VALIDASI QTL DAN APLIKASINYA UNTUK PERBAIKAN SIFAT TOLERAN KERACUNAN Al PADA PADI (Oryza sativa L.)

ABSTRAK   Membuka lahan pertanian baru pada lahan marginal yang merupakan tanah asam adalah salah satu solusi dalam peningkatan produksi beras. Kendala yang dihadapi pada solusi tersebut adalah tanah asam mengandung alumunium terlarut yang dapat meracuni tanaman. Tindakan efektif agar padi tetap dapat diproduksi pada tanah asam adalah dengan penyilangan galur padi yang bersifat toleran yaitu Cabacu dengan padi bersifat peka yaitu IR64 sehingga menghasilkan populasi galur toleran Al. Penelitian ini bertujuan untuk validasi Quantitative Trait Loci (QTL) toleran Al pada galur padi hasil penyilangan Cabacu dengan IR64 pada generasi F7. Metode yang digunakan adalah eksperimen dengan menguji fenotipik menggunakan rancangan acak lengkap pola faktorial dan deskriptif dengan analisis data secara kualitatif menjelaskan hasil isolasi DNA. Data hasil pengamatan fenotipe diolah dengan program SPSS versi 16 untuk melihat sebaran data dan dilakukan uji normalitas menggunakan Uji Shapiro-Wilk. Dilakuan juga uji T untuk melihat keterpautan fenotipe dan genotipe. Hasil penelitian yaitu analisis uji fenotipe menunjukan bawa data distribusi secara normal. Sedangkan analisis marka tunggal menunjukan bahwa sifat toleran alumunium terletak pada kromosom 1 (RM5)     Kata kunci: padi, Quantitative Trait Loci (QTL), toleran alumuniu

RESPON PADI TRANSGENIK CV. NIPPONBARE GENERASI T1 YANG MENGANDUNG GEN Oryza sativa DEHYDRATION-RESPONSE ELEMENT BINDING 1A (OsDREB1A) TERHADAP CEKAMAN SALINITAS

Salinity is one of the abiotic constraints in the cultivation of rice crop. One of the reasons agricultural land becomes saline is due to the intrusion of seawater into the mainland as a result of global climate change. Dehydration-responsive element binding (DREB) gene is a plant -specific transcription factor gene that have important role in regulating plant responses to abiotic stresses, including high salinity. Transgenic rice plants cv. Nipponbare carrying OsDREB1A gene have been generated. However, study of the response of putative transgenic plants to salinity has not been done. The research objective is to study the response of T1 generation Nipponbare-OsDREB1A transgenic rice plants to salinity stress. The result showed that the response of putative transgenic rice Nipponbare-OsDREB1A to salinity stress 25 mM and 150 mM NaCl indicated a level of tolerance varies from highly sensitive to highly tolerance. These variations were possibly occurred because of the segregation state of the T1 generation transgenic rice. Based on damage symptom scoring and PCR analysis provided information that transgenic rice plant cv. Nipponbare-OsDREB1A which showed positive PCR had a very high tolerance to salinity stress 150 mM compared with non-transgenic rice cv. Nipponbare

Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method

Cold 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

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

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

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

Overexpression 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

Construction and Transformation of OsERA1 Gene into Expression Vector and Response of Nipponbare-OsERA1 Transgenic Rice to Drought Stress

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.

Multiplying the efficiency and impact of biofortification through metabolic engineering

Ending all forms of hunger by 2030, as set forward in the UN-Sustainable Development Goal 2 (UN-SDG2), is a daunting but essential task, given the limited timeline ahead and the negative global health and socio-economic impact of hunger. Malnutrition or hidden hunger due to micronutrient deficiencies affects about one third of the world population and severely jeopardizes economic development. Staple crop biofortification through gene stacking, using a rational combination of conventional breeding and metabolic engineering strategies, should enable a leap forward within the coming decade. A number of specific actions and policy interventions are proposed to reach this goal

