Journal Jurnal AgroBiogen-
Jurnal AgroBiogen, Volume 14, pp 75-84; doi:10.21082/jbio.v14n2.2018.p75-84
Abstract:Brown planthopper (BPH) is a major rice pest in Indonesia. The most economical and effective approach to control the insect pest is by using resistant varieties. Exploring for resistance genes is, therefore, a prerequisite for effective breeding program for BPH resistance. This study aimed to map BPH resistance genes in Untup Rajab, an Indonesian local rice variety. Genetic map was constructed using an F2 population from a cross between TN-1 and Untup Rajab, and SNP markers from RiceLD SNP Chip. Phenotyping was performed using bulk seedling test on F2:3 seedlings against two BPH populations, i.e. X1 and S1. Four QTLswere identified on chromosomes 5, 6, 8, and 11 with PVE values of 7.63%, 9.40%, 17.66%, and 3.05%, respectively. Relatively normal distribution of resistance phenotype and the relatively low PVE values indicate that Untup Rajab has a quantitative resistance to BPH with two different resistance loci identified for each BPH test population. The QTL on chromosome 8 overlaps with OsHI-LOX gene, which is associated with resistance to BPH, and adjacent to another QTL for resistance to green leafhopper. The QTL on chromosome 6 was found near OsPLDα4 and OsPLDα5 genes which are related to BPH resistance. Meanwhile, the QTL intervals on chromosome 5 and 11 did not overlap with any known BPH QTLs or genes, which make them attractive candidates for novel BPH resistance gene discovery.
Jurnal AgroBiogen, Volume 14, pp 65-74; doi:10.21082/jbio.v14n2.2018.p65-74
Abstract:Shallot (Allium cepa var. aggregatum) is one of the most important vegetable crops grown in Indonesia. The limited knowledge available on the genetic diversity and the threat of plant disease have been major problems to maintain high shallot production in Indonesia. Development of molecular markers linked to disease resistance is required for molecular breeding activity in this crop. This study aimed to assess the genetic diversity at conserved domain of resistance gene analog (RGA) in a set of 36 Indonesian shallot genotypes to complement morphological characterization. Twelve morphological and fifteen molecular markers traits were investigated in an attempt to characterize and to discriminate the Indonesian shallots genotypes. Characterization at orphological level indicated that phenotypic variance was highest for total bulb weight (TWB, cv = 99.39%) and the least for the plant height (PH, cv = 28.16%). The correlation analysis between traits showed that TWB and number of bulb (NB), TWB and bulb weight per plant (WB), NB and WB, and WB and PH were positively correlated. Molecular analysis revealed a total of 1,512 alleles with an average of 1.946 alleles per locus. The Polymorphism Information Content (PIC) values ranged from 0.253 to 0.676 and six out of 15 RGA markers were highly informative with PIC values ≥0.50. Based on cluster analysis, the 36 Indonesian shallot genotypes were clearly discriminated into six major groups. These results revealed that the RGA-based markers could support the morphological characterization in evaluating the genetic diversity of shallots.
Jurnal AgroBiogen, Volume 14, pp 55-64; doi:10.21082/jbio.v14n2.2018.p55-64
Abstract:Reporter 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.
Jurnal AgroBiogen, Volume 14, pp 37-46; doi:10.21082/jbio.v14n1.2018.p37-46
Abstract:Bioremediation is an inexpensive, easy, and safe technology to rehabilitate agricultural land which is highly polluted with pesticides. The aims of this study were to isolate and characterize profenofos degrading bacteria isolated from Pangalengan soils. The isolation step was carried out by using spread plate method on Nitrate Mineral Salts (NMS) medium containing 100 ppm profenofos. The isolates were selected based on hypersensitive response (HR) and hemolytic test, and ability of the isolates to use and degrade profenofos. The selected isolates were characterized based on the sequence of 16 rRNA and detection of the α and β subunits of terminal deoxygenase and naphtalene dioxygenase encoded genes. Three isolates (CN26, CN44, and CN86), which could use profenofos as the exclusive C source, could degrade more than 86.75% profenofos containing growth medium. Based on the 16S rRNA sequences, the three isolates were closely related to Stenotrophomonas maltophilia (99%), Comamonas terrigena (99%), and Pseudomonas sp. (80%). Pseudomonas CN44 consistenly showed high profenofos degradation activity of up to 91.2% when grown on NMS medium (pH 6.8) for 72 hours. β subunit dioxygenase encoding gene of the isolates were detected using primers Rf2-F/Rf2-R, but optimation of PCR is still needed to detect the α subunit of the gene. Naphtalene dioxygenase gene was detected only from Pseudomonas CN44 using the primer pair 301f/1099r. Based on its biodegradation capability and molecular characteristics, Pseudomonas CN44 is very potential to be developed as a bioremediating agent of profenofos.
