Searching for RHBV Transgenic Resistance

Rice "hoja blanca" virus (RHBV) is a major viral disease of economic importance affecting rice in northern South America, Central America, and the Caribbean. Few genes control resistance to RHBV, but most commercial varieties possessing them are resistant when plants are more than 20 days old.

(A) Disease caused by RHBV in the nontransgenic check CICA 8. (B) At left, a diseased, nontransgenic, inoculated plant of CICA 8; center and right, healthy inoculated transgenic plants. (C) Bottom, typical symptoms of RHBV on CICA 8 leaves; top, hypersensitive reaction of resistance in the transgenic line A3-49-60-12-3-3. (D) Field evaluation of RHBV-resistant transgenic rice.

To ensure stable and durable resistance, additional sources must be identified and incorporated into rice. Transgenic plants with the RHBV nucleoprotein viral gene were generated and are resistant even at 10 days old. The hardy variety CICA 8, used by small farmers in tropical America, is also a suitable gene donor for the breeding program. Field evaluations over two seasons indicated that six, fixed, transgenic lines were more resistant than 'Fedearroz 2000', the most RHBV-resistant commercial variety. The gain obtained with transgenesis is expressed in terms of reaction scores, where CICA 8 obtained the low-resistance score 7-9 versus the high-resistance score of 1-3 for transgenic lines. The transgenic lines express low levels of RNA, which is detectable only by RT-PCR, and the RHBV nucleoprotein is not expressed in these plants, thus suggesting a very low risk, if any, for environmental and food safety concerns. A genome conversion project is in progress, and F5 lines derived from crosses with three other popular commercial varieties indicate that transgenic resistance could be used to complement the natural resistance source. Plants with transgenic-derived RHBV resistance are currently being advanced jointly with agronomic selection traits in the field.This project has been a learning experience on how to scale up the use of genetic transformation at CIAT from the laboratory to the field and then to the farmers. The project also delivered the first field test of transgenic plants developed at a CGIAR center, where the inserted gene had been sequenced and cloned at the center itself. The protocol used for transformation contains innovations applicable to intellectual property rights according to the CIAT IP audit disclosure. The field trials have also become an experiment on how to conduct research on environmental biosafety. The National Biosafety Technical Council of Colombia has suggested using the CIAT experience as a model for its country.


Genetic Introgression from Rice into Wild/ Weedy Relatives:Characterization of Red Rice Populations.


Genetic Introgression from Rice into Wild/ Weedy Relatives: Characterization of Red Rice Populations

Hybridization between crops and their wild relatives sometimes brings genes into wild populations, occasionally resulting in the evolution of aggressive weeds and/ or endangerment of rare species. Transgenic crops may also result in similar outcomes. The likelihood of crop-to-wild hybridization depends on the out-cross rate, and on distance and direction between wild and crop populations. Cultivated rice, O. sativa L., is an autogamous plant, with a low out crossing rate of 0-1%. Rice is an introduced species in the Neotropics from Africa and Asia, but with wild/weedy relatives including wild native species in Central and South America. Hybridization can be expected within the genomic group that includes O. sativa, viz., the AA group. The wild relatives of AA genome, which are found in Central and South America and may hybridize with the rice crop, include O. rufipogon and O. glumaepatula. Red rice (Oryza sativa f. spontanea) is weedy rice with a red pericarp and dark-colored grains. The seeds shatter readily and possess dormancy characteristics. The plants typically are tall, late maturing, and have pubescent leaves and hulls. In contrast to Asia where manual transplanting is still predominant, in tropical America direct seeding of red rice-contaminated seed source is common, making red rice the most serious weed problem. Genes from rice varieties may transfer quickly into red rice (1% to 52% hybridization rate). However, most of the hybridization rate estimates have been done under temperate conditions. This work is part of a project financed by BMZ (Germany) directed to analyze the gene flow from non-transgenic or transgenic rice into wild/weedy relatives in the Neotropics, and its effect(s) on the population genetic structure of the recipient species targeting the development of tools for tracking and monitoring genetic introgression in natural biodiversity, and of management practices for the safe use of transgenic rice in the Neotropics.

Gene Flow Analysis from Rice into Wild/Weedy Relatives in the Neo-Tropics: Morphological and Phenological Characterization of Red Rice.

Tracking and Monitoring Gene Flow into Rice Wild/Weedy Using Molecular Markers

A careful assessment of potential impacts of gene flow from transgenic plants on population genetics of natural crop plant biodiversity is needed in other to design strategies for the safe and durable use of these crops in the Neo-tropics. This work is part of a project financed by BMZ (Germany) directed to analyze the gene flow from non-transgenic or transgenic rice into wild/weedy relatives, and its effect(s) on the population genetic structure of the recipient species. Research is underway on setting up and using molecular markers to track and monitor gene flow from transgenic and non-transgenic rice into wild Oryza species and red rice under controlled confined field plots, and under local agricultural field conditions. A genetic diversity analysis was first conducted in order to determine the genetic structure prior gene flow, and to select the best combinations of transgenic or non-transgenic rice, and wild/weedy populations to assess the gene flow. Specific microsatellite alleles were identified in different commercial varieties, red rice accessions and wild species, and conditions standardized to detect introgression in large number of seed samples..

Molecular characterization of rice and wild/ weedy relatives by microsatellites and their use to assess gene flow in the Poster.

Brachiaria

Somatic Embryogenesis and Plant Regeneration in Brachiaria decumbens Stapf
Comparative analysis of gene insertion and expression patterns in transgenic plants requires the analysis of a number of independent transgenic events, especially when it is applied to breeding programs. One important aspect is the avoidance of genetic instability or variation due to the regeneration pathway used. Plant regeneration via somatic embryogenesis facilitates the maintenance of such genetic true type.Somatic embryogenesis can be described as the process by which haploid or diploid somatic cells develop into differentiated plants through characteristic embryological stages without fusion of gametes. Somatic embryogenesis is a multi-step regeneration process starting with the formation of pro-embryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Plant regeneration in Brachiaria sps, alike other grasses, is. The lack of a reproducible plant regeneration system is a main bottleneck for the development of genetic transformation. An efficient plant regeneration system via somatic embryogenesis from Brachiaria was developed.

Contact: Zaida Lentini