Transcriptomics View over the Germination Landscape in Biofortified Rice

Hidden hunger, or micronutrient deficiency, is a worldwide problem. Several approaches are employed to alleviate its effects (e.g., promoting diet diversity, use of dietary supplements, chemical fortification of processed food), and among these, biofortification is considered as one of the most cost-effective and highly sustainable. Rice is one of the best targets for biofortification since it is a staple food for almost half of the world’s population as a high-energy source but with low nutritional value. Multiple biofortified rice lines have been produced during the past decades, while few studies also reported modifications in germination behavior (in terms of enhanced or decreased germination percentage or speed). It is important to underline that rapid, uniform germination, and seedling establishment are essential prerequisites for crop productivity. Combining the two traits, biofortified, highly-nutritious seeds with improved germination behavior can be envisaged as a highly-desired target for rice breeding. To this purpose, information gathered from transcriptomics studies can reveal useful insights to unveil the molecular players governing both traits. The present review aims to provide an overview of transcriptomics studies applied at the crossroad between biofortification and seed germination, pointing out potential candidates for trait pyramiding

Introduction of cryIB-cryIAa Hybrid Gene Into Rice (Oryza sativa) Genom cv. Rojolele using Agrobacterium-Mediated Transformation

Rojolele is one of Indonesian local variety from Javanica group that susceptible to yellow stem borer (Scirpophaga incertulas). Previous study showed that Rojolele can be cultured and regenerated in vitro. Two cry genes, cryIB-cryIAa were fused and introduced into rice cv. Rojolele in an attempt to improve resistance and to obtain durable resistance rice against the yellow stem borer. Two-week old embryogenic calli of Rojolele rice were inoculated with Agrobacterium tumefaciens harbored with binary vector pCAMBIA 1301, 1303, or 1304 carrying cryIB-cryIAa hybrid gene, hygromycin resistant gene (hpt), and -glucuronidase (gus) gene interrupted with an intron. The transformed calli were selected gradually on medium containing hygromycin (50, 100 mg/l) and regenerated on medium containing 0.5 mg/l IAA and 0.3 mg/l BAP. GUS activity in infected calli was detected by histochemical assay 3 days after inoculation. The highest (100%) transformation efficiency were obtained from calli transformed with pCAMBIA 1303 and 1304. Thirty four out of 77 transformed shoots were tested positive for the cryIB-cryIAa gene using PCR analysis. These shoots were grown in the soil to maturity and to collect the seeds. PCR analysis of the T1 progeny revealed that two out of six lines tested showed a Mendelian segregation pattern. These two lines were also potentially resistant to yellow stem borer based on bioassay in planta. Key words: cryIB-cryIAa hybrid gene, yellow stem bore

Konstruksi Vektor Biner Mengandung Gen Hibrid cryIB-cryIAa untuk Transformasi Agrobacterium Tanaman Padi (Oryza sativa L)

This study aimed to obtain binary vectors carrying  cryIB-cryIAa hybrid gene suitable for Agrobacterium-mediated transformation of two Indonesian rice cultivars, Rajalele (Javanica) and Cisadane (Indica).  Introduction of hybrid Bt toxin, encoded by cryIB-cryIAa hybrid gene with different binding receptor in the insect gut driven by maize ubiquitin promoter, was intended to reduce the possibility of the appearance of stem borer resistant to Bt toxin. A HindIII fragment containing the ubi cryIB-cryIAa hybrid gene was excised from pBKS ubi cryIB-cryIAa and cloned into the HindIII site of binary vector pCAMBIA 1301, 1303, or 1304.  Restriction analysis of recombinant plasmids confirmed the insertion of the gene and demonstrated that the gene inserted in two orientations. The recombinant plasmids were transformed into electrocompetent Agrobacterium tumefaciens EHA105 by electroporation.  Embryogenic calli of Rajalele and Cisadane were co-cultivated with A. tumefaciens EHA 105 harbouring binary vector pCAMBIA 1301, 1303, or 1304 containing ubi cryIB-cryIAa. GUS histochemical assay revealed that high transformation efficiency (75-100%) on both cultivars were obtained from embryogenic calli transformed with pCAMBIA 1303 or 1304

