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Article Excerpt
RICE, the most important cereal grain crop worldwide, is cultivated on 5.9 million hectares spanning 80% of the arable land of Vietnam and providing 80% of the carbohydrate and 40% of the protein intake of the average Vietnamese. Although rice production has increased dramatically in Vietnam in the past 30 yr through the widespread cultivation of modern high yielding rice varieties, the productivity of the traditional photosensitive rice cultivars remained below the national average. This is mainly due to severe crop loss caused by insect pests, of which yellow stem borer is the most devastating.
Yellow stem borer, a lepidopterous insect that feeds inside the rice stem, causes "dead heart" in the vegetative stage, ultimately leading to "white head" in the reproductive stage. At times, severe damage can cause complete crop loss. Chemical control has proven to be ineffective because the insect larvae feed inside the stem pith and remain out of the reach of the pesticide. Moreover, the use of agrochemicals is associated with high cost and the risk of environmental and health hazards. A genotype with built-in resistance (host-plant resistance) is an environment-friendly and cost-effective component of integrated pest management (IPM). But, conventional resistance breeding, particularly for this insect pest, has been handicapped by the lack of a resistant donor in the rice gene pool, despite massive screening of 30 000 rice accessions (Teng and Revilla, 1996). Genetic engineering, on the other hand, holds great promise for transferring genes across taxa for development of transgenics resistant to this insect with the insecticidal crystal protein (ICP) genes (cry) from the soil-borne bacterium Bacillus thuringiensis (Bt). Until now, transgenics have been developed in several crops, including rice, with Bt genes. However, most earlier works document the development of transgenic rice with a single cry gene: cry1A(b) (Ghareyazie et al., 1997; Alam et al., 1998, 1999; Datta et al., 1998, 1999; Husnain et al., 2002; Wu et al., 2002), cry1A(c) (Nayak et al., 1997; Cheng et al., 1998; Khanna and Raina, 2002), cry1B (Breitler et al., 2000; Marfa et al., 2002), or cry2a (Maqbool et al., 1998). Transgenic rice with a single cry1A(b) gene was shown to confer resistance to eight lepidopterans under field conditions (Shu et al., 2000). However, some insect populations develop resistance to a single cry gene (Tabashnik et al., 2000). So, the "high-dose" and "refuge" strategies have been suggested in recent Bt rice research (Cohen et al., 2000). But, at the same time, small holder farmers in Asian countries could hardly devote their land to a refuge, and, moreover, a high dose of a foreign protein could cause a phenotypic trade-off resulting in a yield penalty (Datta et al., 2002b). Hence, efforts are being made to develop two-toxin Bt crops (otherwise known as the "pyramiding" approach), since two-toxin cultivars require smaller refuges to achieve successful resistance management and sustainable field release (Cohen et al., 2000). The use of multiple-toxin genes with different modes of action has been proposed so that cross-resistance is unlikely to occur (i.e., two cry genes for toxins with different receptors or a cry gene in combination with an altogether different unrelated toxin gene, de Maagd et al., 1996; Frutos et al., 1999).
Hybrid toxins produced through inclusion of a domain from another toxin result in increased potency of the fused protein by the shift in receptor binding (Bosch et al., 1994). Alternate receptor-ligand interaction may also be exploited to further broaden the host range of the Bt toxins (Sivasubramanian and Federici, 1994).
The translational fusion gene cry1Ab-1B encodes a single Cry1Ab-Cry1B fusion protein that provides Cry1B and Cry1Ab toxins after proteolysis in the insect midgut. To reconstitute functional Cry1B and Cry1Ab activation sites in the fusion protein, synthetic cry1B and cry1Ab genes were fused at the level of the 28th codon downstream from the Cry1B activation-site codons and the 29th codon upstream from the Cry1Ab activation-site codons (Bohorova et al., 2001). This fusion gene has been reported to confer resistance to southwest corn borer (Diatraea grandiosella Dyar), sugarcane borer [Diatraea saccharalis (Fabricius)], and fall armyworm [Spodoptera frugiperda (JE Smith)] in tropical maize (Bohorova et al., 2001).
The Bt fusion gene cry1Ab/cry1Ac consisted of 1344 bp encoding the N terminus of Cry1Ab and 486 bp encoding the C terminus of Cry1Ac. The efficacy of this fused hybrid Bt gene in transgenic indica rice has been successfully tested under both greenhouse conditions (Wu et al., 1997; Datta et al., 1998; Baisakh, 2000) that showed protection against yellow stem borer and under field conditions where a transgenic hybrid rice (Bt-Sanyou63) also showed an yield advantage of about 28% over the nontransgenic hybrid rice through protection against both yellow stem borer and leaffolder (Tu et al., 2000). Transgenic rice Bt-IR72 with this fusion gene showed consistent resistance against four lepidopteran insects, including yellow stem borer over three generations under both artificial and natural infestations (Ye et al., 2001). Transgenic rice have been produced by cotransfer of two different cry genes with resistance to two different insect pests (Maqbool et al., 2001).
In our study, we report on the resistance of transgenic indica rice cultivars to yellow stem borer conferred by a translational fusion gene, cry1Ab-1B or cry1Ab/cry1Ac, delivered through Agrobacterium- and particle gun-mediated transformations, respectively. This is the first report on the expression of the translational fusion gene cry1Ab-1B in rice.
MATERIALS AND METHODS
Transformation Vectors
For Agrobacterium tumefaciens-mediated transformation, strain LBA4404 harboring a T-DNA containing the translational fusion gene cry1Ab-1B driven by the maize ubiquitin constitutive promoter along with its first intron and untranslated exon (Christensen and Quail, 1996) and bar (coding for phosphinothricin acetyltransferase) as the selectable marker gene, under the control of the 70S promoter (double enhanced constitutive 35S promoter from Cauliflower mosaic virus), (Fig. 1a), was used.
[FIGURE 1 OMITTED]
For biolistic transformation, the plasmid pFWW2 containing the hybrid Bt gene (cry1Ab-cry1Ac)...
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