Tobacco (Nicotiana tabacum cv. Samsun) and apple (Malus x domestica cv. Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation. ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple. Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam. Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam. Tortricidae). More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation. Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days. Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.
The efficacy of four versions of a cry9Aa2 gene was evaluated against larvae of potato tuber moth (PTM; Phthorimaea operculella Zeller) following transfer to potato (Solanum tuberosum L.). A truncated native cry9Aa2 gene and three modified versions (G7, G10 and G14) were used, each with progressively more nucleotide substitutions to produce up to 79 codon changes to more closely resemble the codon bias of dicotyledonous plants. All four cry9Aa2 genes, under the transcriptional control of a CaMV 35S promoter, were independently transformed into the potato S. tuberosum L. cv. Iwa. A total of 100 independently derived cry9Aa2-transgenic plants were regenerated and their transgenic status confirmed by PCR and Southern analysis. Replicated plants were transferred to a containment greenhouse for bioassays against PTM larvae. Higher efficacy of the cry9Aa2 gene against PTM larval growth on greenhouse-grown plants was associated with more changes to the codon usage. This was evident in both the frequency of independently derived lines exhibiting a significant growth inhibition of PTM larvae as well as a lower larval growth index.
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