THE SEGREGATION PATTERN OF INSECT RESISTANCE GENES IN THE PROGENIES AND CROSSES OF TRANSGENIC ROJOLELE RICE

Successful application of genetic transformation technique, especially in developing rice variety resistant to brown plant hopper and stem borer, will depend on transgene being expressed and the gene inherited in a stable and predictable manner. This study aimed to analyse transgene segregation pattern of the progenies and the crosses of transgenic rice cv. Rojolele harboring cry1Ab and gna genes. The third generation (T2) of fivetransgenic Rojolele events containing gna and/or cry1Ab were evaluated for two generations to identify the homozygous lines and to study their inheritance. The homozygous lines were selected based on the result of PCR technique. The segregation patterns of gna and cry1Ab were studied in eight F2 populations derived from Rojolele x transgenic Rojolele homozygous for cry1Ab and or gna and their reciprocal crosses. Data  resulted from PCR of F2 population were analysed using a Chi Square test. The study obtained six homozygous lines for gna, namely A22- 1-32, A22-1-37, C72-1-9, F11-1-48, K21-1-39, K21-1-48, and two homozygous lines for cry1Ab, namely K21-1-39 and K21- 1-48. Both cry1Ab and gna transgenes had been inherited through selfing and crossing with their wild type as indicated from the F1 containing gna and cry1Ab as many as 48.4% and 47.4%, respectively. In six of the eight crosses, gna was inherited in a 3:1 ratio consistent with Mendelian inheritance of a single dominant locus, while in the remaining two crosses, gna was segregated in a 1:1 ratio. The presence of cry1Ab in F2 populations also showed a 3:1 segregation ratio in all crosses. In the F2 population derived from F1 plant containing cry1Ab and gna, both transgenes segregated in a 9:3:3:1 dihybrid segregation ratio. This study will add to the diversity of genetic sources for insect resistance and allow further use of these transgenic lines for pyramiding resistance to brown plant hopper and stem borer or  separately in rice breeding programs whenever the efficacy tests and biosafety requirements have been completed

Comparative Analysis of Rice Transformation Using Agrobacterium tumefaciens and Rhyzobium leguminosarum

This study was aimed to study the effectiveness of Rhizobium transformation system compared to the most widely used Agrobacterium mediated transformation system on three rice cultivars, Ciherang (Indica), Nipponbare (Japonica), and Rojolele (Javanica). Six day old calli induced from immature embryos were inoculated with Rhizobium leguminosarum bv trifolii ANU845 and Agrobacterium tumefaciens LBA288 that harbored with vector pCAMBIA 5106. This plasmid contained a minimum set of transfer machinery genes and had a gusplus and an hptII gene driven by 35S CaMV promoter in the T-DNA. The results showed that the transformation frequencies (number of PCR positive plants per number of calli inoculated) ranging from 0 to 12.05 % depend on the genotype and transfer agent used. The highest transformation frequency (12.05%) was obtained in Ciherang transformed with R. leguminosarum. Most of the transgenic rice obtainedby Rhizobium transformation were normal in morphology and fertile similar to those obtained by Agrobacterium transformation. Integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis in T0 and T1 generations.Key words : Rhizobium leguminosarum, immature embryos, Agrobacterium tumefacien

Comparative Analysis of Rice Transformation Using Agrobacterium tumefaciens and Rhyzobium leguminosarum

