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The present article describes two new applications of plastic-based cell culture systems in the plant biotechnology domain. Different types of bioreactors are used at Nestle R&D Center-Tours for large scale culture of plants cells to produce metabolites or recombinant proteins and for mass propagation of selected plant varieties by somatic embryogenesis. Particularly, recent studies are directed to cut down the production costs of these two processes by developing disposable cell culture systems. For large scale culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 l working volumes, validated with several plant species (“Wave and Undertow” and “Slug Bubble” bioreactors). Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has been recently set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 2.5–3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-l glass bioreactors. An improved process has been developed using a 10-l disposable bioreactor consisting in a bag containing a rigid plastic box (“Box-in-Bag” bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design.

During the 1990s, three major achievements have led to the scaling up of Coffea canephora propagation by somatic embryogenesis: 1) multiplication of embryogenic cells and production of embryos (torpedo stage) in liquid media, 2) pre-germination of embryos by temporary immersion in liquid media (cotyledonary stage), 3) ex vitro germination by sowing directly cotyledonary embryos in the greenhouse. This paper describes the implementation of the pre-germination phase at a commercial scale in the Nestle R&D Center (Tours). The pre-germination is achieved using 10 L-glass jars (up to 20-25,000 pre-germinated embryos per system). The current production capacities are 2.5 M embryos per year, quantity sufficient to potentially regenerate 1.0 M plantlets. Specific disposable plastic bags have also been developed for this purpose and their pros and cons instead of glass jars are discussed.

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 microg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.

An EST database has been generated for coffee based on sequences from approximately 47,000 cDNA clones derived from five different stages/tissues, with a special focus on developing seeds. When computationally assembled, these sequences correspond to 13,175 unigenes, which were analyzed with respect to functional annotation, expression profile and evolution. Compared with Arabidopsis, the coffee unigenes encode a higher proportion of proteins related to protein modification/turnover and metabolism—an observation that may explain the high diversity of metabolites found in coffee and related species. Several gene families were found to be either expanded or unique to coffee when compared with Arabidopsis. A high proportion of these families encode proteins assigned to functions related to disease resistance. Such families may have expanded and evolved rapidly under the intense pathogen pressure experienced by a tropical, perennial species like coffee. Finally, the coffee gene repertoire was compared with that of Arabidopsis and Solanaceous species (e.g. tomato). Unlike Arabidopsis, tomato has a nearly perfect gene-for-gene match with coffee. These results are consistent with the facts that coffee and tomato have a similar genome size, chromosome karyotype (tomato, n=12; coffee n=11) and chromosome architecture. Moreover, both belong to the Asterid I clade of dicot plant families. Thus, the biology of coffee (family Rubiacaeae) and tomato (family Solanaceae) may be united into one common network of shared discoveries, resources and information.

In order to validate the propagation technology of Coffea canephoraPierre var. Robusta via somatic embryogenesis in liquid medium, the clonal fidelity of regenerated trees has been assessed for the first time in large-scale field trials. A total of 5067 trees originating from 5- to 7-month-old embryogenic cell suspension cultures were planted in the Philippines and in Thailand for comparing with control trees derived fromin vitro axillary budding (microcuttings). For the observed morphological traits and the yield characteristics, no significant differences were seen between the somatic seedlings (SS) and the microcutting-derived trees (MC).After three harvests in Thailand, the most productive clones had a cumulative yield higher than 3000 kg of green coffee ha-1. The only detected ‘off-type’ concerns a low vigorous phenotype (2.3% in the Philippines and 3.8% in Thailand), which is probably the consequence of the planting out process as it is also observed in the control trees. The occurrence of some phenotypic variants difficult to visualize or somaclonal variations at the DNA level cannot be excluded. Nevertheless, this study shows that this propagation method can be used for large-scale commercial applications without any negative unforeseen consequences for the grower.

