Printer Friendly

Translational fusion hybrid Bt genes confer resistance against yellow stem borer in transgenic elite Vietnamese rice (Oryza sativa L.) cultivars.

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) under the control of the rice Actin-1 constitutive promoter (Datta et al., 1998) was co-bombarded with pGL2 (Datta et al., 1990) that harbors the hph gene (for hygromycin phosphotransferase) as the selectable marker gene driven by the CaMV 35S constitutive promoter.

Experimental Materials and Transformation

Altogether, four rice cultivars, Nang Huong Cho Dao (NHCD), Mot Bui (MB), and Tai Nguyen (TN) that are popular and adapted to the lowland ecosystem of Vietnam, along with aromatic cultivar Jasmine (J), were used in the present study.

Scutellum-derived embryogenic calli from mature seeds developed on MS-basal callus induction medium (Murashige and Skoog, 1962) supplemented with 2.0 mg [L.sup.-1] 2,4-D and embryogenic cell suspension were used as the target explants for the bombardment as well as Agrobacterium transformation. Embryogenic cell suspension was raised from the embryogenic calli on [R.sub.2] medium containing 20 g [L.sup.-1] maltose, with a 3- to 5-d interval subculture for 1 mo until cytoplasm-rich clusters were obtained

The preparation of explants for bombardment and Agrobacterium infection, selection of the putative transformed calli, regeneration of plantlets, rooting, and transfer of the plants to the soil in the transgenic greenhouse were essentially the same as described before (Datta et al., 1998, 2000). With hph as the selectable marker gene, hygromycin concentration in the selection medium was 50 mg [L.sup.-1] and for bar as the selectable marker gene the tissues were selected on medium supplemented with 3 mg [L.sup.-1] of phosphinothricin (PPT). The regenerants were transferred to rooting medium also containing 3 mg [L.sup.-1 PPT. However, no hygromycin was used in the rooting medium in case of regenerants with hph as the selectable marker gene. The plantlets were transferred to the nutrient culture solution (Yoshida, 1976) for 1 wk and then planted in pots inside the containment facilities of the International Rice Research Institute (IRRI).

Polymerase Chain Reaction (PCR) and Southern Blot Analyses

A rapid microprep method (modified after Kangle et al., 1995) was followed to extract the genomic DNA from 1-mo-old seedlings for the PCR template, and PCR was performed with primers specific for hph, bar, and cry1Ab/cry1Ac. Those primers are HPH F: 5'-tacttctacacagccatc-3', HPH R: tatgtcctgcgggtaaat-3' BAR F: 5'-gtctgcaccatcgtcaacc-3', BAR R: 5'gaagtccagctgccagaaac-3' FWW F: 5'-atcttcacctcagcgtgctt-3', FWW R: 5'-ggcacattgttgttctgtgg-3'.

The PCR conditions were the same as detailed earlier (Baisakh et al., 2000). Plant genomic DNA for Southern analysis was extracted from the freshly collected leaves or freeze-dried leaves of PCR-positive transgenics and the respective nontransformed controls following a modified miniprep method (Dellaporta et al., 1983). For Southern blot analysis, 10 [micro]g of genomic DNA was digested overnight with appropriate restriction enzymes designated for different transgenes (see figures for details). Southern blot transfer, hybridization, washing, X-ray film exposure, and the development were done as described by Baisakh et al. (2001).

HPT, PAT, and Leaf Painting Assay, and BASTA Spray

Total soluble protein extraction from fresh leaves, protein quantification, thin layer chromatography (TLC) assays for hygromycin phosphotransferase (HPT) and phosphinothricin acetyltransferase (PAT) enzymes were performed following the procedures described by Datta et al. (1990, 1992).

A leaf assay for hygromycin resistance of the transgenic plants was done to confirm the functional expression of the hph gene. Leaves (approx. 2-3 cm long) from 1-mo-old transgenie and control plants were excised and placed in assay medium (MS + 0.5 mg [L.sup.-1] BA + 50 mg [L.sup.-1] hygromycin).

Similarly, the leaf assay for the bar gene was done with approximately 2 to 3 cm long leaves placed on the same medium but with PPT (3 g [L.sup.-1]) instead of hygromycin.

