Valentina Talamè

Persistence of Cry toxins and cry genes from genetically modified plants in two agricultural soils

The environmental impact of genetically modified crops has been the subject of intense research in the past decade. Since the introduction of insect-resistant crops in 1996, cultivation of this group of genetically modified crops has grown substantially. Most insect-resistant varieties, including corn and cotton, have been engineered to express crystal (Cry) toxins. Although several studies concerning the environmental fate of this group of insecticidal toxins have been conducted during the past decade, conflicting information exists dealing with the persistence of Cry toxins in soil. In the present investigation, the persistence of antilepidopteran Cry1Ab and Cry1Ac toxins in two different agricultural soils was studied. The potential of cry1Ab genes to persist in soil was also estimated. The results from this laboratory investigation indicated that the two toxins dissipated rapidly in both the sandy and in the clay loam soil. The two toxins showed similar degradation rates in soil. During the 35-day incubation period, more than 92 and 79% of the initial amount of Cry toxins dissipated in the sandy and clay loam soil, respectively. Extractable fractions of the two toxins were lower in the fine-textured soil with respect to the coarse soil. Reduced recovery efficiency from the clay loam soil and thus bioavailability were presumably involved in the lower decline of Cry toxins in this soil. Investigations conducted with an insect-resistant transgenic corn hybrid showed no detectable levels of cry1Ab genes in soil six months after plant harvest.

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.

Identification of root morphology mutants in barley

In this study, a forward-genetics analysis was performed on a portion of TILLMore, a chemically mutagenized population of barley cv. ‘Morex’ (http://www.distagenomics.unibo.it/TILLMore/), to identify root morphology alterations by comparison with ‘Morex’ wild-type. For this purpose, a simple paper-roll approach was performed to identify phenotypic variants at the seedling stage. The analysis of c. 1000 M4 families allowed us to identify c. 70 lines with altered root morphology. A more accurate phenotypic characterization of a portion of the mutant lines has been performed using stereomicroscopy and a scanning electron microscopy approach.

Starch metabolism mutants in barley: A TILLING approach

In this study, the targetting-induced local lesions in genomes approach was used to identify mutants for genes related to starch metabolism in barley. Starch is the major reserve of plants and serves as primary carbohydrate component in human and livestock diets and has also numerous industrial applications. Mutants for biosynthetic or regulatory genes of starch metabolism often produce starch granules with abnormal morphological and molecular features that could be of interest for technological applications. We report the identification of 29 mutations in five starch-related barley genes ( Bmy1 , GBSSI , LDA1 , SSI and SSII ) through the molecular screening of TILLMore, a sodium azide-mutagenized population. Almost all the mutations detected were CG–TA transitions and several ( c. 60%) implied a change in amino-acid sequence and therefore possible phenotypic effects. Four mutants showed non-sense or splice-junction alterations, which could drastically affect the protein function.