Although the Advanced Backcross strategy has proven very useful for QTL detection in tomato, it has been used mainly in identifying QTL for agronomic traits such as yield, color, etc. Tomato flavor is an important quality characteristic, yet it has been difficult to assess flavor or traits that affect it. In this study the AB-QTL strategy was applied to four advanced backcross populations to identify QTL for biochemical properties that may contribute to the flavor of processed tomatoes, such as sugars and organic acids. A total of 222 QTL were identified for 15 traits, including flavor as assessed by a taste panel. Correlations of certain biochemicals with flavor and possible methods of assessing and improving flavor are discussed. In particular, QTL with very significant effects associated with the ratio of sugars/glutamic acid, a trait highly correlated with improved flavor, have been identified as good targets for future work in improving the flavor of tomatoes.

Lycopersicon parviflorum is a sexually compatible, wild tomato species which has been largely unutilized in tomato breeding. The Advanced Backcross QTL (AB-QTL) strategy was used to explore this genome for QTLs affecting traits of agronomic importance in an interspecific cross between a tomato elite processing inbred, Lycopersicon esculentum E6203, and the wild species L. parviflorum (LA2133). A total of 170 BC2 plants were genotyped by means of 133 genetic markers (131 RFLPs; one PCR-based marker, I-2, and one morphological marker, u, uniform ripening). Approximately 170 BC3 families were grown in replicated field trials, in California, Spain and Israel, and were scored for 30 horticultural traits. Significant putative QTLs were identified for all traits, for a total of 199 QTLs, ranging from 1 to 19 QTLs detected for each trait. For 19 (70%) traits (excluding traits for which effects of either direction are not necessarily favourable or unfavourable) at least one QTL was identified for which the L. parviflorum allele was associated with an agronomically favourable effect, despite the overall inferior phenotype of the wild species.

The production of foods for an increasingly informed and selective consumer requires the coordinated activities of the various branches of the food chain in order to provide convenient, wholesome, tasty, safe and affordable foods. Also, the size and complexity of the food sector ensures that no single player can control a single process from seed production, through farming and processing to a final product marketed in a retail outlet. Furthermore, the scientific advances in genome research and their exploitation via biotechnology is leading to a technology driven revolution that will have advantages for the consumer and food industry alike. The segment of food processing aids, namely industrial enzymes which have been enhanced by the use of biotechnology, has proven invaluable in the production of enzymes with greater purity and flexibility while ensuring a sustainable and cheap supply. Such enzymes produced in safe GRAS microorganisms are available today and are being used in the production of foods. A second rapidly evolving segment that is already having an impact on our foods may be found in the new genetically modified crops. While the most notorious examples today were developed by the seed companies for the agro-industry directed at the farming sector for cost saving production of the main agronomical products like soya and maize, its benefits are also being seen in the reduced use of herbicides and pesticides which will have long term benefits for the environment. Technology-driven advances for the food processing industry and the consumer are being developed and may be divided into two separate sectors that will be presented in greater detail: 1. The application of genome research and biotechnology to the breeding and development of improved plants. This may be as an aid for the cataloging of industrially important plant varieties, the rapid identification of key quality traits for enhanced classical breeding programs, or the genetic modification of important plants for improved processing properties or health characteristics. 2. The development of advanced microorganisms for food fermentations with improved flavor production, health or technological characteristics. Both yeasts and bacteria have been developed that fulfill these requirements, but are as yet not used in the production of foods.

