Frederic Georget

Morphohistological study of the different constituents of a banana (Musa 'AAA', cv. 'Grande Naine') embryogenic cell suspension

Abstract Five types of cellular aggregates have been characterised in embryogenic cell suspensions of banana (Musa AAA Grande naine cv.). Type I corresponded to isolated cells or to small cell aggregates. Type II were composed of embryogenic cells. Type III can be distinguished from type II due to the presence of peripheral proliferation zones with embryonic cells. Type IV were composed of protodermic masses histologically comparable to proembryos. Type V were nodules composed of a central zone of meristematic cells and of an external zone of starchy cells. Each culture flask of a cell line contained a majority of one of the above-mentioned aggregate types. Histological studies of somatic embryo developement on semi-solid regeneration medium showed that there were close similarities between the initial steps of ontogenesis of the embryos and the different cell aggregates in liquid multiplication medium. It appeared that aggregates II–IV of the suspension belong to the same development continuum which reproduces the initial phases of somatic embryo ontogenesis on semi-solid medium. Type V resulted from the development of type IV, for which ontogenesis is hindered by direct contact with 2,4-dichlorophenoxyacetic acid and the shaken liquid multiplication medium. Type I aggregates probably do not belong to the development continuum but rather correspond to the degeneration of the other types of aggregates in the suspension. The presence of intermediate types in the liquid medium reinforces the hypothesis of a relationship between the aggregates. The aggregates tended to develop through time from a majority of type II or III at the beginning of their culture to types IV–V for older suspensions.

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.

High genetic and epigenetic stability in coffea arabica plants derived from embryogenic suspensions and secondary embryogenesis as revealed by AFLP MSAP and the phenotypic variation rate

Embryogenic suspensions that involve extensive cell division are risky in respect to genome and epigenome instability. Elevated frequencies of somaclonal variation in embryogenic suspension-derived plants were reported in many species, including coffee. This problem could be overcome by using culture conditions that allow moderate cell proliferation. In view of true-to-type large-scale propagation of C. arabica hybrids, suspension protocols based on low 2,4-D concentrations and short proliferation periods were developed. As mechanisms leading to somaclonal variation are often complex, the phenotypic, genetic and epigenetic changes were jointly assessed so as to accurately evaluate the conformity of suspension-derived plants. The effects of embryogenic suspensions and secondary embryogenesis, used as proliferation systems, on the genetic conformity of somatic embryogenesis-derived plants (emblings) were assessed in two hybrids. When applied over a 6 month period, both systems ensured very low somaclonal variation rates, as observed through massive phenotypic observations in field plots (0.74% from 200 000 plant). Molecular AFLP and MSAP analyses performed on 145 three year-old emblings showed that polymorphism between mother plants and emblings was extremely low, i.e. ranges of 0–0.003% and 0.07–0.18% respectively, with no significant difference between the proliferation systems for the two hybrids. No embling was found to cumulate more than three methylation polymorphisms. No relation was established between the variant phenotype (27 variants studied) and a particular MSAP pattern. Chromosome counting showed that 7 of the 11 variant emblings analyzed were characterized by the loss of 1–3 chromosomes. This work showed that both embryogenic suspensions and secondary embryogenesis are reliable for true-to-type propagation of elite material. Molecular analyses revealed that genetic and epigenetic alterations are particularly limited during coffee somatic embryogenesis. The main change in most of the rare phenotypic variants was aneuploidy, indicating that mitotic aberrations play a major role in somaclonal variation in coffee.

Development of coffee somatic and zygotic embryos to plants differs in the morphological, histochemical and hydration aspects.

In Coffea arabica L., the development of direct sowing of somatic embryos (SE) in planting substrate, with subsequent nursery production of plants, has promoted the industrialization of somatic embryogenesis. However, plant conversion rates are still low and require improvements to enhance the cost-effectiveness of commercial micropropagation. With the aim of improving plant regeneration from SE, we studied the morphological and histological criteria and water characteristics during germination and plant conversion of zygotic embryos (ZE) and SE. At the cotyledonary stage, SE produced in a 1 l RITA(®) temporary immersion bioreactor (area 55.8 cm(2)) were morphologically similar in size (2-3 mm) but abnormal as compared with mature ZE. Protein and starch reserve levels were extremely low throughout germination and conversion to plantlets, while the water status remained steady [water content (WC) from 76 to 87%, Ψ from -0.37 to -0.47 MPa, pressure potential from 0.69 to 0.24 MPa]. In ZE, spectacular hydration occurred during the first 3 weeks (WC from 37 to 75%; Ψ from -6.24 to -1.0 MPa). Cotyledons remained undifferentiated for 10 weeks after sowing. Conversely, after only 3 weeks under germination conditions in a RITA(®) bioreactor, spongy and palisade parenchyma and stomata formed in SE cotyledons. The ZE plant conversion was faster than that of SE (14 vs. 22 weeks) and more efficient (rates 96 vs. 55%), with much more substantial hypocotyl and cotyledon development. The use of a new 5 l MATIS(®) bioreactor (area 355 cm(2)), designed especially to favor embryo dispersion and light transmittance to SE, markedly improved the embryo-to-plantlet conversion rate (91%). These results highlight the morphological heterogeneity and lack of protein reserves in SE at the beginning of the germination phase and marked differences in water characteristics. However, they also reveal high phenotypic plasticity, leading to a highly efficient plantlet conversion rate due to better embryo dispersion and light transmittance in more horizontal bioreactors.

A single-step method for RNA isolation from tropical crops in the field

The RNAzol RT reagent was used to provide pure RNA from human cells. We develop a protocol using RNAzol RT reagent to extract pure RNA from plants tissues and demonstrate that this RNA extraction method works not only at room temperature but also at elevated temperatures and provides the simplest and most effective single-step method to extract pure and undegraded RNA directly from tropical plants in the field. RNA extraction directly in a complex field environment opens up the way for studying gene-environment interactions at transcriptome level to decipher the complex regulatory network involved in multiple-stress responses.