Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? This report serves to document research conducted under a specific cooperative agreement between ARS and Oregon State University. Additional details of research can be found in the report for the parent project 5358-21000-031-00D Improvement of Horticultural Crop Productivity & Quality through Genetics, Physiology, & Biochemistry. T. Chen and colleagues are evaluating methods for developing winter hardy blackberries. Objective: To develop and implement a protocol for inserting genes that increase cold hardiness into 'Marion' and thornless blackberry advanced selections. Progress: Using an in vitro regeneration system optimized for 'Marion' blackberry (Meng et al., 2000, 2004), we have attempted to develop an Agrobacterium- mediated transformation protocol for 'Marion' blackberry, the industry standard. Previously we had tested: (1) Selection agents; (2) Agrobacterium strain and plasmid vector; (3)
Incubation time for explants and Agrobacterium cells in MRM medium; (4) Acetosyringone (AS) concentrations in the incubation medium; (5) AS concentration on the co-cultivation medium; (6) Duration of co-cultivation on transformation efficiency; (7) Vacuum infiltration; (8) Sonication; (9) Leaf explant age (10)Agrobacterium cell density (11)Surfactant concentrations We have continued our efforts to optimize the conditions for 'Marion' transformation and have also examined: 1) Different cocultivation media a) regeneration medium with glucose/acetosyringone (pH 5.2) b) regeneration medium (pH 5.7) While it has been reported that acetosyringone, lower pH, and glucose in the cocultivation medium increase efficiency of T-DNA delivery, we found that this was not true for 'Marion'. These factors can reduce regeneration efficiency after explants are transferred to selection medium, therefore, we simply used regeneration medium as cocultivation medium and found no difference in T-DNA
delivery and regeneration efficiency. 2) Iron chelator and L-cysteine in resuspension medium and cocultivation medium. It has been reported that thiol compounds found in iron chelators and L- cysteine inhibit wound- and pathogen-induced responses, thereby increasing the capacity for Agrobacterium-mediated transformation. Our results showed that iron chelator and L-cysteine in cocultivation medium, but not in resuspension medium, increased the number of transformed cells on leaf explants. 3) Shaking time after sonication (30s, 1min, 2min, 4min, 8min, 16min) Shaking time had no significant effect on T-DNA delivery. 4) Pretreatment effects. a) We inoculated 1-2 week old plantlets (TDZ pretreated) then grew them in TDZ medium for 1-2 weeks, hoping the transformed cells would divide and grow into a large area of transformed leaf tissue. When these leaves are cut for regeneration, they are expected to have higher chance to recover to real transformants than those obtained by single step
TDZ pretreatment. With this method, we have obtained putative transgenic 'Marion'. PCR will be applied to verify if they are real transgenic plants. b) Three-week-old plants were pretreated with BMM for three days (instead of TDZ) to obtain leaf explants for cocultivation. Plants were then cultivated in TDZ medium containing hydromycin (selection agent) and claforan for four days prior to transfer to a selection medium. We hope by using this method, we can reduce chimera frequency and obtain real transgenic 'Marion'. We have obtained putative transgenic 'Marion'. PCR will be applied to verify if they are real transgenic plants. 5) Claforan's concentration (0, 250, 500, 750, 1000 mg/L). Optimum claforan concentration will minimize Agrobacterium contamination and regeneration inhibition. We found that 250-500 mg/L is the optimum concentration for recovery of transformants. 6) Gelling agents. We found gelling agents (phytagel and agar) have no effect on T-DNA delivery. By combining
the best conditions for each parameter we examined, the frequency of transformation, expressed as number of transformed cell per explant, is about two times higher than what we obtained last year. We are currently using the improved protocol to select for permanent transformants.
Impacts
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Progress 09/12/00 to 09/30/04
Outputs
4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Oregon State University. Additional details of research can be found in the report for the parent project 5358-21000-036-00D Physiology, Biochemistry, and Genetic Improvement of Small Fruit Crops. This project has been completed. There were funds remaining to be disbursed at the end of the project last year, but no further progress will be reported.
Impacts
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Publications
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