The aim of this study was to determine the genetic components controlling yield in an F1 cacao cross between Catongo and Pound 12 clones. Genetic maps were constructed for the two parents using molecular markers which detected 158 polymorphic loci covering 772 cM for the heterozygous genotype Pound 12 and only 4 loci representing 16.9 cM of a linkage group which indicated a high level of homozygosity of Catongo. Yield was recorded twice a month during 15 years on 55 individuals from this segregating population. Ten yield QTL were detected on eight linkage groups. Some of these QTL were frequently detected over 15 years of production, while others were specific for a given year. Total yield genetic variance, on a yearly basis, ranged from 0 to 56%. Two major QTL (E and I) each explained approximately 20% of the total variance of the average yield over 15 years. The analysis of potential cacao yield components, such as pod index and trunk diameter, suggested that some regions of the genome exert effects on more than one trait, providing a possible genetic explanation for the correlations detected between some of title traits studied. Data showed that correlation between successive annual yield decreased when the lag between corresponding seasons increased. When separated by more than 10 years, annual yields were no longer correlated. The utilisation of molecular markers alone or in combination with phenotypic selection showed an advantage in the early selection of the best cacao producer trees. Further use of molecular markers in breeding programs is discussed with a view to reducing the generation time of a selection procedure.

The accumulation of the terpenes ginkgolides and bilobalide in Ginkgo biloba was reported in plants as well as in plant cell cultures. Several hundred plants cultivated under controlled conditions in the field have been analyzed for their terpene production over many years. Cross-pollination experiments were performed with mature trees and the terpene content of the progeny was analyzed. The age of the tree is the main factor influencing the terpene content of the leaves as the level always decreases dramatically between young and old trees. 80 cell culture strains have been established and ginkgolides analyzed by GC/MS. These cell cultures reveal very low amounts of terpenes (1 microgram g-1 D.W. or less). On the contrary, isolated in vitro root cultures accumulate terpenes at the same concentration as the young plant leaves (4 mg g-1 D.W.). Attempts to obtain rapid growing roots or even hairy-roots did not succeed but the possibility to transform Ginkgo cell strains has been demonstrated.

In order to define better the endosperm protein content of commercial coffee species Coffea arabica (Arabica) and C. canephora(Robusta), the principal storage protein of coffee grains has been analysed by 2-dimensional electrophoresis (2DE) and amino acid microsequencing. The most abundant polypeptide spots observed on mature coffee grain 2DE profiles were found to be subunits of the same protein, which exists as multiple isoforms with varying pIs. Strong sequence similarity was found to the 11S family of plant storage proteins. The structure is typical of the 11S type, which occurs as a precursor of 55 kDa, and is observed under denaturing and reducing conditions on 2DE profiles in the form of cleavage products at approximately 20 kDa (b arms) and 32 kDa (a arms). Differences between Arabica and Robusta 2DE profiles indicate a secondary 11S protein family in some varieties of the latter. The existence of multiple pI forms may indicate that a multigene family encodes for these proteins. We estimate that the protein accounts for approximately 45 % of total grain protein. A cloned full-length cDNA of 1 706 bp coding for one of the isoforms is described and discussed in relation to other coffee storage protein sequences. © Elsevier, Paris Coffea arabica / Coffea canephora / 11S storage protein / endosperm / full-length cDNA / storage protein / two dimensional electrophoresis 2DE, immobilized pH gradient 2-dimensional electrophoresis / CHAPS, 3-(3(cholamidopropyl)-dimethylammonio)-1- propanesulfonate / DTT, dithiothreitol / SDS-PAGE, SDS polyacrylamide gel electrophoresis / TCA, trichloroacetic acid / WAF, weeks after flowering

The protein content of cacao (Theobroma cacao) beans was studied by quantitative two-dimensional electrophoresis (2-DE) and by measuring total and protein nitrogen by the Kjeldahl method from the unfermented stage up to the seventh day of fermentation. The major trends in evolution of protein concentration were followed by measuring the intensities of some of the most abundant bean polypeptides. During fermentation a biphasic proteolytic process was observed. Protein degradation was detected after two days of fermentation, and was most pronounced during the third day. Following the initial phase of degradation until the end of fermentation, very little further protein degradation was observed, possibly due to the release of polyphenolic compounds and their subsequent complexing with the remaining proteins. Total protein estimated by the Kjeldahl method decreased to 57% of the initial value during the fermentation period. The process of degradation is selective, with some polypeptides resisting more than others. The evolution of total protein and non-protein nitrogen content is also described. © 1999 Society of Chemical Industry

High concentrations of sucrose are often used to cryopreserve regenerable plant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80 % of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was restored by addition of 1 μM abscisic acid in the pretreatment medium. Using these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in relation to acquisition of cryopreservation tolerance. Only boiling-stable protein patterns showed some changes: five polypeptides accumulated in 0.4 M sucrose-pretreated embryos or in embryos pretreated by media containing abscisic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides was highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance. Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos appeared more active than those extracted from control embryos in protecting in vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These results suggest that these polypeptides may be involved in a mechanism of protection needed for cell survival during freezing stress.

