes in rice anther culture of F1 populations derived from indica x indica crosses and obtain spontaneous DH lines to be used further for variety development program. Anther donor plant used for the experiment consisted of four kinds of F1 populations derived from single crosses, i.e. IR85/I-5, BioR-81/I-5, Bio-R81/O18, and BioR-82/O-18. The result indicated that kindy of all F1 plants showed similar response to callus induction, but significatly different responses to plant regeneration. Genotypes of anther donor plants (F1) significantly affected the percentage of calli producing green plantlet, but not significantly affected the percentage of calli producing albino plantlet. Anther culturability of anther donor plants (F1) was relatively high, ranged from 2.0 to 7.0%. A total of 73 first generation of spontaneous DH lines (38.8%) from the grew out green plantlets was obtained from this study. The resulted DH lines are readily available to be selected and be used in rice cultivar development program