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An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called "high frequency" embryogenic callus of Coffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures. Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1-1 in a modified MS medium containing 4.5 #M 2,4-dichlorophenoxyacetic acid, under 3 #mol m -2 s-1 illumination, and subcultured every 7-10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250-1000 #m in diameter) at an inoculum density of 5 g 1-1, with medium renewed every 3-4 weeks. Under these conditions, embryogenic potential of C. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro-and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1-1. Under these conditions of culture, 1 g of C. canephora or Arabusta callus produced 1.2 and 0.9 × 105 somatic embryos, respectively, after 8-10 weeks in liquid regeneration medium. This was an overall reduction of 4--6 months from explant to regenerant, when compared with other procedures.

A culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Mtill. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4-dichlorophenoxyacetic acid and N6-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development ( + 60%) and epicotyl emergency (+ 35%), combined with increased synchronization and a substantially reduced workload.

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~UMMARYIn order to improve the late phases of Theobroma cacao L. embryogenesis from tissues of maternal origin, zygotic embryogenesis and somatic embryogenesis were compared, with respect to morphological, histological, and physiological parameters. Zygotic embryogenesis could be divided into three steps: (a) embryogenesis sensu stricto, (b) a growth period in which cotyledonary embryos reached their final dimensionsl and (c) a maturation period in which embryos accumulated protein and starch reserves, dehydrated to a water content equal to 30%, and underwent a modification in soluble sugar composition. Monosaceharides and sucrose contents decreased to the benefit of the oligosaccharides raffinose and stachyose. The formation of somatic embryos by use of basic protocols was studied to define the limiting factors that could lie behind their poor development. Morphological abnormalities of somatic embryos, which represented 80% of the total population, were described. A histological study showed that somatic embryos lacked starch and protein reserves; moreover, their water content was much higher than that of their zygotic counterparts. Introducing a growth period into the culture protocol made for better embryo development. Adding sucrose and abscisic acid to the maturation medium was effective in increasing reserve synthesis and resulted in higher germination, conversion, and acclimatization rates.

Somatic embryogenesis from Theobroma cacao L. flower buds, as previously reported on five Forastero hybrid genotypes, was tested on several other genotypes, belonging to the three cocoa-tree groups: Forastero, Trinitario and Criollo. The results gave evidence of genotypic efficiencies. Explants were cultivated under two successive conditions: callogenesis and expression media. The morphological and histological responses were different for embryogenic or non-embryogenic genotypes. For embryogenic genotypes, only staminodes and stamen filaments were able to produce somatic embryos: after a few days on the expression medium, groups of callus cells went through the meristematic and then embryonic stages, and finally formed somatic embryos. Many of them showed abnormalities. Simultaneously, some embryogenic cells were visible. These started to divide to form pro-embryos which however were unable to evolve into proper somatic embryos.

Some conditions related to the transient expression of β-glucuronidase in biolistically-treated Coffea spp. tissues were investigated, and subsequently used in a promoter study. Bombardments were performed on different types of tissue (leaves, somatic embryos and suspension cultures) of genotypes of C. arabica, C. canephora and Arabusta, using 4 different promoter sequences. Tobacco leaves were used as a comparison. In general, similar large variation and mean values of transient expression were observed between coffee and tobacco leaves. With regard to the coffee tissues effect, transient expression was best detectable and most frequently observed with bombarded leaves of microcuttings. Disturbing endogenous light blue staining was found with control treatments of somatic embryos. For the three coffee species tested, the most effective promoter was the EF1α-A1 promoter of Arabidopsis thaliana.

Leaf explants of Coffea canephora (P. ex Fr.) produced a friable yellow callus when they were cultured on a conditioning basal medium with 2.2 txM 2,4-D, 2.4 ~ IBA and 9.8 txM 2iP for 4 weeks then on an induction basal medium with 4.4 IxM 2,4-D and 17.8 ~tM BA for 10 weeks. This callus could be maintained by means of regular subcultures or it could give rise to somatic embryos depending on the culture medium. Cytological studies documented somatic embryogenesis and embryo development.