Advanced backcross QTL (AB-QTL) analysis is a new strategy for studying the effect of unadapted alleles on the agronomic performance of elite cultivated lines. In this paper we report results from the application of the AB-QTL strategy to cultivated tomato using the wild species Lycopersicon hirsutum LA1777 as the donor parent. RFLP genomic fingerprints were determined for 315 BC2 plants and phenotypic data were collected for 19 agronomic traits from approximately 200 derived BC3 lines which were grown in replicated field trials in three locations worldwide. Between 1 and 12 significant QTLs were identified for each of the 19 traits evaluated, with a total of 121 QTLs identified for all traits. For 25 of the QTLs (20%) corresponding to 12 traits (60%), the L. hirsutum allele was associated with an improvement of the trait from a horticultural perspective, despite the fact that L. hirsutum is overall phenotypically inferior to the elite parent. For example, L. hirsutum has fruit that remains green when ripe (lack of red pigment) yet alleles were found in this species that significantly increase red color when transferred into cultivated tomatoes. Wild alleles were also associated with increases in total yield and soluble solids (up to 15%) and brix×red yield (up to 41%). These results support the idea that one cannot predict the genetic potential of exotic germplasm based on phenotype alone and that marker-based methods, such as the AB-QTL strategy, should be applied to fully exploit exotic germplasm.

Improved-processing tomato lines were produced by the molecular breeding strategy of advanced backcross QTL (AB-QTL) analysis. These near-isogenic lines (NILs) contained unique introgressions of wild alleles originating from two donor wild species, Lycopersicon hirsutum (LA1777) and L. pimpinellifolium (LA1589). Wild alleles targeted for trait improvement were selected on the basis of previously published replicated QTL data obtained from advanced backcross populations for a battery of important agronomic traits. Twenty three NILs were developed for 15 genomic regions which were predicted to contain 25 quantitative trait factors for the improvement of seven agronomic traits: total yield, red yield, soluble solids, brix×red yield, viscosity, fruit color, and fruit firmness. An evaluation of the agronomic performance of the NILs in five locations worldwide revealed that 22 out of the 25 (88%) quantitative factors showed the phenotypic improvement predicted by QTL analysis of the BC3 populations, as NILs in at least one location. Per-location gains over the elite control ranged from 9% to 59% for brix×red yield; 14% to 33% for fruit color; 17% to 34% for fruit firmness; 6% to 22% for soluble-solids content; 7% to 22% for viscosity; 15% to 48% for red yield, and 20% to 28% for total yield. The inheritance of QTLs, the implementation of the AB-QTL methodology for characterizing unadapted germplasm and the applicability of this method to other crops are discussed.

A pair of processing tomato (Lycopersicon esculentum Mill.) lines, nearly isogenic for the Tm-2a gene for resistance to tobacco mosaic virus, were grown in replicated trials under commercial production conditions in five locations worldwide. The lines were evaluated for 17 processing traits including fruit yield, size, soluble solids concentration, color, firmness, and viscosity. Eight of those traits differed significantly among the nearly-isogenic lines (NILs). Most notably, the NIL heterozygous for Tm-2a yielded, on average, 16% more than the NIL homozygous for the susceptible allele and 33% more than the NIL homozygous for Tm-2a. Viscosity was lower in the heterozygous NIL and the homozygous and heterozygous resistant NILs had softer fruit with larger stem scars compared to the homozygous susceptible NIL. These results indicate the presence of the Tm-2a locus affects many traits of importance for processing tomatoes and may be used, in the heterozygous state, to significantly increase yield. Whether the observed effects are due to the Tm-2a gene itself or genes associated via linkage drag could not be determined.

