Jurnal AgroBiogen
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Genome-Edited Plants and the Challenges of Regulating Their Biosafety in Indonesia
Get PDFGenome editing is a precise breeding technique to improve plant properties by editing specific genes that regulate desired trait. Genome editing techniques can be designed so that the resulting plant does not contain foreign genes and the resulting changes in DNA sequences cannot be distinguished from products obtained by conventional gene mutations which have been considered as safe and therefore unregulated. Thus, genome editing products in some countries are also not specifically regulated as GM products even though their assembly process uses recombinant DNA and genetic transformation. Brazil, likeIndonesia ratified the Cartagena Protocol, but it issued a special regulation that provides dispensation for several types of genome editing products and exempts them from regulations that apply to transgenic plants. The steps taken by other countries in regulating genome editing products can be taken into consideration in drafting regulations in Indonesia, in order to create a conducive environment that supports the use of this potential technology while at the same time provides assurance regarding its safety to human health and the environment. The purpose of this review was to provide information onthe development of genome editing technologies in plant breeding, analyze its risks compared to that of conventional breeding, and compare its biosafety regulation in various countries to provide some considerations for drafting regulations on the risk assessment of genome editing products in Indonesia, as a ratifying country of the Cartagena Protocol
Molecular Breeding for Developing A New Upland Rice Variety ‘Bio Patenggang Agritan’
Get PDFSitu Patenggang is an elite upland rice variety well-accepted by farmers, but recently this variety started to be attacked by blast disease (Pyricularia grisea) that causes yield loss up to 50%. To overcome this problem, Bio Patenggang variety has been developed through molecular breeding using blast-resistant monogenic lines as donor parents. The aim of this research was to present molecular breeding approach of Bio Patenggang development, including steps from the phenotypic selection on candidate lines to the genotypic analysis assisted by molecular markers. The phenotypic performances were assessed using Distinctness, Uniformity, and Stability (DUS) testing by following the standard method stated in the rice test guidelines. The genotypic analyses were performed using foreground and background markers. Four BC3F7 lines were selected as the candidates of the Situ Patenggang-derived variety. Based on ANOVA and Principal Component Analysis (PCA), phenotypic performances of the four lines demonstrated no significant differences with that of Situ Patenggang. The selected lines have also passed the foreground analysis confirming that the lines contained Pita, Pii, Pik-p, and Pia last resistance genes. Background analysis showed that the selected lines demonstrated agronomic perfomances very similar to that of Situ Patenggang. Association analyses showed that 14 markers were associated with the target traits and 10 out of the 14 markers were identified as co-segregation markers. The four selected lines, therefore, were proposed to be released as Situ Patenggang-derived variety. One of the lines (Sta-8-S15-TB16) has been approved to be released as a new variety, namely ‘Bio Patenggang Agritan’
Expression and Characterization of Recombinant β-1,3-Glucanase of Burkholderia cepacia (BiogenCC E76) Expressed in Escherichia coli Expression Systems
Get PDFBurkholderia cepacia (Bcc) BiogenCC E76 isolate is an endophytic bacterium producing cell wall degrading enzyme, glucanase, and antagonistic to fungal pathogens, such as Magnaporte grisea and Colletotrichum gloeosporioides. The glucanase is able to lyse fungal cell walls composed of glucan causing disintegrity of mycelia and fungi fail to infect plants. The purpose of this study was to clone, express, and characterize 48 kDa subunit of β-1,3-glucanase from Bcc isolate BiogenCC E76 using the Escherichia coli expression system. The 1,300 bp of the β-1,3-glucanase gene was constructed using the pET-32b vector in BamHI-HindIII restriction sites to generate the pET-Glu plasmid. The gene was fused with nucleotides sequence encoding Trx-tag, His-tag, and S-tag producing 65 kDa of recombinant β-1,3-glucanase. Gene expression in the construct was controlled by the T7 promoter and Trx-tag start codon through IPTG induction. The recombinant β-1,3-glucanase was then purified and its activities were tested at different pH and temperature conditions. Results showed that E. coli carrying pET-Glu overexpressed a 65 kDa protein in induced culture as a soluble protein that was expressed in periplasm. Purification result of the crude extract of the recombinant protein obtained 27% pure enzymes with a specific activity of 1,207.976 U/mg and purity level of 3.9 fold. This recombinant glucanase demonstrated optimal activity at 40°C and pH 5–7. A deeper study is needed to understand the role of 48 kDa subunit of β-1,3-glucanase has in antagonistic mechanism of Bcc against pathogenic fungi