Jurnal AgroBiogen, Volume 14, pp 1-10; doi:10.21082/jbio.v14n1.2018.p1-10
Abstract:Indonesia is rich in rice genetic resources, however, only a small number has been used in variety improvement programs. This study aimed to determine the genetic diversity of Indonesian rice varieties using 6K SNP markers. The study was conducted at ICABIOGRAD for DNA isolation and IRRI for SNP marker analysis. Genetic materials were 53 rice genotypes consisting of 49 varieties and 4 check genotypes. SNP markers used were 6K loci. Results showed that among the markers analyzed, only 4,606 SNPs (76.77%) were successfully read. The SNP markers covered all twelve rice chromosomes of 945,178.27 bp. The most common allele observed was GG, whereas the least allele was TG. Dendrograms of the 53 rice varieties analyzed with 4,606 SNPs demonstrated several small groups containing genotypic mixtures between indica and japonica rice, and no groups were found to contain firmly indica or japonica type. Structure analysis (K = 2) with value of 0.8 showed that the 53 rice varieties were divided into several groups and each group consisted of 4 japonica, 2 tropical japonica, 46 indica, and 1 aus rice type, respectively. IR64 and Ciherang proved to have an indica genome, while Rojolele has japonica one. Dupa and Hawara Bunar, usually grouped into tropical japonica rice, were classified as indica type, and Hawara Bunar has perfectly 100% indica type. The results of this study indicated that rice classification (indica-japonica) which is usually classified based only on morphological characters, e.g. grain and leaf shapes, is not enough and classification based on SNP markers should be considered for that purpose.
Jurnal AgroBiogen, Volume 14, pp 23-36; doi:10.21082/jbio.v14n1.2018.p23-36
Abstract:Drought stress is a major constrain which could influence rice productivity. Enhanced Response to ABA1 (ERA1) gene encoding a β-subunit farnesyltransferase enzyme plays a role to control sensitivity of the guard cells to abscisic acid (ABA), hence regulating drought stress response in plant species including rice. This study aimed to clone the OsERA1 gene into expression vector, introduce it into rice plant, and confirm the positive OsERA1-rice plants conferring drought tolerance. This study was initiated by isolation of the OsERA1 gene from rice cDNAs and cloned it to an expression vector cassette, pCAMBIA1301. The cassette harboring OsERA1 gene was introduced into rice plant cv. Nipponbare mediated by Agrobacterium tumefaciens strain LBA4404.Putative transgenic lines were detected using PCR and Southern blot analyses to confirm the inserted transgene and the positive lines were assayed their tolerance to drought. The OsERA1 gene was successfully isolated and constructed into expression vector to generate pCAMBIA1301-OsERA1. Introduction of the gene into Nipponbare has produced nine putative transgenic rice lines, of which, six lines harbored OsERA1 gene. Southern blot analysis of sixteen T2 plants from two PCR-positive transgenic lines revealed1–3 copies of transgene were integrated into rice genome of transgenic lines. Five transgenic lines of Nipponbare-OsERA1 showed better response to drought at vegetative phase compared to control in term of recovery ability. At generative phase, the five transgenic lines yielded less unfilled grains compared to control. Overall, the transgenic lines obtained from this study could bepotential candidates for developing rice varieties tolerant to drought.
