Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).
Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) 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.
Zheng
Si-Jun
SJ
Plant Research International, Wageningen University and Research Center, P O Box 16, 6700 AA Wageningen, The Netherlands.
Henken
Betty
B
de Maagd
Ruud A
RA
Purwito
Agus
A
Krens
Frans A
FA
Kik
Chris
C
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D030841:Food, Genetically Modified; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D019697:Onions / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D018411:Spodoptera / Q000187:drug effects*; D046509:Tissue Culture Techniques; D014162:Transfection
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012273:Ribulose-Bisphosphate Carboxylase