Genetic Diversity of Jatropha spp. Germplasm Based on Morphological and Molecular Markers
Get PDFGermplasm of Jatropha spp. with high genetic diversity is needed to develop a new superior variety of Jatropha spp. Morphological and molecular characterizations are important to support development of their superior hybrid varieties. The aims of this study were to identify Jatropha spp. accesion potential for genetic improvement using morphological characters and analyze their genetic diversity using SSR markers. A total of eight genotypes of Jatropha spp. originated from several localities in Indonesia and Thailand was observed. Results showed that accessions of Jatropha spp. were varied in morphological and molecular characters. Based on principle component analysis, characters of stem color, leaf veins, leaf shapes, flower position, total branch number, productive branch number, petiole color, and petal color contributed most to the total diversity. Based on oil seed content, potential accessions identified for further genetic improvement were J. podagrica (34.63%) and J. curcas (29.64%). The results of molecular analysis showed that high allele variation (3–7 alleles) was observed among Jatropha spp. accessions with an average allele number of 4.12 and the average Polymorphism Information Content (PIC) value was 0.57 (0.46–0.77). Three SSR markers showed PIC value >0.5 indicating that these markers were informative for genetic diversity detection of Jatropha spp. The phylogenetic analysis showed that seven accessions of Jatropha spp. could be divided in two groups at similarity coefficent of 0.53. Results of genetic diversity analysis in this study should be useful for proper identification and selection for appropriate parents to assist in breeding of Jatropha spp. in Indonesia
Increasing the Growth and Quality of Red Chili with Growth Hormone from Endophytic Bacteria
Get PDFRed chili is a very important horticultural commodity in Indonesia having low productivity and quality. Cultivation method needs to be improved including the use of exogenous growth hormones. The purpose of this study was to determine (1) the optimum concentration of IAA and GA growing hormones from isolate B6.2 in stimulating plant growth and improving the quality of large red chili fruit; (2) molecular identity of the B6.2 bacterial isolate. The growth hormone content of B6.2 isolates using HPLC obtained 0.49 ppm IAA and 64.53 ppm GA. The growth hormone potential test on the growth and quality of chili was carried out with a concentration of 1, 3, 5 ml/l, while water and synthetic hormones was used as negative and positive control, respectively. The experimental design used was a Complete Random Design with the foliar spray application to the plant canopy three times during the growth period. The results showed the best concentration in increasing plant height, fruit weight, shooth wet, and dry weight compared to controls at the age of 76 days after planting (dap) was a concentration of 5 ml/l, with the values of 71.7±0.9 cm , 94.7±0.3 g, 11.5±0.43 g, and 1.4±0.09 g, respectively. The molecular identification showed that B6.2 isolate was classified as Bacillus vallismortis with 100% homology. The growth hormone from isolate B6.2 has the potential to increase growth and production of red chili plants
Genetic Diversity Analysis of 14 Potato Genotypes Based on Morphological Characters and SSR Markers
Get PDFPotato is one of important carbohydrate sources used as an alternative crop in Indonesia. The challenges in national potato breeding program included low productivity, less tolerance to environmental stresses, and narrow genetic diversity. The purpose of this study was to analyze genetic diversity of 14 potato genotypes based on morphological characters and SSR markers newly developed from genome sequences of Indonesian potato genotypes. Principal component analysis of morphological data was done using program XLSTAT. DNA of 14 potato genotypes were assayed using 22 SSR markers. Phylogenetic tree was constructed using program NTSYS version 2.1. The PCA showed that leaf shape, leaf color, tuber shape, tuber skin color, and tuber color contributed most to the total diversity. SSR polymorphism analysis demonstrated that as many as 196 alleles were detected in this study. The average allele number was 8.9 ranged from 5 to 13 alleles per locus. The average major allele frequencies was 22% ranged from 14 to 43%. Gene diversity ranged from 0.70 to 0.92 with the average of 0.86, meanwhile the heterozigosity observed ranged from 0 to 0.71 with the average of 0.05. Phylogenetic analysis generated two main clusters in the coefficient of similarity 0.77. The first cluster consisted of three genotypes while the second cluster consisted of eleven genotypes. The new developed SSR markers used in this study were able to differentiate potato accessions having similar morphological characters but were different genetically. The results of this study should be useful in assessing genetic materials in potato cultivar development program
Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco
Get PDFReporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct
