Hervé Etienne

Temporary immersion systems in plant micropropagation

Temporary immersion systems for plant micropropagation have been described and grouped into 4 categories according to operation: tilting and rocker machines; complete immersion of plant material and renewal of the nutrient medium; partial immersion and a liquid nutrient renewal mechanism; complete immersion by pneumatic driven transfer of liquid medium and without nutrient medium renewal. The positive effects of temporary immersion on micropropagation are indicated for shoot proliferation and microcuttings, microtuberization and somatic embryogenesis. Immersion time, i.e. duration or frequency, is the most decisive parameter for system efficiency. Optimizing the volume of nutrient medium and the volume of the culture container also substantially improves efficacy, especially for shoot proliferation. Temporary immersion also generally improves plant material quality. It results in increased shoot vigour and in the frequency of morphologically normal somatic embryos. Hyperhydricity, which seriously affects cultures in liquid medium, can be eliminated with these culture systems or controlled by adjusting the immersion times. Plant material propagated by temporary immersion can perform better during the acclimatization phase than material obtained on semi-solid or in liquid media. Successful regeneration of plants, after direct sowing on soil of Solanum tuberosum microtubers and Coffea arabica somatic embryos produced in temporary immersion bioreactors, has been demonstrated. As could be expected when using liquid medium for micropropagation, several estimations confirm large gains in efficacy from temporary immersion. The parameters most involved in reducing production costs include: (1) the drastic reduction in work; (2) reduction in shelving area; (3) reduction in the number of containers used; (4) better biological yields. Scaling-up somatic embryogenesis and shoot proliferation procedures involving temporary immersion systems in order to commercialize this process are now taking place.

Direct sowing of Coffea arabica somatic embryos mass-produced in a bioreactor and regeneration of plants

Abstract The effect of germination conditions on the morphology of Coffea arabica L. somatic embryos mass-produced in a 1-l temporary immersion bioreactor (RITA®) was studied with emphasis on direct sowing in soil. Using germinated embryos, direct sowing resulted in a highly successful conversion of embryos into plants. A culture density above 1600 embryos per 1-l bioreactor positively affected embryo morphology by causing higher embryonic axis elongation (+4–5 mm). At this density, the addition of a high concentration of sucrose (234 mM) 2 weeks before sowing promoted an increase in effective plant conversion in soil (78%) and a vigorous vegetative growth of the resulting plants. Furthermore, direct sowing reduced handling time to 13% and shelving area requirements to 6.3% of the values obtained by conventional acclimatization of plants developed on gel media.

Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

SummaryA culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. 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.

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.

Temporary immersion system: a new concept for use liquid medium in mass propagation

Mass propagation of plants by tissue culture is labour intensive and costly. Gelling agents have many drawbacks: they are not inert medium components and do not enable easy automation for commercial mass propagation. So liquid culture systems are considered to have advantages, e.g. culture conditions are much more uniform, media can be changed easily. The use of liquid medium for in vitro culture has many advantages and has been the subject of many studies over many years. It has also frequently been considered an ideal technique for mass production as it reduces manual labor and facilitates changing the medium composition. Techniques and culture vessels of varying complexity have been developed as a result of studies.

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.

BIOTECHNOLOGICAL APPLICATIONS FOR THE IMPROVEMENT OF COFFEE (COFFEA ARABICA L.)

