Apple scab, caused by the ascomycete Venturia inaequalis, is the most damaging fungal disease of commercial apple orchards. Functional scab resistance genes are present in some wild Malus species. The HcrVf2 gene, derived from the Vf-region of the wild apple Malus floribunda 821 and encoding a receptor-like protein, has proved to confer scab resistance in a transgenic susceptible cultivar. In order to minimize nonplant DNA in genetically modified apple and to go a step toward the development of cisgenic apples, we have studied the capability of the HcrVf2 gene to confer apple scab resistance when it is controlled by its own promoter. Three promoter deletion constructs containing 115, 288, and 779 bp of the 5′ untranslated region and the HcrVf2 gene were used to transform the scab susceptible apple cvs. 'Gala' and 'Elstar.' The influence of the promoter length on both the HcrVf2 expression level and the response to V. inaequalis was analyzed in different transgenic lines. Promoter length was found to influence both the constitutive transcription levels of HcrVf2 in transgenic lines and the resistance level. Highly scab resistant 'Elstar' and 'Gala' plants were obtained, proving that the HcrVf2 gene controlled by its native promoter is effective in conferring resistance to V. inaequalis similarly as Vf introgressed in apple cvs. through classical breeding.
In order to compare transcription profiles in cultivars of Malus domestica that are differentially sensitive to apple scab (Venturia inaequalis), two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method. Subtraction hybridization was performed between cDNAs from uninfected young leaves of the resistant cultivar Remo and the susceptible Elstar. In total, 480 EST clones were obtained: 218 (ELSTAR) clones represent transcripts that are preferentially expressed in Elstar, while the other 262 (REMO) are derived from RNAs that are more highly expressed in Remo. The putative functions of about 50% of the cloned sequences could be identified by sequencing and subsequent homology searches in databases or by dot-blot hybridization to known targets. In the resistant cv. Remo the levels of transcripts encoding a number of proteins related to plant defense (such as beta-1,3-glucanase, ribonuclease-like PR10, cysteine protease inhibitor, endochitinase, ferrochelatase, and ADP-ribosylation factor) or detoxification of reactive oxygen species (such as superoxide dismutase) were highly up-regulated relative to the amounts present in cv. Elstar. Most surprising was the large number of clones derived from mRNAs for metallothioneins of type 3 (91 out of 262) found in the REMO population. The corresponding transcripts were only present in small amounts in young uninfected leaves of the cv. Elstar, but were up-regulated in the susceptible cultivar after inoculation with V. inaequalis. These results indicate that constitutively high-level expression of PR proteins may protect cv. Remo from infection by different plant pathogens.
A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a beta-glucuronidase (GUS) gene was transferred into apple cv. 'Holsteiner Cox' via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1-2 g l(-1) mannose in combination with 30 g l(-1) sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l(-1) and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.
Frutos de um pomar comercial composto por progênies de duas plantas matrizes foram avaliados, com o objetivo de caracterizar a variabilidade fenotípica. As características peso de fruto, diâmetro e sólidos solúveis totais apresentaram diferenças estatísticas significativas entre as médias de famílias em pelo menos dois dos anos avaliados, enquanto para comprimento e rendimento de polpa a diferença não foi significativa. Houve diferença significativa entre médias de anos, dentro de cada família, para todas as características, com exceção das médias de peso de fruto entre 1998 e 1999 nas duas famílias e as médias de sólidos solúveis totais entre 1998 e 1999 em uma família e entre 1999 e 2000 na outra família. As correlações e regressões entre características produtivas mais relevantes foram obtidas entre peso de fruto e peso de casca, peso de fruto e comprimento, peso de fruto e diâmetro, peso de casca e comprimento, peso de casca e diâmetro e comprimento e diâmetro.
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