A BC3 population previously developed from a backcross of Lycopersicon peruvianum, a wild relative of tomato, into the cultivated variety L. esculentum was analyzed for QTLs. Approximately 200 BC4 families were scored for 35 traits in four locations worldwide. One hundred and sixty-six QTLs were detected for 29 of those traits. For more than half of those 29 traits at least 1 QTL was detected for which the presence of the wild allele was associated with an agronomically beneficial effect despite the inferior phenotype of the wild parent. Eight QTLs for fruit weight could be followed through the BC2, BC3, and BC4, generations, supporting the authenticity of these QTLs. Comparisons were made between the QTLs found in this study and those found in studies involving two other wild species; the results showed that while some of these QTLs can be presumed to be allelic, most of the QTLs detected in this study are ones not previously discovered.

Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection.

The quality of Ecuadorian cacao is presently threatened by the introduction of hybrid material. An estimation of genetic diversity in Ecuador is required in order to avoid the loss of fine flavored cocoa. Genetic variability amongst 60 Ecuadorian genotypes of Theobroma cacao has been evaluated using molecular and phenotypic markers. The two distance matrices derived from the molecular and phenotypic data were found to be correlated (R2 = 0.5). Dynamic clustering analyses classified the genotypes in two or three groups depending on the markers used. The genotypes coming from Sebasti{an Arteaga (SA) and Balao Chico (BCH) plantations appeared related to each other suggesting a common genetic origin. They also may be considered as a distinct group with high RFLP homozygosity. The EETP (Estaci}on Experimental Tropical Pichilingue of Ecuador) collection was comprised of more variable genotypes possessing variable heterozygosity levels. The low heterozygous genotypes may be genetically related to SA and BCH trees, whereas the higher heterozygous genotypes may have resulted from hybridizations between original Nacional material of Ecuador and genotypes imported from Trinidad at the beginning of the century. Thus genetic introgression may have occurred giving rise to a range of variation between Nacional and hybrid forms.

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 1∶1 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC1 generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P

Approximately 170 BC2 plants from a cross between an elite processing inbred (recurrent parent) and the wild species Lycopersicon pimpinellifolium LA1589 (donor parent) were analyzed with segregating molecular markers covering the entire tomato genome. Marker data were used to identify QTLs controlling a battery of horticultural traits measured on BC2F1 and BC3 families derived from the BC2 individuals. Despite its overall inferior appearance, L. pimpinellifolium was shown to possess QTL alleles capable of enhancing most traits important in processing tomato production. QTL-NIL lines, containing specific QTLs modifying fruit size and shape, were subsequently constructed and shown to display the transgressive phenotypes predicted from the original BC2 QTL analysis. The potential of exploiting unadapted and wild germplasm via advanced backcross QTL analysis for the enhancement of elite crop varieties is discussed.

Tomato (Solanum lycopersicum) and coffee (Coffea canephora) belong to the sister families Solanaceae and Rubiaceae, respectively. We report herein the mapping of a common set of 257 Conserved Ortholog Set II genes in the genomes of both species. The mapped markers are well distributed across both genomes allowing the first syntenic comparison between species from these two families. The majority (75%) of the synteny blocks are short (

The presence of totipotent and non-totipotent cells in embryogenic carrot cell suspension cultures was examined by cloning of cell microclusters. Forty clones were isolated and the distribution of their embryogenic potential was studied. Nonembryogenic, weakly and highly embryogenic cell lines were selected. After one year of subculture a second cloning round showed that the highly embryogenic and the non-embryogenic cell lines were homogenous and stable. A measurement of ploidy levels of clones by flow cytometry showed that the embryogenic clones were all diploid whereas the non-embryogenic were diploid or tetraploid. Hence, for our strain, there was a strict relationship between the tetraploid state and the inability to produce somatic embryos.

Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-L-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2(1)2(1)2(1) for XMT and C222(1) for DXMT. X-ray diffraction to 2.8 A for XMT and to 2.5 A for DXMT have been collected on beamline ID23-1 at the ESRF.

We report herein the application of a set of algorithms to identify a large number (2869) of single-copy orthologs (COSII), which are shared by most, if not all, euasterid plant species as well as the model species Arabidopsis. Alignments of the orthologous sequences across multiple species enabled the design of “universal PCR primers,” which can be used to amplify the corresponding orthologs from a broad range of taxa, including those lacking any sequence databases. Functional annotation revealed that these conserved, single-copy orthologs encode a higher-than-expected frequency of proteins transported and utilized in organelles and a paucity of proteins associated with cell walls, protein kinases, transcription factors, and signal transduction. The enabling power of this new ortholog resource was demonstrated in phylogenetic studies, as well as in comparative mapping across the plant families tomato (family Solanaceae) and coffee (family Rubiaceae). The combined results of these studies provide compelling evidence that (1) the ancestral species that gave rise to the core euasterid families Solanaceae and Rubiaceae had a basic chromosome number of x = 11 or 12.2) No whole-genome duplication event (i.e., polyploidization) occurred immediately prior to or after the radiation of either Solanaceae or Rubiaceae as has been recently suggested.

Somatic embryos of Daucus carota L. and Coffea canephora L. (var. Robusta) were dehydrated under a 43 % relative humidity then placed in the hopper of a precision seeding system used in the transplant industry. The seeder was adjusted to distribute the embryos onto horticultural trays, each one containing 240 cells filled with soil. As a preliminary result, 72 % and 88 % of the individual cells received a single embryo, in Daucus and Coffea respectively. The embryo-to-plantlet conversion rate was not affected either by the vibration of the hopper or by the nozzles. In carrot 66 % of the embryos germinated after the use of the seeding system (62% germination for the control). Sorting methods traditionally used for the seeds (e.g. air column, vibrating table) can also be used. Such an approach, based on desiccation as a key step, has the potential for a complete automation of the large-scale handling and delivery of somatic embryos.

[alpha]-D-Galactosidase ([alpha]-Gal; EC 3.2.1.22) is one of three principal enzymes involved in the modification or degradation of plant cell wall galactomannans. In the present paper it is shown that [alpha]-galactosidase activities in field-grown coffee beans are variable amongst cultivars of the two species investigated (Coffea arabica and C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beans from greenhouse-cultivated C. arabica as a model, we showed that [alpha]-Gal activity was undetectable in the bean perispem tissue, but increased gradually during the endosperm development, to reach a peak at approximately 30 weeks after flowering (WAF) which coincided with the hardening of the endosperm. [alpha]-Gal-specific transcripts detected at 22 and 27 WAF accompanied the peak of [alpha]-Gal activity, but were reduced to be undetectable in mature beans at 30 WAF, while [alpha]-Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crude protein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoforms accumulated in a linear fashion throughout grain maturation. [alpha]-Gal activity was also observed to increase to high levels during in vitro germination of coffee beans suggesting an important function of this enzyme in this process. [alpha]-Gal cDNA sequences from Arabica and Robusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysis suggests that the enzyme was encoded by at least two genes in C. arabica that could explain the existence of the two isoforms identified in 2-DE profiles.