This study was aimed to study the effectiveness of Rhizobium transformation system compared to the most widely used Agrobacterium mediated transformation system on three rice cultivars, Ciherang (Indica), Nipponbare (Japonica), and Rojolele (Javanica). Six day old calli induced from immature embryos were inoculated with Rhizobium leguminosarum bv trifolii ANU845 and Agrobacterium tumefaciens LBA288 that harbored with vector pCAMBIA 5106. This plasmid contained a minimum set of transfer machinery genes and had a gusplus and an hptII gene driven by 35S CaMV promoter in the T-DNA. The results showed that the transformation frequencies (number of PCR positive plants per number of calli inoculated) ranging from 0 to 12.05 % depend on the genotype and transfer agent used. The highest transformation frequency (12.05%) was obtained in Ciherang transformed with R. leguminosarum. Most of the transgenic rice obtainedby Rhizobium transformation were normal in morphology and fertile similar to those obtained by Agrobacterium transformation. Integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis in T0 and T1 generations.Key words : Rhizobium leguminosarum, immature embryos, Agrobacterium tumefacien

Keefektivan Padi Transgenik terhadap Hama Penggerek Batang Padi Kuning Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae).

Transformation two cry genes (cryIB-cryIAa) and transformation with a single the cry1B gene under the control of a wound-inducible maize proteinase inhibitor gene (mpi) promoter were two approaches that were used to get resistant rice to the rice stemborer which may be had a durable resistance. To obtain information on the effectiveness of seven transgenic rice lines to the rice yellow stemborer S. incertulas, a test was conducted in greenhouse. The seven lines were 1). line 4.2.3 and 2) line 4.2.4 both contain fusion of two cry genes (cryIB-cryIAa); 3) line 3R9 and 4) line 3R7 lines both contain of mpi-cryIB gene; and 5) line 6.11 contains of cryIAb gene by particle bombardment, 6) line DT-cry (Azygous) that do not contain cry gene (null), and 7) DT-cry line contains cryIAb gene by Agrobacterium, and as a negative control, we used three non transgenic rice varieties i.e., Rojolele, Cilosari, and Ciherang. The result showed that transgenic rice lines, except DT-cry and DT-cry (Azygous) lines were effective to suppress damage by the insect, and showed an inhibition effect on the growth of S. incertulas, and had a high level ofresistance than non transgenic rice varieties had. There were differences on resistance value/level among transgenic rice lines. Based on the resistance value, 6.11 line was the highest (scale 0) followed by 4.2.4 line and 3R7 line, these lines were categorized as high resistance (scale 1). Transgenic rice-4.2.3 line and 3R9 line were categorized as moderat resistance (scale 3). DT-cry and DT-cry (Azygous) lines were susceptible (scale 7-9). KEY WORDS: Effectiveness, resistance value, transgenic rice, S. incertulas

Keefektivan Padi Transgenik terhadap Hama Penggerek Batang Padi Kuning Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae).

Transformation two cry genes (cryIB-cryIAa) and transformation with a single the cry1B gene under the control of a wound-inducible maize proteinase inhibitor gene (mpi) promoter were two approaches that were used to get resistant rice to the rice stemborer which may be had a durable resistance. To obtain information on the effectiveness of seven transgenic rice lines to the rice yellow stemborer S. incertulas, a test was conducted in greenhouse. The seven lines were 1). line 4.2.3 and 2) line 4.2.4 both contain fusion of two cry genes (cryIB-cryIAa); 3) line 3R9 and 4) line 3R7 lines both contain of mpi-cryIB gene; and 5) line 6.11 contains of cryIAb gene by particle bombardment, 6) line DT-cry (Azygous) that do not contain cry gene (null), and 7) DT-cry line contains cryIAb gene by Agrobacterium, and as a negative control, we used three non transgenic rice varieties i.e., Rojolele, Cilosari, and Ciherang. The result showed that transgenic rice lines, except DT-cry and DT-cry (Azygous) lines were effective to suppress damage by the insect, and showed an inhibition effect on the growth of S. incertulas, and had a high level ofresistance than non transgenic rice varieties had. There were differences on resistance value/level among transgenic rice lines. Based on the resistance value, 6.11 line was the highest (scale 0) followed by 4.2.4 line and 3R7 line, these lines were categorized as high resistance (scale 1). Transgenic rice-4.2.3 line and 3R9 line were categorized as moderat resistance (scale 3). DT-cry and DT-cry (Azygous) lines were susceptible (scale 7-9). KEY WORDS: Effectiveness, resistance value, transgenic rice, S. incertulas