The 2- to 3-mo-old transgenics and nontransformed control plants in the greenhouse were sprayed with 0.5% (v/v) of the commercial basta [ammonium 2-amino-4-(hydroxy-methylphosphoryl)-butanoate] solution and observation was recorded after 1 wk.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The total RNA was extracted from the leaves of the PCR and/or Southern positive plants carrying transgene cry1Ab-1B with the RNeasy plant minikit (Qiagen, Valencia, CA). Two micrograms of total RNA were used for the RT-PCR. The primers were designed with the forward primer (5'-gatagcaggacctatccaat-3') residing in the activation site of cry1Ab region and the reverse primer (5'-gccgaagagggaggcgtcaa-3') in the activation site of cry 1B region. The PCR profile was same as described in K Datta et al. (2002). The PCR products were resolved in a 1.5% (w/v) 1X TAE-agarose gel.

Immunoblot Analysis

For immunoblot analysis, the total protein was isolated as per Datta et al. (1998), quantified with Pierce's bicinchoninic acid (BCA) Protein Quantitation Assay kit (Pierce Products, Rockford, IL, USA) with bovine serum albumin (BSA) as standard. Fifty micrograms of total protein was separated with 12% (w/v) SDS-PAGE gel and transferred to a nitrocellulose membrane followed by blocking, hybridization, and immunodetection. The rabbit anti-Bt Cry1Ac and Cry1Ab-1B antibody was used as the primary antibody for Cry1Ab/Cryl Ac and Cry1Ab-1B, respectively, and detection was done by an IgG anti-rabbit conjugated horseradish peroxidase following the procedures detailed earlier (Datta et al., 1998). The amount of Bt toxin was measured with an ELISA kit (Envirologix Inc., Portland, ME, USA) as per the manufacturer's instruction manual.

Quick Dip Stick Detection

Quick detection of the hybrid/fused Bt protein was done using the "cry1Ab/cry1Ac lateral flow Quickstix Strip" as per the manufacturer's instructions (Envirlogix Inc., USA). The presence of a test line (second line) on the membrane strip between the control line (common to all, including the nontransformed control) and the protective tape would indicate the expression of foreign Bt protein in the transgenics.

Insect Bioassay

The [T.sub.0] and [T.sub.1] transgenic plants from all the cultivars expressing the transgene at the maximum tillering stage were bioassayed for resistance to neonate larvae of yellow stem borer using the cut-stem method (Datta et al., 1998). Three to five stems (including sheath) of 8 cm in length were collected at the booting stage. These were placed on a moistened filter paper disc in a 90-ram-diameter Petri dish and infested with six neonate larvae of yellow stem borer. Petri dishes were incubated at 28[degrees]C in the dark. The percentage larval mortality was based on dead and alive larvae 4 d after their release; the missing ones were considered dead.

RESULTS AND DISCUSSION

Plant Transformation

A total of 76 transgenics were obtained from the different cultivars with different cry genes by biolistic-and Agrobacterium-mediated transformation methods (Table 1).

No definitive comparison could be made between the two methods of gene transfer since the cultivars responded differently under basta and hygromycin selection systems. The transformation frequency [determined on the basis of the number of the PCR (or Southern)positive plants to total number of plants regenerated from selected putatively transformed independent calli] was 85% (17/20) for Agrobacterium method with bar as the selectable marker gene and 73% (59/81) for biolistic method with hph as the selectable marker gene.

PCR Analysis and HPT and PAT Assay

The PCR was performed on the DNA from the shoots when the putatively transformed planters were transferred to the nutrient culture solution to detect the presence of the transgene(s) and or selectable marker gene(s). The presence of the 1.1-, 0.73-, and 0.5-kb amplicons in the transformants but not in the nontransformed control indicated the transgenic status of the plants for cry1Ab/ cry1Ac, hph, and bar genes, respectively (Fig. 2 a, b, and c).

[FIGURE 2 OMITTED]

Similarly, thin layer chromatography (TLC) assays of crude leaf proteins from young seedlings showed the expression of the selectable marker genes, hph and bar in the transgenics. The [sup.32]P-[gamma]ATP phosphorylated and the [C.sup.14]-1abeled acetylated products of hygromycin and phosphinothricin, respectively were detected due to the action of the HPT and PAT, respectively, on the substrates, which were missing in the nontransgenic controls (data not shown).