Two bacterial strains, Serratia liquefaciens and Pseudomonas maltophilia , were isolated from orris rhizomes ( Iris pallida ) which were cultu

A methodology for flavor and composition assessment of processed tomato juice samples was developed using a wide range of commercial processing tomato varieties (Lycopersicon esculentum) grown in Spain and the United States. Fruitiness intensity was found by a trained panel to best describe overall tomato flavor. For two consecutive years, fruitiness intensity was significantly dependent on growing location and variety, and it was consistently linked to increased levels of glucose and reducing sugars and decreased glutamic acid content. Using the same procedure on a population of 176 breeding lines derived from the wild species of Lycopersicon pimpinellifolium, it was shown that tomato fruitiness intensity was significantly correlated to reducing sugars/glutamic acid ratio and glucose and glutamic acid contents. The definition of markers for tomato flavor of processed juice can provide the tomato breeder and processor with reliable analytical tools that can be applied in a straightforward way for the identification of raw materials that can be processed into juice with predictably high or low fruitiness.

In this paper, we present the complete nucleotide sequence of the csp1 gene from Coffea arabica coding for the 11S-globulin seed storage protein. To investigate the sequences responsible for the regulated expression of this seed-specific coffee storage protein gene, about 1 kb of the 5'-upstream region from the csp1 gene was isolated using inverse polymerase chain reaction (IPCR) and then sequenced. Several DNA boxes were found in this coffee sequence that had similarity to those previously identified as being essential for grain (endosperm) specific expression in other plants. To study the ability of this sequence to direct grain-specific expression, the whole fragment, as well as a series of 5' deletions, was fused to the reporter gene β-glucuronidase (uidA) and analysed in transgenic Nicotiana tabacum plants. GUS measurements showed that all the deletions of the csp1 promoter directed the expression of the reporter gene in tobacco grain but not in the other tissues examined. GUS activities also revealed that the csp1 promoter constructs function as very strong promoters by comparison to the strength of the cauliflower mosaic virus (CaMV) 35S promoter. Therefore, this 11S promoter could represent a useful tool to change the expression of targeted genes in the grain of transgenic coffee plants.

Les essais de transformation génétique ont été conduits sur des embryons somatiques de Coffea canephora Pierre, Coffea arabica L. et de l'hybride interspécifique Arabusta, en utilisant Agrobacterium rhizogenes A4 ou Agrobacterium tumefaciens LBA4404. Deux plasmides ont été utilisés, portant l'un les gènes uidA (GUS) et NPTII ou l'autre, le gène csr 1-1 conférant la résistance à un herbicide (chlorsufuron) et le gène cryIA(c) codant l'endotoxine de Bacillus thuringiensis conférant la résistance à la mineuse des feuilles. Selon l'espèce Agrobacterium, on obtient des cals transformés, des racines ou directement des embryons somatiques qui peuvent être regénérés sur milieu sélectif ou non. Les embryons somatiques secondaires sont regénérés et se développent en plantules. L'intégration des gènes NPTII, uidA ou cryIA(c) est démontrée par PCR et le test d'activité de la bêta-glucuronidase a montré l'expression du gène GUS. Plusieurs de ces plantules transformées sont transférées en serre er apèrs floraison, il sera possible de suivre la transmission de l'ADN étranger dans la descendance. Les analyses "southern blot" ont confirmé l'intégration des gènes spécifiques uidA et Bt dans le génome des plantules. Les spécificités différentes ainsi que les limites des deux souches Agrobacterium rhizogenes et A. tumefaciens sont discutées. Des travaux sont en cours pour transformer Coffea arabica

Une carte de liaison génétique du caféier (#Coffea canephora$) totalisant 1402 cM a été établie sur la base d'une population de plantes haploïde-doublées. Des marqueurs RFLP comme des marqueurs utilisant la PCR (RAPD) ont permis la construction de 15 groupes de liaison. Des clones de DNA génomiques de caféier ainsi que des DNAc ont été utilisés. Au total, 47 RFLP et 100 RAPD sont positionnés sur la carte de liaison. Un niveau relativement faible de polymorphisme est observé. 81 % des marqueurs RAPD et 85 % des marqueurs RFLP présentent une ségrégation significativement non différente (P < 0.01) du ration 1:1. La disponibilité d'une carte de liaison moléculaire du caféier offre de nombreuses perspectives tant pour des études génétiques que pour l'amélioration des espèces de caféiers qui constituent une culture de grande importance économique. (Résumé d'auteur)