Jurnal AgroBiogen, Volume 14, pp 11-22; doi:10.21082/jbio.v14n1.2018.p11-22
Abstract:Genetic diversity analysis using molecular markers is an important step for selecting appropriate parents in a soybean breeding program. The aims of this study were to (1) analyze genetic diversity of 29 soybean genotypes assessed with 27 SSR markers for selecting appropriate parents and (2) develop F2 populations to be used for breeding long juvenile (LJ) trait in soybean tobe cultivated in short photoperiod condition. The soybean genotypes used consisted of 11 Indonesian soybean genotypes and 18 genotypes introduced from the USA. F2 populations were developed by crossing Grobogan with three introduced genotypes carrying LJ character. The PIC values of the 27 SSR markers ranged from 0.87 to 0.96. Cluster analysis resulted in three mainclusters at coefficient similarity of 0.76. The five LJ introduced accessions and the nine Indonesian genotypes showed high genetic distances and are useful as parent pairs for developing breeding populations. The F1 progeny phenotypicperformances of the cross far exceeded the performaces of both parents. Three F2 populations were developed by crossing the distantly related soybean genotypes. The F2 populations were verified by using SSR markers and it was found that they segregated in a 1:2:1 ratio confirming the segregation ratio of codominant SSR markers. The F2 populations should be useful for breeding LJ characters to improve soybean productivity in low latitude tropical countries such as Indonesia, which has day length of approximately 12 h all year round.
Jurnal AgroBiogen, Volume 14, pp 47-54; doi:10.21082/jbio.v14n1.2018.p47-54
Abstract:Aspergillus spp. (A. niger, A. flavus, and A. fumigatus) contaminate food commodities through production of secondary metabolites (mycotoxins) and aspergillosis, and thus pose severe hazard to human and animal health. Hence, the inhibition of mycotoxin-producing fungi on agricultural storage commodities needs to be considered. The aims of this study were to evaluate endophytic bacteria isolated from rice tissues that inhibit Aspergillus spp. growth, as well as to characterize the selected isolates morphologically and biochemically. Dual culture and disk diffusion method tests on 155 endophytic bacteria obtained three isolates, i.e. FB-Endo 65, FB-Endo 73, and FB-Endo 95, which showed inhibition zone from 13 to 17 mm against Aspergillus spp. growth. The inhibition zone and quantity of antifungal compounds increased positively with the length of incubation periods from 0 to 6 days. Antifungal compounds from the three isolates were insoluble in ethyl acetate, but soluble in methanol. The methanol soluble substance(s) from FB-Endo 73 showed higher inhibition zone than that of the other isolates. This result indicated that all three isolates produced strong antifungal activity. Morphological and biochemical identifications of the isolates revealed that all isolates belonged to the genus Bacillus sp. Further studies include identification and production methods of antifungal compounds of those endophytic bacteria and their application on stored seeds.
Jurnal AgroBiogen, Volume 13, pp 35-42; doi:10.21082/jbio.v13n1.2017.p35-42
Abstract:Regenerasi tanaman merupakan tahapan penting yang perlu dikuasai sebelum diaplikasikan untuk perakitan varietas unggul secara kultur in vitro. Penelitian bertujuan mengoptimasi teknik regenerasi in vitro beberapa varietas padi indica melalui jalur organogenesis. Percobaan menggunakan Rancangan Acak Lengkap yang disusun secara faktorial. Materi yang dicobakan, yaitu Ciherang, Inpari 13, Inpara 3, Pokkali, dan IR29. Perlakuan yang diberikan untuk induksi kalus organogenik adalah 2,4-D (0, 1, 3, 5, 7 mg/l) + kasein hidrolisat 3 g/l, untuk regenerasi kalus membentuk tunas adventif adalah BA (0, 1, 5 mg/l) + zeatin (0, 0,1, 0,3 mg/l) + prolin 100 mg/l, untuk multiplikasi tunas adalah MS + thidiazuron/TDZ (0, 0,1, 0,3 mg/l) dan untuk perakar-an adalah IBA (0, 1, 2, 3 mg/l). Hasil penelitian menunjukkan media terbaik untuk induksi kalus untuk padi varietas Ciherang, Inpari 13, dan Pokkali adalah MS + 2,4-D 3 mg/l, sedangkan untuk varietas Inpari 3 dan IR 29 adalah MS + 2,4-D 5 mg/l. Media terbaik untuk regenerasi tunas untuk varietas Ciherang adalah BA 5 mg/l + zeatin 0,1 mg/l + prolin 100 mg/l, untuk Inpari 13 adalah MS + BA 3 mg/l + zeatin 0,3 mg/l + prolin 100 mg/l, untuk Inpara 3 adalah BA 5 mg/l + zeatin 0,3 mg/l + prolin 100 mg/l dan untuk Pokkali dan IR29 adalah MS + BA 3 mg/l + zeatin 0,1 mg/l. Media terbaik untuk multiplikasi tunas adalah MS + TDZ 0,3 mg/l dan untuk induksi perakaran adalah MS + IBA 2 mg/l. Setiap tahapan kegiatan pembentukan planlet membutuhkan formulasi media yang berbeda.