Southern Analysis

The Southern analyses of PCR-positive plants revealed the integration pattern of the transgenes in the genome. As expected from Agrobacterium-mediated transformation, the transgenics from both cultivars, NHCD and MB, showed a simple integration pattern with a single 6.0-kb fragment expected for cry1Ab-1B for most cases. However, a very faintly hybridizing band smaller in size (~2.0 kb) was observed in some of the transgenics (Fig. 3a). The simple integration pattern of Agrobacterium-mediated gene transfer confirmed that of many earlier published results (Datta et al., 2000; Baisakh et al., 1999; Tinland, 1996; Khanna and Raina, 1999). The lower and faint fragment in Nab3, Nab4, Nab6, Mab2, and Mab4 might be a result of the transfer of a truncated T-DNA that had less homology with the intact Bt gene used as the probe in this case. The Southern blot analysis with HindIII (that cuts at one end of the coding sequence of the fusion Bt gene) and PstI (that does not cut inside the coding sequence of the T-DNA but cuts once in the transformation vector) showed only one hybridization band, meaning that the transgene had been integrated at only one site in the genome in all the transgenics produced through Agrobacterium-mediated transformation (data not shown). Further, the Southern analysis for the bar gene invariably showed only one expected size fragment (Fig. 3b). This indicates that the transgenics, where there were two copies of the Bt gene (one intact and another truncated), had an additional T-DNA insertion that contained a truncated fragment of the Bt gene without the bar gene, which is near the right border of the T-DNA. A similar type of T-DNA insertion with a truncated copy of the hph gene was observed earlier (Datta et al., 2000). Moreover, the insertion of the full-length fusion Bt gene (including the truncated one) at a preferred site in the genome supports the prediction of microhomology-mediated gene transfer into the genome (Kohli et al., 1999) as observed in transgenics derived from biolistic transformation. Nevertheless, transgene integration still remains largely a random phenomenon.

In the case of biolistic transformation, a few transgenics showed two different integration pattern (Fig. 3c). NB 6, NB7, MB3, MB6, MB7, MB8, MB9, MB10, TN3, and J2 transgenics contained an additional lower-size truncated fragment (~1.5 kb) apart from the expected size 1.8 kb for the hybrid Bt gene (cry1Ab/cry1Ac), whereas others had 1.8-kb hybrid Bt gene integrated into their genome. In biolisitic transformation, integration of single and intact copy transgene(s) are reported in many species (Altpeter et al., 2005). However, the independent status of the transgenics was evident when the same Southern filters were reprobed with the hph gene as the probe. Some transgenics having an identical banding pattern for the Bt gene showed a differential pattern with an additional fragment (mostly deleted) for hph, thus indicating independent transformation events (data not shown). This could be possible during the bombardment process, in which an intact copy of both the Bt and hph gene along with a few deleted fragments of hph (possibly from mechanical shearing before integration) were targeted to the tissues, or rearrangements and deletion of the hph gene under selection pressure. Rearrangement and multiple copy number of transgene(s), especially of the selectable marker gene, have been widely shown in the biolistic transformation of rice (Christou et al., 1991; Baisakh et al., 2001).

Immunoblot Analysis

The western blot analysis showed the presence of a 66-kDa protein in the transgenics containing cry1Ab-1B, which was absent in the nontransformed control (Fig. 4a). This confirmed the expression of the translational fusion gene (cry1Ab-1B). A 66 kDa protein expected for the cry1Ab-1B fusion gene, was also demonstrated by Bohorova et al. (2001) in transgenic maize plants. In Nab4-8-1 and Nab4-8-1-3 plants (lanes 2 and 4) there was an additional band of size ~56 kDa, which might have been resulted from the proteolytic degradation of the protein during processing.

The cry1A(b)/cry1A(c) expression was also shown by the lateral flow Quickstix Strip (Cat # AS 003 BG, Envirologix Inc., USA). Transgenic plants showed the second line (test line; arrow marked) on the membrane strip between the control line and the protective tape, whereas the control plants showed the absence of this test line (Fig. 4b). This method could be successfully used for quick detection of the protein expression at the early stage of seedling growth immediately after performing the PCR to detect the presence or absence of the transgene.

Further, the western blot analysis showed the expression of a 60-kDa protein in the transgenics obtained from biolistic transformation expected for the hybrid Bt gene (cry1Ab/cry1Ac). Apart from the 60-kDa band, an additional band of about 42 kDa was also observed in the transgenics (Fig. 4c). This further substantiated the Bt protein expression in the transgenic plants. The 60-kDa band corresponded to the expected protein encoded by the cry1AB/cry1Ac gene and the additional lower band might be the result of either truncated mRNA transcribed by the rearranged fragments of the cry1Ab/cry1Ac gene or the posttranslational modifications of the protein by the plants' endogenous proteases. Similar cases of proteolytic degraded proteins have been reported earlier in many of our transgenic rice lines (Datta et al., 1998; Tu et al., 1998; Baisakh, 2000).

