Benoît Bertrand

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.

Cycle characteristics in a temporary immersion bioreactor affect regeneration, morphology, water and mineral status of coffee (Coffea arabica) somatic embryos

Mass regeneration of Coffea arabica L. somatic embryos using a temporary immersion bioreactor was improved by optimizing the immersion cycles, i.e. both the duration and the frequency of immersions. It was demonstrated that increasing the frequency of short immersions (1xa0min immersions every 24, 12 and 4xa0h) stimulated embryo production (480, 2,094 and 3,081 embryos/1-l bioreactor, respectively) and improved quality (60, 79 and 85 of torpedo shaped embryos, respectively). On the other hand, an increase in the immersion duration (1, 5 and 15xa0min) inhibited embryo regeneration (from 2,094 to 428 embryos per 1-l bioreactor) and negatively affected their morphological quality (from 79 to 49 torpedo-shaped embryos) and the conversion of embryos into plants (from 70 to 33). A 15xa0min immersion duration applied every 4xa0h produced hyperhydric symptoms in 90 of the embryos. Hyperhydric embryos were characterized by higher fresh weight and water content, more negative values for water potential and higher K+ content when compared to normal torpedo-shaped embryos. Micrographs showed structural problems in the globular stage, such as the existence of an irregular epidermis and an absence of reserves. Whatever the immersion cycle used, the somatic embryos exhibited water and mineral characteristics very different from those of their zygotic counterparts. The use of 1xa0min immersions every 4xa0h led to the production of the largest quantities of torpedo-shaped embryos without hyperhydricity that succeeded in regenerating plants (75 conversion).

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

BackgroundFollowing genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.ResultsWe identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.ConclusionMost progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

Agrobacterium rhizogenes-transformed Roots of Coffee (Coffea arabica): Conditions for Long-term Proliferation, and Morphological and Molecular Characterization

BACKGROUND AND AIMSnThe aims of this study were to set up proliferation conditions for hairy roots of Coffea arabica regenerated after transformation by Agrobacterium rhizogenes strain A4-RS, and to carry out the morphological and molecular characterization of hairy root clones maintained over the long term.nnnMETHODSnAuxin supply, light conditions and sucrose concentration were modified with the aim of establishing efficient root proliferation conditions. The morphological variability among 62 established hairy root clones was phenotyped by scanning the roots and analysing the images using whinRHIZO software procedures. PCR analysis of integration in transformed root cells of rol and aux oncogenes from the T-DNA of the Ri plasmid was used to study the molecular variability among clones.nnnKEY RESULTSnAuxin supply was necessary to obtain and stimulate growth and branching, and IBA applied at 0.5 microm was the most efficient auxin. Significant differences were shown among the 62 clones for total root length and for the percentage of fine roots. These variables were stable across subcultures and could hence be used for efficient characterization of hairy root clones. The majority of hairy root clones (86 %) exhibited non-significant phenotype differences with non-transformed roots. Eight clones were significantly different from the non-transformed controls in that they possessed a low proportion of fine roots. Two other hairy root clones grew significantly faster than the other clones. The PCR analysis revealed a low variability in the integration of rol and aux oncogenes in transformed root cells. The T(R)-DNA was never integrated as aux1 and aux2 genes were not found, although rolB and rolC genes from the T(L)-DNA were always present.nnnCONCLUSIONSnThe discovery of low morphological variability among coffee hairy roots together with the identification of morphological variables allowing easy identification of phenotypically altered clones represent two important results. They make hairy roots a possible, and efficient, tool for functional-genomic studies of coffee root genes.

The morphological variability within a population of coffee somatic embryos produced in a bioreactor affects the regeneration and the development of plants in the nursery

A 1-liter bioreactor was used to obtain approximatively 800 Coffea arabica somatic embryos, 86% of which reached the `germinated stage but with morphological heterogeneity. The population was sub-divided into three categories according to cotyledon area: small, medium and large, that amounted to 32%, 36% and 4.5%, respectively. The effect of embryo morphology on plantlet conversion after direct sowing in soil and on plant development in the nursery was investigated. Somatic embryos with large cotyledons had only a 25% plantlet conversion rate, whereas somatic embryos with small to medium-sized cotyledons had conversion rates of 47% and 63%, respectively. The vigour of the aerial and root systems of regenerated plantlets at the end of the plant conversion stage was also affected as the embryos with small, medium and large cotyledon mostly regenerated small plantlets (0.5–1.5 cm), medium plantlets (1.5–2.5 cm) and large plantlets (2.5–5 cm), respectively. When transplanted in plastic bags, these 3 populations of plantlets exhibited distinct development rates. They had an initial slow growth phase, which was much longer for the small plantlets, followed by a rapid growth phase. After 40 weeks in the nursery, an analysis of the growth parameters of aerial and radical systems showed that the vigour of the plants was strongly related to the vigour of the plantlets transplanted. The heterogeneity of somatic embryos in the bioreactor affected both the plant conversion efficiency in soil and the plant growth in nursery, where it mainly resulted in retarded growth, primarily in plantlets derived from the somatic embryos with small cotyledons.