SummaryThe important advances in coffee biotechnological techniques which have been made particularly during the last 10yr could benefit the coffee breeder in practice and open new perspectives for the development of new varieties. The molecular phylogeny of Coffea species has been established using DNA sequence data. The molecular markers have revealed an extremely reduced genetic diversity in Coffea arabica L. in comparison to C. canephora. However, wild accessions collected in the Ethiopian highlands appeared to constitute a valuable gene reservoir. A complete genetic linkage map of C. canephora was reported and additional ones are being constructed, particularly on C. arabica. The integration of Molecular Assisted Selection in coffee breeding promises to drastically increase the efficiency of breeding programs. Economically important genes of the caffeine biosynthetic pathway or genes encoding for seed storage proteins have been isolated. The high performance already achieved in the in vitro propagation process by somatic embryogenesis offers the possibility to mass propagate superior hybrids in different countries of both C. arabica (selected F1 hybrids) and C. canephora (rootstock variety). Pilot productions by somatic embryogenesis currently permit preparation for commercial application. Somaclonal variation was observed. The percentage of the off-types can vary between 3 and 10% depending on the genotype. Seed cryopreservation enables a routine use for long-term conservation of coffee genetic resources. Transgenic plants have been obtained for the C. arabica and C. canephora cultivated species through Agrobacterium-mediated transformation which constitutes the technique now currently used to transfer directly genes in coffee plants.

Relations between exogenous growth regulators and endogenous indole-3-acetic acid and abscisic acid in the expression of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.)

Abstract Endogenous IAA (indole-3-acetic acid) and ABA contents were measured during the somatic embryogenesis process from Hevea brasiliensis (Mull. Arg.) callus. The decrease in 3,4-D (3,4-dichlorophenoxyacetic acid) and BAP (6-benzylaminopurine) supply in the culture medium and the addition of ABA both stimulated embryogenic induction. But only exogenous ABA sustained embryogenic expression of the callus. The two operations resulted in a decrease in endogenous ABA and an increase in IAA in callus. Low endogenous ABA levels (2–4 nmol/g dry wt.) appear to be necessary for the acquisition and conservation of a embryogenic state. Non-embryogenic calli accumulated high levels of ABA (25–40 pmol/g dry wt.). This accumulation confirm the hypothesis that non-embryogenic Hevea calli were water stressed.

Somatic embryogenesis in rubber ( Hevea brasiliensis Müll. Arg.)

Natural rubber, produced by Hevea brasiliensis (Mull. Arg.) has been known since ancient times and the Mayas and Aztecs used it in their daily life and religious ceremonies (Serier, 1993). In forests, this tropical tree can reach 40 m in height and 5 m in circumference. It is much smaller in plantations, where twice-weekly tapping of the tree bark considerably reduces its growth. Paradoxically, cultivation of this tree is very recent, beginning in Southeast Asia at the turn of the century, i.e., outside its original zone in the Amazonian basin, where “South American Leaf Blight”, caused by the fungus Microcyclus ulei is rife. The normal cultivation zone lies between 20° N and 20° S, apart from plantations in China and northern India (Compagnon, 1986). The areas planted cover around 7 million hectares, primarily in Thailand, Indonesia and Malaysia, but also in China, India, Africa and Latin America.

Callus friability and somatic embryogenesis in Hevea brasiliensis

The influence of plant growth regulators, sucrose, calcium and various macronutrient media on callus friability and somatic embryogenesis was investigated inHevea brasiliensis Müll. Arg. Friable and embryogenic calli were spontaneously formed in two rubber tree clones (PR 107 and RRIM 600) on the Medium for Hevea (MH), with 3,4-dichlorophenoxyacetic acid (3,4-d), kinetin and sucrose, while compact embryogenic calli were enhanced in three other clones (PB 260, PB 235 and GT1). Callus friability was enhanced in clone PB 260 when the concentration of one growth factor (3,4-d or kinetin) was reduced from 4.5 μLM to 0.45 μM during the first culture, or when high sucrose or calcium levels 351 mM and 12 mM, respectively) were maintained during subcultures. The different macronutrient media did not alter callus texture but only use of MH and Murashige and Skoog (MS) media led to somatic embryogenesis. Friable calli obtained by modifying the auxin/cytokinin balance lost their embryogenic potential. In contrast, those obtained on media with high sucrose or calcium concentrations were mainly composed of embryogenic cells embedded in a mucilaginous matrix. Such calli could be of potential interest for establishing embryogenic cell suspension cultures.