The amount of Bt protein was found to range from 0.8 to 1.3% of the total soluble protein in different transgenic lines with cry1Ab-1B, whereas this value was from 0.1 to 0.9% for transgenics with cry1Ab/cry1Ac (Chandel et al., unpublished). These variations in protein expression levels in different transgenic lines are quite frequent and common due to genotypic, developmental, and environmental control. Even such variations could be observed among the progenies of a single parental line with an identical pattern of transgene integration grown under the same environment (Aguda et al., 2001; Alinia et al., 2001; Datta et al., 1998, 2002a; Husnain et al., 2002; Mafia et al., 2002).

Insect Bioassay

The cut-stem bioassay results showed that yellow stem borer neonate larval mortality after feeding for 4 d reached 100% in more than 95% of the tested Southern-and western-positive To plants, whereas the mortality was 0 to 16.6% in the nontransgenic control plants (Fig. 5). It is worthwhile to note that Bt protein even at the level of 0.1% of total soluble protein was sufficient to cause 100% mortality. The higher efficacy of the cry1Ab/ cry1Ac gene was shown earlier in several different rice cultivars (Datta et al., 1998; Tu et al., 1998; Baisakh, 2000; Balachandran et al., 2002). Similarly, the fusion gene cry1Ab-1B was shown to be effective against the European shoot borer in corn (Bohorova et al., 2001). The cry1B gene itself gave a higher level of protection against the striped stem borer in transgenic Mediterranean rice (Marfa et al., 2002). The fusion of cry1B with cry1Ab seemed to increase the insecticidal property of the hybrid gene as evidenced from the complete protection of all the transgenic rice plants (Nab4 and Mab2) produced including the progenies against yellow stem borer, whereas transgenic rice with cry1Ab have been shown to confer varied protection ranging from 66.7 to 100% (Datta et al., 1998). Similarly, the transgenic plants produced with the construct cry1Ab/cry1Ac (30 plants from each NB2, NB3, NB4, NB5, MB2, MB3, MB4, TN1, and TN2) also showed protection within the range of 89.9 to 100%. Each individual progeny plant was considered a line and therefore statistical analysis was not performed over the number of progenies rather was based on the number of larvae dead or alive.

Detached Leaf Assay for HPT and PPT and Whole-Plant Assay for Basta Resistance

A leaf assay for hygromycin resistance of the transgenic plants was done to confirm the functionality of the hygromycin gene. Leaf tips of control plants showed necrosis and dark brown stripes after 2 d and uniform necrosis after 7 d, whereas transgenic leaves remained healthy and green in the assay medium (MS with 50 mg [L.sup.-1] hygromycin) after 7 d, showing the resistance capacity of the plants to hygromycin.

Detached-leaf assays of these plants in PPT (3 mg [L.sup.-1]) showed resistance of the transgenics that remained green depending on the expression level of PAT, whereas the control plants started yellowing in 2 to 3 d after treatment and ultimately turned completely yellow within 1 wk of the experiment. These plants were also sprayed with 0.5% (v/v) solution of the PPT-based herbicide Basta. The subsequent growth and development of the resistant plants after spraying appeared normal, with green leaves and stem, whereas the nontransformed plants turned yellow within 3 d of spray and ultimately died within 1 wk of spraying (data not given).

Progeny Analysis

The [T.sub.1] seed progenies from different independent transgenics with 100% protection against yellow stem borer were grown for inheritance and functionality studies of the transgene(s)-selectable marker gene(s).

At the seedling stage, the transgenics were screened by PCR followed by Southern blot analysis for the segregation pattern of the transgenes. Inheritance data (Table 2) showed that in all the transgenics from Agrobacterium transformation, Mendelian segregation (3:1) was observed, which indicates that the transgenes were inserted into one locus in the genome and this was already shown by the Southern analysis for the site of integration analysis of the primary (To) transgenics. But in the case of transgenics derived from biolistic transformation, some transgenics (in TN and J) showed a segregation ratio other than 3:1, although in most transgenics, the transgene followed the Mendelian monogenic segregation ratio. The segregation data also correlated to the germination percentage of the seeds of transgenics in MS basal medium supplemented with PPT at 3 mg [L.sup.-1] or hygromycin at 50 mg [L.sup.-1]. Further Southern analysis confirmed the stable integration of the Bt genes without undergoing any change in the first selling generation. Agrobacterium-mediated transformation had the advantage of simple integration with a single or few copies of transgene at a single site (Tinland, 1996), as has been found in this study. This helped in isolating the homozygous lines from the progenies of a parental line in [T.sub.2] generation (Fig. 6). Interestingly, in particle bombardment although multiple copies with complex rearrangements of the transgenes are not rare, several transformation events showed only the expected-size fragment without any rearrangements. We also had a few transgenics from TN and J that had rearranged copies of transgenes apart from the expected size. The nonMendelian segregation of the transgenes in these particular transgenics and the differential banding pattern among the progenies of a single parental line (data not shown) confirmed their integration in more than one locus. Such rearrangements and segregation patterns in biolistic transformation have earlier been reported from our laboratory and from many others (Bohorova et al., 2001; Datta et al., 1998; Maqbool et al., 1998), which might occur either due to mechanical shearing during bombardment process and/or during the transgene integration.

