Development Of Transgenic Rice Plants Research Articles

Development of Bt rice potential for yellow stem borer control

Rice (Oryza sativa L.) production is always threatened by biotic and abiotic stresses. Both stresses can reduce rice productivity and quality. Yellow stem borer (YSB; Scirpophaga incertulas Walker) is one of the biotic stresses and is reported as the most destructive pest of tropical rice insects. The application of pesticides is less effective since the insect larvae live and feed inside the stem, thus inhibiting pesticides to reach the larvae. Planting YSB-resistant cultivars is a good strategy besides environmentally friendly. However, no sufficient level of resistance to YSB has been identified among rice germplasm collection, making the use of conventional breeding methods is difficult. Bacillus thuringiensis (Bt) is a Gram-positive bacterium produces insecticidal proteins, Cry toxins that are toxic to YSB. The introduction of cry genes from Bt into rice plants by using genetic engineering has been widely and successfully carried the out. A number of strategies have been conducted to increase the expression level and to prolong the effectiveness of Bt toxins in transgenic rice, including plant codon usage-optimized genes, the use of strong promoters and wound-inducible promoters and gene stacking. Insect bioassays under laboratory, greenhouse and field conditions showed that transgenic rice plants harboring and expressing cry genes are highly resistant to YSB compared to the original cultivars from which transgenic plants were developed. Thus, the development of transgenic rice plants harboring and expressing cry genes from Bt is a good strategy to build plant resistance against YSB.

Simultaneous resistance against the two viruses causing rice tungro disease using RNA interference

Rice tungro is the most important viral disease affecting rice in South and Southeast Asia, caused by two viruses rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Transgenic resistance using RNA-interference (RNAi) has been reported individually against RTBV and RTSV earlier. Here we report the development of transgenic rice plants expressing RNAi against both RTBV and RTSV simultaneously. A DNA construct carrying 300 bp of RTBV DNA and 300 bp of RTSV cDNA were cloned as the two arms in hairpin orientation in a binary plasmid background to generate RNAi against both viruses simultaneously. Transgenic rice plants were raised using the above construct and their resistance against RTBV and RTSV was quantified at the T1 plants. Levels of both the viral nucleic acids showed a fall of 100- to 500-fold in the above plants, compared with the non-transgenic controls, coupled with the amelioration of stunting. The transgenic plants also retained higher chlorophyll levels than the control non-transgenic plants after infection with RTBV and RTSV. Small RNA analysis of virus inoculated transgenic plants indicated the presence of 21 nt and 22 nt siRNAs specific to RTBV and RTSV. The evidence points towards an active RNAi mechanism leading to resistance against the tungro viruses in the plants analysed.

Synthetic control of flowering in rice independent of the cultivation environment.

For genetically homogeneous crops, the timing of flowering is determined largely by the cultivation environment and is strongly associated with the yield and quality of the harvest1. Flowering time and other agronomical traits are often tightly correlated, which can lead to difficulty excluding the effects of flowering time when evaluating the characteristics of different genetic varieties2. Here, we describe the development of transgenic rice plants whose flowering time can be controlled by specific agrochemicals. We first developed non-flowering rice plants by overexpressing a floral repressor gene, Grain number, plant height and heading date 7 (Ghd7)3,4, to inhibit any environmentally induced spontaneous flowering. We then co-transformed plants with a rice florigen gene, Heading date 3a (Hd3a)5, which is induced by the application of specific agrochemicals. This permitted the flowering time to be experimentally controlled regardless of the cultivation environment: some transgenic plants flowered only after agrochemical treatment. Furthermore, plant size and yield-related traits could, in some cases, be increased owing to both a longer duration of vegetative growth and an increased panicle size. This ability to control flowering time experimentally, independently of environmental variables, may lead to production of crops suitable for growth in different climates and facilitate breeding for various agronomical traits.

MYB-mediated upregulation of lignin biosynthesis in Oryza sativa towards biomass refinery.

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five Arabidopsis thaliana (Arabidopsis) and one Oryza sativa (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice (O. sativa L. ssp. japonica cv. Nipponbare). Among them, three Arabidopsis MYBs (AtMYB55, AtMYB61, and AtMYB63) in transgenic rice T1 lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in AtMYB61-overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of AtMYB61 in rice increased lignin content mainly by enriching syringyl units as well as p-coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as Sorghum, switchgrass, Miscanthus and Erianthus.

Rice endosperm produces an underglycosylated and potent form of the HIV-neutralizing monoclonal antibody 2G12.

Protein microbicides against HIV can help to prevent infection but they are required in large, repetitive doses. This makes current fermenter-based production systems prohibitively expensive. Plants are advantageous as production platforms because they offer a safe, economical and scalable alternative, and cereals such as rice are particularly attractive because they could allow pharmaceutical proteins to be produced economically and on a large scale in developing countries. Pharmaceutical proteins can also be stored as unprocessed seed, circumventing the need for a cold chain. Here, we report the development of transgenic rice plants expressing the HIV-neutralizing antibody 2G12 in the endosperm. Surprisingly for an antibody expressed in plants, the heavy chain was predominantly aglycosylated. Nevertheless, the heavy and light chains assembled into functional antibodies with more potent HIV-neutralizing activity than other plant-derived forms of 2G12 bearing typical high-mannose or plant complex-type glycans. Immunolocalization experiments showed that the assembled antibody accumulated predominantly in protein storage vacuoles but also induced the formation of novel, spherical storage compartments surrounded by ribosomes indicating that they originated from the endoplasmic reticulum. The comparison of wild-type and transgenic plants at the transcriptomic and proteomic levels indicated that endogenous genes related to starch biosynthesis were down-regulated in the endosperm of the transgenic plants, whereas genes encoding prolamin and glutaredoxin-C8 were up-regulated. Our data provide insight into factors that affect the functional efficacy of neutralizing antibodies in plants and the impact of recombinant proteins on endogenous gene expression.

Infection of Early and Young Callus Tissues of Indica Rice BPT 5204 Enhances Regeneration and Transformation Efficiency

A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture (LBA4404: pSB1, pCAMBIA1301) and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6- and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants (T0) revealed the presence of 1–2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants.

Development of Low Phytate Rice by RNAi Mediated Seed-Specific Silencing of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase Gene (IPK1)

Phytic acid (InsP6) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP6 is not utilized efficiently by monogastric animals due to lack of phytase enzyme. Furthermore, due to its ability to chelate mineral cations, phytic acid is considered to be an antinutrient that renders these minerals unavailable for absorption. In view of these facts, reducing the phytic acid content in cereal grains is a desired goal for the genetic improvement of several crops. In the present study, we report the RNAi-mediated seed-specific silencing (using the Oleosin18 promoter) of the IPK1 gene, which catalyzes the last step of phytic acid biosynthesis in rice. The presence of the transgene cassette in the resulting transgenic plants was confirmed by molecular analysis, indicating the stable integration of the transgene. The subsequent T4 transgenic seeds revealed 3.85-fold down-regulation in IPK1 transcripts, which correlated to a significant reduction in phytate levels and a concomitant increase in the amount of inorganic phosphate (Pi). The low-phytate rice seeds also accumulated 1.8-fold more iron in the endosperm due to the decreased phytic acid levels. No negative effects were observed on seed germination or in any of the agronomic traits examined. The results provide evidence that silencing of IPK1 gene can mediate a substantial reduction in seed phytate levels without hampering the growth and development of transgenic rice plants.

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance

The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R0C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R0 generation, suggesting gene silencing. Preliminary screening of these R1 plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance.

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.

Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.).

To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph genes revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.