#Coffea arabica# hybrid performance for yield, fertility and bean weight

The performance of F1 hybrid plants derived from crosses between traditional varieties of Coffea arabica of Latin America with a “wild” collection of Sudan–Ethiopian origin were studied for yield, fertility and bean weight. Sudan–Ethiopian material possesses resistance to certain diseases, and possibly, better beverage quality. The objective of this breeding programme is to widen the very narrow genetic base of Central American coffee, even while increasing quality and productivity. The hybrid plants were obtained from two factorial crosses made and evaluated in two locations. They were compared in the two field trials using the six maternal lines as controls. Observations were taken on vegetative characters, annual and cumulated yield, dry weight of 100 beans, extent of early abortion as measured by the fraction of ‘peaberries’ and post-zygotic ovule fertility as measured by the fraction of mature no-floating berries in water (FF). The F1 hybrid population were compared to the populations of maternal lines for the aforementioned variables. An index-based selection was done in the hybrid populations employing three traits, yield, 100-bean weight and the post-zygotic fertility (FF). The performance of the selected hybrids was then compared to those of the best parental ‘control’ lines in each trial. The hybrid populations yielded 22–47% more than the maternal lines, but hybrids showed significantly more sterility than the parental control lines. Selection in the hybrid populations using the three selected traits led to significant genetic gain for yield and dry weight of 100 beans, and insignificant gain for fertility (FF). When selected on the basis of fertility alone, increase in yield and 100-bean weight were not obtained within the hybrid populations. By applying selection on yield and 100-bean weight, the selected hybrids produced 11–47% higher yields than the best line along with significantly higher or identical 100-bean weight and performed identically for fertility. The yield performance of hybrids between the Latin American material and the “wild’’ Sudan–Ethiopian material calls for further selection effort for improving beverage quality.

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.

#Coffea# and #Psilanthus#

Coffee production relies on two species, Coffea arabica and C. canephora, grown in Latin America, Africa and Asia, but up to 126 species have been described in the Coffeae tribe. Traditional cultivars of the main cultivated species – C. arabica – derive from two narrow genetic bases and consequently present a homogeneous agronomic behavior characterized by high susceptibility to many pests and diseases. The low genetic diversity displayed by wild C. arabica genotypes has considerably limited the success of breeding programs based on intraspecific hybrids. Therefore, breeders have focused on the transfer of traits from other species. To date, C. canephora has been the main source of resistance traits not found in C. arabica and few species have been involved in breeding programs. However, the coffee genetic resources represent valuable sources for introducing new traits in cultivars of both cultivated species since the genome of coffee species presents strong affinities, allowing production of interspecific hybrids. This chapter summarizes available information on taxonomy, biology including geographical distribution and habitat, evolution and conservation of coffee species. The recent results achieved in studying coffee genomes are also presented here as well as the genomic resources that have been developed worldwide. The role of wild coffee in breeding programs is described considering the use of wild C. arabica as cultivars or parents of F1 hybrids, and the use of other species as donors of alien genes. The transfer of desirable genes into C. arabica cultivars can be efficiently controlled using molecular marker-assisted selection. The advent of large-scale molecular genomics will soon provide access to previously inaccessible sources of genetic variations that could be exploited in breeding programs.

Influence of Environmental Conditions and Genetic Background of Arabica Coffee (C. arabica L) on Leaf Rust (Hemileia vastatrix) Pathogenesis

Global warming is a major threat to agriculture worldwide. Between 2008 and 2013, some coffee producing countries in South and Central America suffered from severe epidemics of coffee leaf rust (CLR), resulting in high economic losses with social implications for coffee growers. The climatic events not only favored the development of the pathogen but also affected the physiological status of the coffee plant. The main objectives of the study were to evaluate how the physiological status of the coffee plant modified by different environmental conditions impact on the pathogenesis of CLR and to identify indicators of the physiological status able to predict rust incidence. Three rust susceptible genotypes (one inbred line and two hybrids) were grown in controlled conditions with a combination of thermal regime (TR), nitrogen and light intensity close to the field situation before being inoculated with the rust fungus Hemileia vastatrix. It has been demonstrated that a TR of 27-22°C resulted in 2000 times higher sporulation than with a TR of 23–18°C. It has been also shown that high light intensity combined with low nitrogen fertilization modified the CLR pathogenesis resulting in huge sporulation. CLR sporulation was significantly lower in the F1 hybrids than in the inbred line. The hybrid vigor may have reduced disease incidence. Among the many parameters studied, parameters related to photosystem II and photosynthetic electron transport chain components appeared as indicators of the physiological status of the coffee plant able to predict rust sporulation intensity. Taken together, these results show that CLR sporulation not only depends on the TR but also on the physiological status of the coffee plant, which itself depends on agronomic conditions. Our work suggests that vigorous varieties combined with a shaded system and appropriate nitrogen fertilization should be part of an agro-ecological approach to disease control.