Stability of Expression in the Progeny

The functional expression of the transgenes showed stability in terms of the Bt protein produced as well as the insect control efficiency, with expected variation among the progenies. The RT-PCR resulted in amplification of an expected 1.2-kb fragment that confirmed the expression of crylAb-lB gene in all the progenies carrying the fusion gene (Fig. 7). The transgenics were similar to the nontransformed control plants with respect to the morphology and seed setting. The [T.sub.2] progenies from selected lines are being grown to obtain homozygosity.

Gene pyramiding in transgenic plants has been used as a strategy for delaying the insect pest evolution showing resistance against single cry genes (Cao et al., 2002; Zhao et al., 2003; Greenplate et al., 2000) as well as providing protection against two or more different insect pests or insect and disease pests (Datta et al., 2002a). In our study, we have shown the efficacies of two fusion genes under maize ubiquitin and rice Actin-1 promoters that protect the plants completely against yellow stem borer under containment facilities. However, the results of the transgenics in open field conditions need to be carefully evaluated although whole plant bioassay results showed 100% mortality of the both yellow and striped stem borers (data not shown). The fusion genes with two-toxin strategy (such as, cry1Ab-1B used in this study) or the receptor-binding domain of one and the toxicity of another (as in the case of cry1Ab/cry1Ac) could serve very well for deployment as the important component of IPM, which would eventually delay the evolution of resistance by insect pests.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Mike Cohen, IRRI for his help in the insect bioassays and G. Chandel, IRRI for providing the ELISA data. NHH was supported by a grant from the Rockefeller Foundation, USA. We thank Dr. Bill Hardy for his editorial assistance.

REFERENCES

Aguda, R.M., K. Datta, J. Tu, S.K. Datta, and M.B. Cohen. 2001. Expression of Bt genes under control of different promoters in rice at vegetative and flowering stages. Int. Rice Res. Notes 26:26-27.

Alam, M.F., K. Datta, E. Abrigo, A. Vasquez, D. Senadhira, and S.K. Datta. 1998. Production of transgenic deepwater indica rice plants expressing a synthetic Bacillus thuringiensis crylA(b) gene with enhanced resistance to yellow stem borer. Plant Sci. 135:25-30.

Alam, M.F., K. Datta, E. Abrigo, N. Oliva, J. Tu, S.S. Virmani, and S.K. Datta. 1999. Transgenic insect-resistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Rep. 18:572-575.

Alinia, F., B. Ghareyazie, L. Rubia, J. Bennett, and M.B. Cohen. 2001. Expression of effect of plant age, larval age, and fertilizer treatment on resistant of a cry1Ab-transformed aromatic rice to lepidopterans stem borers and foliage feeders. J. Econ. Entomol. 93:484-493.

Altpeter, F., N. Baisakh, R. Beachy, R. Bock, T. Capell, P. Christou, H. Daniell, K. Datta, S. Datta, P.J. Dix, C. Fauquet, N. Huang, A. Kohli, H. Mooibroek, L. Nicholson, T.T. Nguyen, G. Nugent, K. Raemakers, A. Romano, D.A. Somers, E. Stoger, N. Taylor, and R. Visser. 2005. Particle bombardment and the genetic enhancement of crops: Myths and realities. Mol. Breed. 15:305-327.

Baisakh, N., K. Datta, N. Oliva, I. Ona, G.J.N. Rao, T.W. Mew, and S.K. Datta. 2001. Rapid development of homozygous transgenic rice using anther culture harboring rice chitinase gene for enhanced sheath blight resistance. Plant Biotechnol. 18:101-108.

Baisakh, N., K. Datta, H. Rashid, N. Oliva, and S.K. Datta. 2000. Agrobacterium tumefaciens-mediated transformation of an elite indica rice maintainer line IR68899B with a reconstructed T-DNA carrying multiple genes. Rice Genet. Newsl. 17:122-125.

