We introduced a truncated delta-endotoxin gene, cry1Ab of Bacillus thuringiensis which has a specific biological activity against lepidopteran insects into chrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitamura]. The chrysanthemum cultivar 'Shuho-nochikara' was transformed using a disarmed strain of Agrobacterium tumefaciens, LBA4404, carrying a binary vector, pIAbT1 that harboured a cry1Ab gene encoding an insecticidal crystal protein fragment of B. thuringiensis var. kurstaki HD-1. Leaf discs were cocultured with Agrobacterium and thereafter cultured on the callus induction medium containing G418. A total of 92 shoots were regenerated from 1,760 leaf discs on the regeneration medium (5.2 %). The cry1Ab gene was detected in all the regenerated plantlets by Southern blot analysis. The accumulation of Cry1Ab protein in 20 transformed lines, selected at random, was confirmed by Western blot analysis. The level of accumulation of Cry1Ab protein ranged from 4.5 ng to 40 ng per 50 µg total soluble protein (from 0.009 to 0.08 % of the total protein). Insect bioassay was conducted using tobacco budworm (Helicoverpa armigera) larvae. On the lines showing a high expression of Cry1Ab (more than 32.5 ng per 50 µg of total soluble protein), a significantly higher feeding inhibition and/or growth inhibition of the insects was observed, compared to those on the non-transformed control plants.Key Words: chrysanthemum, Agrobacterium tumefaciens, transformation, Bacillus thuringiensis, insect resistance.
IntroductionChrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitamura] was introduced into Japan from China during the Nara Era (A.D.710-794), and is now one of the important ornamental plants characterized by conventional vegetative propagation by stem cutting, adventitious shoot formation from various tissues and calli (Hill 1968, Iizuka et al. 1973, Earle and Langhans 1974, Khalid et al. 1989.Insect damage is one of the serious problems in chrysanthemum cultivation resulting in yield loss and high pesticide cost. For controlling lepidopteran insect pests, synthetic chemical insecticides are applied several times during the plant growing season. The use of chemical insecticides is limited by the cost, pollution of the environment, and development of resistance in insects resulting in a low effectiveness.Bacillus thuringiensis (Bt), a Gram-positive bacterium, produces insecticidal crystal proteins upon sporulation. These proteins called delta-endotoxins or Bt-toxins, are highly toxic to lepidopteran (Dulmage 1981), dipteran (Goldberg et al. 1977) and coleopteran insects (Krieg et al. 1983). Bt-toxins are considered to act as 'biorational' control agents due to the lack of toxicity to vertebrates and nontarget insects (Parrott et al. 1994). Microbial formulations have been used safely for almost 30 years. However, the limited field stability is a major constraint on the use of commercial Bt-toxin preparations. Also the time of application is critical because the susceptibility to Bt-toxins decreases as larvae mature.Rec...