Edgardo Alpizar

Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots.

The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The A. rhizogenes strain A4RS proved to be the most virulent. High transformation efficiencies (70%) were obtained using a 2-week co-cultivation period at a temperature of 15–18°C. Using a p35S-gusA-int construct inserted in the pBIN19 binary plasmid, we could estimate that 35% of transformed roots were GUS positive (co-transformed). Using the GUS assay as visual marker, 40% composite plants bearing a branched co-transformed rootstock could be obtained after only 12 weeks without selection with herbicides or antibiotics. Transgenic coffee roots obtained with A. rhizogenes did not exhibit the ‘hairy’ disturbed phenotype and were morphologically similar to normal roots. PCR analyses demonstrated that all co-transformed roots were positive for the expected rolB and gusA genes. Transformed and non-transformed root systems from both susceptible and resistant varieties were inoculated with Meloidogyne exigua nematode individuals. Inoculation of composite plants from the Caturra susceptible variety resulted in the normal development of nematode larvae. Numbers of extracted nematodes demonstrated that transformed roots retain the resistance/sensibility phenotype of varieties from which they are derived. These results suggest that composite plants constitute a powerful tool for studying nematode resistance genes.

Performance of Coffea arabica F1 hybrids in agroforestry and full-sun cropping systems in comparison with American pure line cultivars

Coffea arabica F1 hybrids derived from crosses between wild Sudan-Ethiopian and American cultivars and propagated by somatic embryogenesis have been obtained in Central America. These new hybrids considerably enhanced the genetic diversity of coffee in the region. We conducted 15 trials to assess whether using hybrids represents substantial genetic progress in terms of productivity in agroforestry and full-sun cropping systems. The new germplasm was grown in the same conditions as the best American cultivar (homozygous pure lines). The results showed that yields of hybrids were earlier and superior to those of American cultivars. The hybrids were also more stable than the American cultivars in all environments. In the agroforestry system, the mean yield of hybrids was 58% higher than that of the American cultivars, while the mean yield of hybrids in the full-sun system was 34% higher. Coffee-based agroforestry systems (AS) are considered effective in protecting the environment in the volcanic cordilleras of Central America. We found that introducing hybrids in coffee-based AS can considerably increase productivity. This finding could be a convincing argument to encourage coffee growers who have adopted the full-sun cropping system to return to agroforestry cropping systems. Finally, the conditions for large-scale dissemination of those new hybrids—which represent a major innovation for C. arabica cropping—was analysed.