Marc Berthouly

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.

High frequency somatic embryogenesis from coffee leaves : Factors influencing embryogenesis, and subsequent proliferation and regeneration in liquid medium

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 ofCoffea 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 μM 2,4-dichlorophenoxyacetic acid, under 3 μmol 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 μm in diameter) at an inoculum density of 5 g 1-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. 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 ofC. 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.

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.

High frequency somatic embryogenesis in Coffea canephora: induction conditions and histological evolution

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 μM 2,4-D, 2.4 μM IBA and 9.8 μM 2iP for 4 weeks then on an induction basal medium with 4.4 μM 2,4-D and 17.8 μM BA for 10 weeks. This calus 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.

Histology of somatic embryogenesis from floral tissues cocoa

Somatic embryogenesis fromTheobroma 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.

A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants

SummaryIn 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 dimensions, 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. Monosaccharides 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.

Transient expression of β-glucuronidase following biolistic delivery of foreign DNA into coffee tissues.

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.

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.

Glufosinate as an efficient inhibitor of callus proliferation in coffee tissue

SummaryTo improve selection of transgenic Coffea spp. tissue after transformation treatments, the effects of the selective agents chlorsulfuron, glufosinate, glyphosate, hygromycin, and kanamycin were studied on callus development from leaf explants (from greenhouse-grown plants and somaplants) and in embryogenic suspension cultures. Studied genotypes were from C. arabica, C. canephora, and the interspecific hybrids Arabusta and Congusta. A culture system based on “direct” somatic embryogenesis from C. canephora leaf explants proved to be more sensitive to selective agents than high frequency somatic embryogenesis from C. arabica or Arabusta leaf explants. With respect to the selective effect, chlorsulfuron and hygromycin provoked strong inhibition and severe necrosis, whereas glyphosate and kanamycin showed variable inhibition. Glufosinate appeared to efficiently inhibit growth of both leaf callus and callus suspensions of all genotypes tested without inducing necrosis. These properties may make the use of glufosinate advantageous in a selective growth system for detection of transformed coffee tissues.

Effect of strictly plant-related factors on the response ofHevea brasiliensis andTheobroma Cacao nodal explants culturedIn vitro

SummaryNodal cuttings (primary nodal explants) were used to initiate microcutting cultures ofHevea brasiliensis andTheobroma cacao. The physiological stage of growth reached by the branches from which the explants were taken had a considerable influence on culture initiation and response. In both species, the buds of the nodal explants taken from dormant branches (stages 11, 12 and 13 inTheobroma; stage D inHevea) revealed greater opening abilityin vitro and shoot elongation than buds taken from branches in a phase of active meristem growth inTheobroma (stages F1 and F2) or during a foliar growth phase inHevea (stage C). InHevea, the branch growth phase effect persisted into the multiplication phase when shoots developedin vitro were used as the source of secondary nodal explants. However, it disappeared during subcultures of primary nodal explants. In both species, the position of the nodes on the branch with respect to the apex also had an effect on culture initiation. The buds of nodes furthest from the apex were more likely to develop into shoots. InHevea, the position of secondary nodal explants obtained from the primary shoot was seen to vary in effect depending on growth regulator applications during primary culture. These results are analyzed in terms of variations in the balance between endogenous growth promoters/inhibitors in the buds, depending on morphogenic stages and the bud position on the branch.