Irones are violet-scented ketonic compounds contained in the rhizome of certain species of iris. As cultivation of the iris tends to decrease, a selection program has been initiated to find the best performing clones in terms of growth and yield. Parallel to this selection, in vitro regeneration studies have been carried out in order to multiply interesting clones. A method of rapid multiplication by somatic embryogenesis associated with multibudding was developed. Callus was obtained from leaf bases, flower pieces or rhizome apices; the best explants were flower pieces. The induction media used to obtain embryogenic callus were Murashige & Skoog (1962) media. Assays with adding of proline in these media have showed that it could double the yield of embryogenic callus. The embryogenic expression medium was the Knudson's orchid agar (Knudson 1946) medium. Conformity of the plants obtained was checked by comparing their chemotypes with those of the mother plants.

Rhizomes of Iris species used for perfume production do not contain scented irones immediately after harvest, precluding early selection of potentially high-producing genotypes. A recently developed technique involving a bacterial treatment (Rahnella aquatilis Izard, Gavini, Trinel, and Leclerc) of fresh rhizomes shortened the maturation time from 3 years to a few days. Variability in irone content among freshly harvested Iris species (76 clones) was evaluated, and three high-producing clones of Iris pallida Lam. were selected. Significant variability among clones was observed for irone content, growth, and yield.

Biotransformation of tryptamine to serotonin by cell cultures of Peganum harmala was performed in 250 ml conical flaskes or 10 l bioreactor with high serotonin yields (2.5 g/l of culture and 20% of the biomass dry weight). The specific biotransformation rate reached more than 100 mg/g dry weight/day. The influence of pH, auxin concentration, and temperature were studied. Phenobarbital stimulated the reaction. Immobilized cells showed a lower biotransformation rate than cell suspensions. The stability of the cell line after cryostorage (growth and biotransformation capability) was established.

ResumeLa conservation a long terme de cultures de cellules vegetales productrices de metabolites a deux objectifs principaux: 1 Eviter des modifications de souches pouvant etre dues a une evolution du degre de ploidie, a des mutations ou a des variations epigenetiques.2 Diminuer les risques de contaminations et alleger les charges de travail que represente le repiquage regulier des souches. Deux voies sont envisageables: ralentir ou bien arreter la croissance des tissus.Les techniques de croissance ralentie sont principalement representees par le stockage des tissus a des temperatures relativement basses non gelantes (de 4°C a 15°C) et par l'hypoxie sous une couche d'huile de paraffine.L'arret de la croissance des tissus et du metabolisme cellulaire dans son ensemble est essentiellement obtenu par la conservation des cellules dans l'azote liquide a—196°C.Si ces trois techniques se sont revelees efficaces pour la conservation du phenotype de plusieurs souches, aucune a ce jour n'est cependant «universelle»...

α,β-Unsaturated ketones were reduced by various plant cells grown under different cultural conditions. The stereochemistry of the reduction of (-)-carvone by Medicago sativa was found to be identical to that obtained with other organisms.

Plant tissue cultures from various species were stored at low temperatures or under mineral oil overlay for 4 to 6 months without subcultures. After transfer to normal culture conditions, it was checked, with 3 strains, that growth characteristics and secondary metabolite production were preserved. The storage with a mineral oil overlay (easy to run and economical method) could be a possible alternative to cryogenic or low temperature storage for a large number of strains.

The ability of plant cell cultures to produce useful compounds (pharmaceutical, food additives, cosmetics) with large-scale production has been investigated by numerous authors from scientific and economical points of view. (For reviews see: Zenk and Deus 1982, Kurz and Constabel 1983, Rosevear 1984. Yamada and Hashimoto 1984, Brodelius 1985, Dougall 1985, Sahai and Knuth 1985, Collinge 1986, Fowler 1986a, Fujita and Tabata 1986, Mac Laren 1986.)