Identification of Quantitative Trait Loci (QTL) for Awn, Incomplete Panicle Exertion and Total Spikelet Number in an F2 Population Derived from A Backcross Inbred Line, Bio-148, and the Recurrent Parent, IR64

An F2 rice population developed from a cross between a backcross inbred line (BIO-148) and its recurrent parent (IR64) was used to identify quantitative trait loci (QTL) for awn, panicle exertion and total spikelet number. BIO-148 is a BC2F8 line derived from a cross between IR64 (a high-yielding lowland rice variety) and Gajah Mungkur (an upland tropical japonica rice variety). Two hundred plants were grown in the greenhouse, and their DNAs were isolated for genotyping using SSR markers. Panicle exertion was observed during the grain-filling stage. The awn length of the seed and the total spikelet number per panicle were observed after harvesting. A total of four QTLs were identified using single-marker regression with LOD>3, explaining 8.4-18.1% of phenotypic variation. A QTL for awn was identified on Chromosome 8. A QTL for incomplete panicle exertion was identified on Chromosome 4. Two QTLs for total spikelet number were identified on Chromosome 4, in which the BIO-148 allele contributed to a higher number of spikelets per panicle. The QTLs identified in this study will be useful in the improvement of yield potential for modern lowland indica rice varieties by harnessing the hidden useful alleles from upland tropical japonica rice varieties

Identification of Quantitative Trait Loci (QTL) for Awn, Incomplete Panicle Exertion and Total Spikelet Number in an F2 Population Derived from A Backcross Inbred Line, Bio-148, and the Recurrent Parent, IR64

An F2 rice population developed from a cross between a backcross inbred line (BIO-148) and its recurrent parent (IR64) was used to identify quantitative trait loci (QTL) for awn, panicle exertion and total spikelet number. BIO-148 is a BC2F8 line derived from a cross between IR64 (a high-yielding lowland rice variety) and Gajah Mungkur (an upland tropical japonica rice variety). Two hundred plants were grown in the greenhouse, and their DNAs were isolated for genotyping using SSR markers. Panicle exertion was observed during the grain-filling stage. The awn length of the seed and the total spikelet number per panicle were observed after harvesting. A total of four QTLs were identified using single-marker regression with LOD>3, explaining 8.4-18.1% of phenotypic variation. A QTL for awn was identified on Chromosome 8. A QTL for incomplete panicle exertion was identified on Chromosome 4. Two QTLs for total spikelet number were identified on Chromosome 4, in which the BIO-148 allele contributed to a higher number of spikelets per panicle. The QTLs identified in this study will be useful in the improvement of yield potential for modern lowland indica rice varieties by harnessing the hidden useful alleles from upland tropical japonica rice varieties

Identification of A Major Quantitative Trait Locus for Grain Weight In Rice Using Microsatellite Marker

Rice is one of the major staple foods in the world, especially in Asia. Improving yield potential of superior cultivars is important to meeting the demand for rice production, which is increasing due to human population increase, climate change, and degradation of agricultural resources, such as land and water. In this study, a BC3F2 population developed from an intraspecific cross between Ciherang and a new plant type line (B11143D) was used in a quantitative trait locus (QTL) analysis. Ciherang is a high yielding rice cultivar with good grain quality which has been planted in 37% of the irrigated rice area in Indonesia. The objective of this study was to identify QTL(s) for yield components on chromosome 12, which can be used to improve the elite cultivar Ciherang or other popular cultivars through marker-assisted breeding. A total of two hundred BC3F2 lines were evaluated in the greenhouse during this study. The population was observed for eight agronomic traits including days to heading (dth), plant height (ph), flag leaf length (fll), panicles per plant (ppl), panicle length (pl), grains per panicle (gpp), 1000-grain weight (gw), and yield (yld). Four simple sequence repeats (SSR) markers (RM3472, RM28048, RM28195, and RM1986) were used for targeted mapping on chromosome 12. Linkage analysis identified a QTL for 1000-grain weight located on chromosome 12 at position 53.5 cM–73 cM