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Title:   Author  item
                                            Rajasekaran, Kanniah

                                                         - *Rajah*item
                                            Ulloa, Mauricioitem
                                            HUTMACHER, BOB - UC DAVIS, CAitem
                                            Cary, Jeffreyitem
                                            JAYNES, JESSE - TUSKEGEE UNIV, ALitem
                                            Cleveland, Thomas
  Submitted to: Aflatoxin Workshop
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 12/8/2005
                                    Publication  Date: 12/15/2005
                                    Citation: Rajasekaran, K., Ulloa, M., Hutmacher, B., Cary, J.W., Jaynes, J.M., Cleveland, T.E. 2005. Genetic engineering of cotton for resistance to phytopathogens including Aspergillus flavus [abstract]. 18th Aflatoxin Elimination Workshop, p. 111.Interpretive Summary:
                                Technical Abstract: Fertile, transgenic cotton plants expressing the synthetic antimicrobial peptide, D4E1, were produced through Agrobacterium-mediated transformation (Rajasekaran et al. 2005).  PCR products and Southern blots confirmed integration of the D4E1 gene, while RT-PCR of cotton RNA confirmed the presence of D4E1 transcripts.  In vitro assays with crude leaf protein extracts from T0 and T1 plants confirmed that D4E1 was expressed at sufficient levels to inhibit the growth of Fusarium verticillioides and Verticillium dahliae compared to extracts from negative control plants transformed with pBI-d35S'-uidA-nos (CGUS).  Although in vitro assays did not show control of pre-germinated spores of Aspergillus flavus, bioassays with cotton seeds in situ or in planta, inoculated with a GFP-expressing A. flavus, indicated that the transgenic cotton seeds inhibited extensive colonization and spread by the fungus in cotyledons and seed coats.  In planta assays with the fungal pathogen, Thielaviopsis basicola, which causes black root rot in cotton, showed typical symptoms such as black discoloration and constriction on hypocotyls, reduced branching of roots in CGUS negative control T1 seedlings, while transgenic T1 seedlings showed a significant reduction in disease symptoms and increased seedling fresh weight, demonstrating tolerance to the fungal pathogen.  Field evaluation of T2 progeny for Fusarium wilt (Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans) race 1 was carried out in sandy soil that also exhibited presence of  root-knot nematodes (Meloidogyne incognita).  R2 progenies of four independent transformation events expressing the antifungal peptide D4E1, a transgenic control entry with the GUS marker gene and the original non-transgenic variety (Coker 312), along with commercial Acala (G. hirsutum) and Pima (G. barbadense) cultivars were included in the field evaluation.  Entries were planted in a randomized complete block design with four replications on 10 feet long plots.  Plant survival rate, foliage damage symptoms, vascular root staining, presence of root-knot and agronomic data have been collected for these entries.  Preliminary observations indicated that the transgenic entries showed a healthy, higher germination stand (up to 68%) than the controls (43%).  We hope to complete the initial field evaluation this year (2005) and the promising lines will be re-tested for pathogen resistance including pre-harvest-resistance to A. flavus.


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