Baisakh, N. 2000. Improvement of rainfed lowland indica rice through in vitro culture and genetic engineering. Ph.D. Thesis (Utkal Univ.), India.

Baisakh, N., K. Datta, N.P. Oliva, and S.K. Datta. 1999. Comparative molecular and phenotypic characterization of transgenic rice with chitinase gene developed through biolistic and Agrobacterium-mediated transformation. Rice Genet. Newsl. 16:149-152.

Balachandran, S., G. Chandel, M.E Alam, J. Tu, S.S. Virmani, K. Datta, and S.K. Datta. 2002. Improving hybrid rices through Anther Culture and transgenic approaches. In Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi. 4-17 May 2002.

Bohorova, N., R. Frutos, M. Royer, E Estanol, M. Pacheco, Q. Rascon, S. Mclean, and D. Hoisington. 2001. Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor. Appl. Genet. 103:817-826.

Bosch, D., B. Schipper, H. van der Kleij, R. de Maagd, and W.J. Stiekema. 1994. Recombinant Bacillus thuringiensis crystal proteins with new properties: Possibilities for resistance management. Biotechnology 12:915-918.

Breitler, J.C., V. Marfa, M. Royer, D. Meynard, J.M. Vassal, B. Vercambre, R. Frutos, J. Messeguer, R. Gabarra, and E. Guiderdoni. 2000. Expression of a Bacillus thuringiensis cry1B synthetic gene protects Mediterranean rice against the striped stern borer. Plant Cell Rep. 19:1195-1202.

Cao, J., J.Z. Zhao, A.M. Shelton, and E.D. Earle. 2002. Broccoli plants pyramided with cry1Ac and cry1C Bt genes control diamondback oths resistant to Cry1A and Cry1Ac proteins. Theor. Appl. Genet. 105:258-264.

Cheng, X., R. Sardana, H. Kaplan, and I. Altosaar. 1998. Agrobacterium-transformed rice plants expressing synthetic cry1A(b) and cry1A(c) genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl. Acad. Sci. USA 95:2767-2772.

Cohen, M.B., G. Gould, and J.S. Bentur. 2000. Bt rice: Practical steps to sustainable use. Int. Rice Res. Notes 25:4-10.

Christensen, A.H., and P.H. Quail. 1996. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5: 213-218.

Christou, P., T. Ford, and M. Kofron. 1991. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Biotechnology 9:957-962.

Datta, K., N. Baisakh, K.M. Thet, J. Tu, and S.K. Datta. 2002a. Pyramiding transgenes for multiple resistance in rice against bacterial blight, stem borer and sheath blight. Theor. Appl. Genet. 106:1-8.

Datta, K., Z. Koukolikova-Nicola, N. Baisakh, N.P. Oliva, and S.K. Datta. 2000. Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems. Theor. Appl. Genet. 100:832-839.

Datta, K., A. Vasquez, G.S. Khush, and S.K. Datta. 1999. Development of transgenic New Plant Type rice for stem borer resistance. Rice Genet. Newsl. 16:143-144.

Datta, K., A. Vasquez, J. Tu, L. Torrizo, M.F. Alam, N. Oliva, E. Abrigo, G.S. Khush, and S.K. Datta. 1998. Constitutive and tissue-specific differential expression of Cry1A(b) gene in transgenic rice plants conferring enhanced resistance to insect pests. Theor. Appl. Genet. 97:20-30.

Datta, S.K., N. Baisakh, S. Balachandran, L.B. Torrizo, G. Chandel, M. Arboleda, E.M. Abrigo, N.P. Oliva, J. Tu, and K. Datta. 2002b. Development of transgenic Bt rice and its deployment in Asia. p. 151-153. In A.J. Akhrust et al. (ed.) Biotechnology of Bacillus thuringiensis and its environmental impact. CSIRO, Canberra.

Datta, S.K., K. Datta, N. Soltanifar, G. Donn, and I. Potrykus. 1992. Herbicide-resistant indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplast. Plant Mol. Biol. 20:619-629.

Datta, S.K., A. Peterhans, K. Datta, and I. Potrykus. 1990. Genetically engineered fertile indica-rice plants recovered from protoplasts. Biotechnology 8:736-740.

Dellaporta, S.L., J. Wood, and J.B. Hicks. 1983. A plant DNA Mini preparation Version II. Plant Mol. Biol. Rep. 1:19-21.

de Maagd, R.A., H. van der Kleij, P.L. Bakker, W.J. Stiekema, and D. Bosch. 1996. Different domains of Bacillus thuringiensis [delta]-endotoxins can bind to insect midgut membrane proteins on ligand blots. Appl. Environ. Microbiol. 62:2753-2757.