Jurnal AgroBiogen, Volume 13, pp 1-16; doi:10.21082/jbio.v13n1.2017.p1-16
Abstract:Sebagai salah satu komoditas tanaman pangan penting di Indonesia setelah padi dan jagung, kedelai memerlukan upaya peningkatan keragaman genetik dengan cara introduksi aksesi dari negara lain terutama Cina sebagai salah satu negara asal kedelai di dunia. Marka simple sequence repeat (SSR) dapat digunakan untuk analisis keragaman genetik antaraksesi kedelai introduksi. Tujuan penelitian ini adalah mempelajari keragaman genotipik dan fenotipik 48 aksesi kedelai introduksi asal Cina menggunakan 15 marka SSR. Analisis DNA dilakukan menggunakan PCR dan data hasil PCR menggunakan marka SSR dianalisis menggunakan perangkat lunak XLSTAT, NTSYS, dan PowerMarker. Data karakter morfologis diperoleh dari basis data Germplasm Resources Information Network (GRIN), United States Department of Agriculture (USDA) (www.ars-grin.gov). Data ini digunakan sebagai data keragaman fenotipik yang diperlukan untuk menunjang hasil karakterisasi molekuler. Hasil penelitian menunjukkan bahwa terdapat keragaman karakter morfologis dan molekuler antaraksesi kedelai yang dipelajari. Berdasarkan hasil analisis komponen utama, karakter tinggi tanaman, bobot 100 biji, hasil biji, warna pusar biji, warna trikoma, warna bunga, dan warna polong berkontribusi besar terhadap keragaman total. Analisis molekuler menggunakan marka SSR menunjukkan bahwa terdapat variasi alel yang cukup tinggi (9–25 alel) di antara aksesi kedelai dengan rerata jumlah alel 15,6, sedangkan rerata nilai Polymorphism Information Content (PIC) sebesar 0,89 (0,84–0,94). Seluruh marka SSR memiliki nilai PIC>0,5 yang menunjukkan bahwa marka tersebut informatif untuk studi keragaman genetik kedelai dengan rerata nilai diversitas gen sebesar 0,90. Hasil analisis filogenetik dan analisis koordinat utama menunjukkan bahwa 48 aksesi tersebut mengelompok menjadi tiga dengan koefisien kemiripan 0,84. Pada penelitian ini dilakukan pula uji asosiasi antara marka SSR dan karakter morfologis. Asosiasi yang signifikan ditemukan pada tujuh lokus marka SSR. Persentase keragaman total yang dapat dijelaskan oleh marka SSR tersebut, yaitu 17,25–78,45%. Marka GMES2225 dan Sat_286 berasosiasi dengan warna kulit biji, sedangkan marka GmF35H berasosiasi dengan tinggi tanaman. Informasi keragaman genetik akan sangat bermanfaat sebagai langkah awal untuk kegiatan seleksi tetua persilangan dengan sifat yang diinginkan dalam membantu program pemuliaan kedelai di Indonesia.