Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)

Abstract

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hu¨bner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.Abbreviations: AL-PCR -adaptor ligation PCR; Bt -Bacillus thuringiensis; cry1Ca -a synthetic gene from Bacillus thuringiensis; GUS -b-glucuronidase; H04 -a hybrid gene from B. thuringiensis; hpt -hygromycin phosphotransferase gene; LB -T-DNA left border; RB -T-DNA right border; gusA -b-glucuronidase gene.

Development of insect‐resistant transgenic rice with Cry1C*‐free endosperm

“…Zheng et al 32 expressed Cry1Ca in transgenic shallots under the control of a chrysanthemum Rubisco small subunit promoter, and the transgenic plants showed high resistance to beet armyworm. In the present study, a modified novel cry1C * gene19 was driven by the rice rbcS promoter,31 then introduced into Zhonghua 11 ( Oryza sativa L. ssp.…”

Aminopeptidase N1 is involved in Bacillus thuringiensis Cry1Ac toxicity in the beet armyworm, Spodoptera exigua

“…In agreement with this hypothesis, cross-resistance was observed between Cry1Ab and Cry1Ca in S. exigua larvae after selection with toxin31. The possible reason of this cross-resistance might be that the two toxins share same binding site of SeAPN1 , while further work would be needed to test this hypothesis, sharing of binding proteins between Cry1 and Cry1Ca proteins would have important consequences for resistance management tactics, as these proteins have been proposed as candidates for gene pyramiding in transgenic crops3233.…”

Endeavours of RuBisCO small subunit promoter as a tool of green tissue specific expression

“…In contrast, this pigeon pea promoter sequence was more similar to that of spinach and rice rbcs promoters. Zheng et al (2005) applied Agrobacteriummediated genetic transformation to produce beet armyworm (spodoptera exigua Hubner) resistant tropical shallots (Allium cepa L. group Aggregatum). Cry1ca or h04 hybrid gene from Bacillus thuringiensis, driven by Chrysanthemum rbcs promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was used for genetic transformation.…”