Frutos, R., C. Rang, and M. Royer. 1999. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit. Rev. Biotechnol. 19:227-276.

Ghareyazie, B., F. Alinia, C. Menguito, L.G. Rubia, J.M. de Palma, E.A. Liwanag, M.B. Cohen, G.S. Khush, and J. Bennett. 1997. Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cry1Ab gene. Mol. Breed. 3:401-404.

Greenplate, J.T., S.R. Penn, Z. Shappley, M. Oppenhuizen, J. Mann, B. Reich, and J. Osborn. 2000. Bollgard II efficacy: Quantification of total lepidopteran activity in a two-gene product. Proc. Beltwide Cotton Conf. 2:1041-1043.

Husnain, T., J. Asad, S.B. Maqbool, S.K. Datta, and S. Riazuddin. 2002. Variability in expression of insecticidal cry1Ab gene in indica Basmati rice. Euphytica 128:121-128.

Kangle, Z., P.K. Subudhi, J. Domingo, G. Magpantay, and N. Huang. 1995. A rapid DNA isolation protocol for marker assisted selection in rice breeding. Rice Genet. Newsl. 12:255-258.

Khanna, H.K., and S.K. Raina. 2002. Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas). Transgenic Res. 11:411-423.

Khanna, H.K., and S.K. Raina. 1999. Agrobacterium-mediated transformation of indica rice cultivars using binary and super binary vectors. Aust. J. Plant Physiol. 26:311-324.

Kohli, A., S. Griffiths, N. Palacios, R.M. Twyman, E Vain, D.A. Laurie, and P. Christou. 1999. Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology-mediated recombination. Plant J. 17:591-601.

Marfa, V., E. Mele, R. Gabarra, J.M. Vassal, E. Guiderdoni, and J. Messeguer. 2002. Influence of the developmental stage of transgenic rice plants (cv. Senia) expressing the cry1B gene on the level of protection against the striped stem borer (Chilo suppressalis). Plant Cell Rep. 20:1167-1172.

Maqbool, S.B., P. Christou, S. Riazuddin, N.T. Loc, A.M.R. Gatehouse, and J.A. Gatehouse. 2001. Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Mol. Breed. 7:85-93.

Maqbool, S.B., T. Husnain, S. Riazuddin, L. Masson, and P. Christou. 1998. Effective control of yellow stem borer and rice leaf folder in transgneic rice indica varieties Basmati 370 and M7 using the novel delta endotoxin cry2A Bt gene. Mol. Breed. 4:501-507.

Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bio-essays with tobacco tissue cultures. Physiol. Plant. 15:473-497.

Nayak, P., D. Basu, S. Das, B. Asitava, D. Ghosh, N. Ramakrishnan, M. Ghosh, and S. Sen. 1997. Transgenic elite indica rice plants expressing cry1Ac [alpha]-endotoxin of Bt are resistant against yellow stem borer. Proc. Natl. Acad. Sci. USA 94:2211-2216.

Shu, Q., G. Ye, H. Cui, X. Cheng, Y. Xiang, D. Wu, M. Gao, Y. Xia, C. Hu, R. Sardana, and I. Altosaar. 2000. Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species. Mol. Breed. 6:433-439.

Sivasubramanian, N., and B.A. Federici. 1994. Method and means of extending the host range of insecticidal proteins. US Patent 5143905.

Tabashnik, B.E., A.L. Patin, T.J. Dennehy, Y.B. Liu, S. Carriere, M.A. Sims, and L. Antilla. 2000. Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm. Proc. Natl. Acad. Sci. USA 97:12980-12984.

Teng, P.S., and I.M. Revilla. 1996. Technical issues in using crop loss for research prioritization, p. 261-275. In R.E. Evenson et al. (ed.) Rice research in Asia: Progress and priorities. CAB International in association with the International Rice Research Institute, Wallingford, UK.

Tinland, B. 1996. The integration of T-DNA into plant genomes. Trends Plant Sci. 1:178-184.

Tu, J., G. Zhang, K. Datta, C. Xu, Y. He, Q. Zhang, G.S. Khush, and S.K. Datta. 2000. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis [delta]-endotoxin. Nat. Biotechnol. 18:1101-1104.

