Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic cry1Ab gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F1 plants obtained through crossing of T0 plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.

[1]  J. Valkonen,et al.  dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris) , 2006, Molecular Breeding.

[2]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[3]  K. Shimamoto,et al.  Insect Resistant Rice Generated by Introduction of a Modified δ-endotoxin Gene of Bacillus thuringiensis , 1993, Bio/Technology.

[4]  S. E. Wilhite,et al.  Inhibition of aspartyl and serine proteinases in the midgut of sugarbeet root maggot with proteinase inhibitors , 2000 .

[5]  James B. Hicks,et al.  A plant DNA minipreparation: Version II , 1983, Plant Molecular Biology Reporter.

[6]  R. Miller,et al.  Nutrient requirements of suspension cultures of soybean root cells. , 1968, Experimental cell research.

[7]  B. Sangwan-Norreel,et al.  Factors influencing T-DNA transfer in Agrobacterium-mediated transformation of sugarbeet , 1993, Plant Cell Reports.

[8]  THH. Chen,et al.  Activation of Two Osmotin-Like Protein Genes by Abiotic Stimuli and Fungal Pathogen in Transgenic Potato Plants , 1995, Plant physiology.

[9]  J. Valero,et al.  Expression of a Bacillus thuringiensis cry1Ab gene in transgenic white spruce and its efficacy against the spruce budworm (Choristoneura fumiferana) , 2007, Tree Genetics & Genomes.

[10]  A. Smigocki,et al.  Identification of highly regenerative plants within sugar beet (Beta vulgaris L.) breeding lines for molecular breeding , 2005, In Vitro Cellular & Developmental Biology - Plant.

[11]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[12]  Daniel Schubert,et al.  Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. , 2003, The Plant journal : for cell and molecular biology.

[13]  C. Koncz,et al.  Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae , 1996, Applied and environmental microbiology.

[14]  F. Bigler,et al.  Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis , 2005 .

[15]  A. Smigocki,et al.  Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves , 2005, Plant Cell Reports.

[16]  Jörg Romeis,et al.  Transgenic crops expressing Bacillus thuringiensis toxins and biological control , 2006, Nature Biotechnology.

[17]  M. Peferoen.  Progress and prospects for field use of bt genes in crops , 1997 .

[18]  L. Crossland,et al.  Field Performance of Elite Transgenic Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis , 1993, Bio/Technology.

[19]  M. Matzke,et al.  Listening to the silent genes: transgene silencing, gene regulation and pathogen control. , 1999, Trends in plant science.

[20]  P. Sanders,et al.  Expression of bacterial genes in plant cells. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Cai-guo Xu,et al.  Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin , 2000, Nature Biotechnology.

[22]  W. K. Roberts,et al.  Thaumatin-like pathogenesis-related proteins are antifungal , 1992 .

[23]  P. Christou,et al.  Transgenic Plants for Insect Pest Control: A Forward Looking Scientific Perspective , 2006, Transgenic Research.

[24]  F. Bigler,et al.  Uptake of Bt‐toxin by herbivores feeding on transgenic maize and consequences for the predator Chrysoperla carnea , 2002 .

[25]  C. Jung,et al.  Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.) , 2002, Applied Microbiology and Biotechnology.

[26]  M. Montagu,et al.  Transgenic plants protected from insect attack , 1987, Nature.

[27]  J. Opabode.  Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency , 2006 .

[28]  M. Cohen,et al.  Effect of Plant Age, Larval Age, and Fertilizer Treatment on Resistance of a cry1Ab-Transformed Aromatic Rice to Lepidopterous Stem Borers and Foliage Feeders , 2000, Journal of economic entomology.

[29]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[30]  B. San Segundo,et al.  Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions. , 2004, Plant biotechnology journal.

[31]  S. Jansens,et al.  Transgenic corn expressing a Cry9C insecticidal protein from Bacillus thuringiensis protected from European corn borer damage , 1997 .

[32]  P. Tenning,et al.  Reduced titer of BNYVV in transgenic sugar beets expressing the BNYVV coat protein , 2004, Euphytica.

[33]  P. Norouzi,et al.  Using a competent tissue for efficient transformation of sugarbeet (Beta vulgaris L.) , 2007, In Vitro Cellular & Developmental Biology - Plant.

[34]  S. Asano,et al.  High frequency Agrobacterium-mediated transformation and plant regeneration via direct shoot formation from leaf explants in Beta vulgaris and Beta maritima , 2004, Plant Cell Reports.

[35]  Z. Yin,et al.  Transgene inheritance in plants. , 2004, Journal of applied genetics.

[36]  G. W. Snyder,et al.  Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet (Beta vulgaris L.) by Agrobacterium or particle bombardment , 1999, Plant Cell Reports.

[37]  Y. Li,et al.  Inheritance and expression of the cry1Ab gene in Bt (Bacillus thuringiensis) transgenic rice , 2002, Theoretical and Applied Genetics.

[38]  I. Kovalchuk,et al.  High frequency Agrobacterium tumefaciens-mediated plant transformation induced by ammonium nitrate , 2009, Plant Cell Reports.

[39]  Gurdev S. Khush,et al.  Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene , 1997, Molecular Breeding.

[40]  F. Skoog,et al.  A revised medium for the growth and bioassay with tobacco tissue culture , 1962 .

[41]  I. Altosaar,et al.  Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[42]  L. Owens,et al.  A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid. , 1997, Molecular plant-microbe interactions : MPMI.

[43]  M. Matzke,et al.  How and Why Do Plants Inactivate Homologous (Trans)genes? , 1995, Plant physiology.