Tu, J., K. Datta, M.F. Alam, Y. Fan, G.S. Khush, and S.K. Datta. 1998. Expression and function of a hybrid Bt toxin gene in transgenic rice conferring resistance to insect pest. Plant Biotechnol. 15:195-203.

Wu, C., Y. Fan, C. Zhang, N. Oliva, and S.K. Datta. 1997. Transgenic fertile japonica rice pants expressing modified cry1A(b) gene resistant to yellow stem borer. Plant Cell Rep. 17:129-132.

Wu, G., H. Cui, G. Ye, Y. Xia, R. Sardana, X. Cheng, Y. Li, I. Altosaar, and Q. Shu. 2002. Inheritance and expression of the cry1Ab gene in Bt (Bacillus thuringiensis) transgenic rice. Theor. Appl. Genet. 104:727-734.

Ye, G.Y., J. Tu, C. Hu, K. Datta, and S.K. Datta. 2001. Transgenic IR72 with fused Bt gene cry1A (b)/cry1A(c) from Bacillus thuringiensis is resistant against four lepidopteran species under field conditions. Plant Biotechnol. 18:125-133.

Yoshida, H. 1976. Routine procedures for growing rice plants in culture solution, p. 61-66. In Laboratory manual for Physiological studies of rice. International Rice Research Institute, Los Banos, The Philippines.

Zhao, J.Z., J. Cao, Y.X. Li, H.L. Collins, R.T. Roush, E.D. Earle, and A.M. Shelton. 2003. Plants expressing two bacillus thuringiensis toxins delay insect resistance evolution. Nat. Biotechnol. 21: 1493-1497.

doi: 10.2135/cropsci2005.06-0111

N. H. Ho, ([dagger]) N. Baisakh, * ([dagger]) N. Oliva, K. Datta, R. Frutos, and S. K. Datta

N.H. Ho, Plant Biotechnology, National Centre for Natural Sciences and Technology, Institute of Tropical Biology, No 1 Mac Dinh Chi St., 1st Ho Chi Minh, Viet Nam; N. Baisakh, Agronomy and Environment Management, Louisiana State University AgCenter, 104 Madison, Sturgis Hall, Baton Rouge, LA 70803; N. Oliva, K. Datta, and S.K. Datta, Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Makati, Metro Manila, Philippines; R. Frutos, EMVT CIRAD, TA 30/D, Campus International de Baillar-guet, Montpellier 34398, France. ([dagger]) NHH and NB contributed equally to this paper. Received 13 June 2005. * Corresponding author (nbaisakh@ agctr.lsu.edu).
Table 1. Transgenics developed with different cry genes in different
indica rice cultivars.

                                                  Selectable marker
Cultivar    Method ([dagger])        Gene                gene

NHCD                A            cry1Ab-1B               bar
                    B            cry1Ab/cry1Ac           hph
MB                  A            cry1Ab-1B               bar
                    B            cry1Ab/cry1Ac           hph
TN                  B            cry1Ab/cry1Ac           hph
J                   B            cry1Ab/cry1Ac           hph

                                                 Number of plants
Cultivar    Number of plants regenerated     PCR/S+([double dagger])

NHCD                     14                             12
                         13                              6
MB                        6                              5
                         31                             24
TN                       30                             26
J                         7                              3

([dagger]) A = Agrobacterium; B = biolistic.

([double dagger]) S+ = Southern blot analysis positive.

Table 2. Inheritance studies in the T1 progenies of the transgenic
lines.

              Total number of                    [chi square] fitness
Transgenic   progenies analyzed   PCR/Southern    test for 3:1 ratio
Line

Nab4                 30                22          0.444 *
Mab2                 30                19          2.177
NB                   30                18          3.6
MB                   30                20          1.110
TN                   23                12          6.393 ([dagger])
11                   30                28          8.377 ([dagger])

* Values insignificant at P [greater than or equal to] 0.05 and values
do not fit 3:1 ratio at P [greater than or eqaul to] 0.05.

([dagger]) Progenies were analyzed for transgenes by PCR and subsequent
Southern blot analyses.
COPYRIGHT 2006 Crop Science Society of America
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2006 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Ho, N.H.; Baisakh, N.; Oliva, N.; Datta, K.; Frutos, R.; Datta, S.K.
Publication:Crop Science
Geographic Code:9VIET
Date:Mar 1, 2006
Words:6632
Previous Article:Virulence of two Russian wheat aphid biotypes to eight wheat cultivars at two temperatures.
Next Article:Risk assessment for transgenic sorghum in Africa: crop-to-crop gene flow in Sorghum bicolor (L.) moench.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters