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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 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.

The major disadvantage of a liquid medium is hyperhydricity, which is a severe physiological disorder. So we considered that to compensate for this problem it would be necessary to expose the plant to the liquid medium intermittently rather than continuously. For this the bioreactors previously developed are not suitable as they are mainly adapted to bacterial culture and do not take into account the specific requirements of plant cells and tissues, such as sensitivity to shear forces, mechanical damages or foam formation in bubble aerated bioreactors.

So temporary immersion systems for plant micropropagation have been described and grouped into 4 categories according to operation: i) tilting and rocker machines, ii) complete immersion of plant material and renewal of nutrient medium, iii) partial immersion and a liquid nutrient renewal mechanism, iiii) 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 critical parameter for system efficiency. Optimizing the volume of nutrient medium and the volume of container also substancially improves efficiency, especially for shoot proliferation. Temporary immersion also generally improves plant tissue quality. It results in increased shoot vigour and quantity of morphologically normal somatic embryos. Hyperhydricity, which seriously affects cultures in liquid medium, is eliminated with these culture systems or controlled by adjusting the immersion times.

Plant material propagated by temporary immersion performs better during the acclimatization phase than material obtained on semi-solid or liquid media. Successful regeneration of microtubers and somatic embryos produced in temporary immersion bioreactors after direct sowing on soil has been demonstrated. As was predicted, when using liquid medium for micropropagation, several investigations have confirmed large gains in efficiency from temporary immersion. The parameters most involved in reducing production costs are, firstly a large reduction in labour, followed by a reduction in shelving area requirement and the number of containers used, along with better biological yields. Scaling up embryogenesis and shoot proliferation procedures involving temporary immersion systems are now taking place, in order to commercialize this process. To improve this system as well in research as in commercial production, CIRAD has developed a new simple and specific apparatus for plant tissue culture using temporary immersion in liquid medium.

Temporary immersion systems (TIS) have been described for in vitro multiplication of a wide range of tropical crops. Laboratory protocols are available for shoot multiplication, somatic embryo and microtuber production. Seven species are now commercially propagated by this culture technique ( sp., sp., ) with different regeneration pathways and a variety of TIS designs. Beside the development of methods for producing somatic embryos in TIS, shoot multiplication protocols are the most applied from the commercial point of view. RITA® proved to be a suitable tool for research and laboratory scale, but for commercial application larger vessels are frequently used. Most important tropical species are commercially propagated in TIS using twin flasks ranging from 5–10 litres.

Production strategies for plant propagation in TIS, either by organogenesis or somatic embryogenesis are discussed and examples are given to illustrate the different possibilities for TIS integration in propagation of . and for shoot multiplication and microtuber production. For sugarcane ( sp.) somatic embryo production in bioreactors, embryo germination in TIS and field performance of regenerated plants are described.

Temporary immersion culture (TIS) offers several advantages over solid medium for banana shoot multiplication, e.g. TIS results in an increase in the multiplication rate and improves the quality of the plantlets. For a commercial application of this technique large vessels are required. When using 10-litre culture vessels an excessive growth of the shoots (leaves and pseudostem) was obtained, which limited the final number of shoots to be produced per flask and reduced the production capacity in the growth room. Labor costs also increased, since handling is more difficult when dividing and subculturing large shoots during the multiplication stage. The effect of growth retardants ancymidol (ANC), paclobutrazol (PBZ) and daminozide (DAM) in liquid shake cultures and TIS was investigated in order to reduce the size of the shoots and allow a better use of the space inside the culture vessel. In liquid shake cultures ANC and PBZ, independently of the tested concentrations, promoted bud cluster formation with reduced size and compact shape. Shoots multiplied with ANC or PBZ (2.5 mg l) after five subcultures, recovered their normal morphology after transfer to a hormone-free medium without growth retardants. However, during the acclimatization stage, plants multiplied in ANC (2.5 mg l) containing media showed reduced height in comparison with control plants and plants multiplied in PBZ and DAM containing medium. The application of PBZ and ANC in TIS (1-litre flasks) stimulated bud proliferation. Both compounds were also effective in controlling the excessive growth of the shoots and in inducing the formation of compact bud clusters. Shoots multiplied in TIS in presence of PBZ (2.5 mg l) were successfully transferred to semisolid or liquid rooting media in traditional culture vessels or TIS. The developed protocol was further scaled up in 10-litre TIS vessels.

Germination of somatic embryos and development to plants is recognized as one of the most critical stages in the process of plant propagation via somatic embryogenesis. The objective of the study was to investigate the germination of somatic embryos of Psidium guajava cv. Cuban Red Dwarf EEA 18–40 in the RITA® system and on semisolid medium. Somatic embryos were obtained from immature zygotic embryos which were cultured on the major salts of MS medium at half strength, supplemented with 400 mg l L-glutamine, 100 mg l ascorbic acid, 60 g l sucrose and 1 mg l 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos at the heart and torpedo stages were transferred for germination into RITA® vessels containing liquid half strength MS medium of the major salts supplemented with 0.25 mg l 6-benzylaminopurine (6-BAP), 10 µg l Biobras-6 (brassinosteroid analogue) and 20 g l sucrose or to semiliquid medium of the same composition (solidified with 2.5 g l Gellum Gum, Spectrum®) in 250 ml glass vessels. The germination percentage, fresh weight and number of somatic embryos with complete germination were determined. After 10 weeks of culture the highest germination percentage (91%) and fresh weight (1.22 g) were achieved in the temporary immersion system, being statistically superior to those obtained from semisolid culture medium (81.79% and 1.03 g respectively).

A temporary immersion system consisting of a series of five-litre twin glass vessels was successfully used for adventitious shoot multiplication and rooting of . Highest shoot multiplication rate of 25.4 was achieved after twelve weeks when eight immersions per day, of ten minutes each, were used. Fresh medium containing 0.5 mg l TDZ was supplemented every two weeks. The different periods of TDZ exposure affect the multiplication rate, fresh weight rate and shoot size. Seven-week culture on TDZ-containing medium, followed by five weeks on TDZ-free medium, resulted in 18 % of shoots smaller than 1 cm, 56 % were of 1–3 cm and 26 % exceeded 3 cm in length, respectively. Small shoots are suitable as inoculum for the next multiplication cycle, whereas shoots larger than 1 cm can be rooted on cytokinin-free medium. Highest percentage of rooted shoots (93.8 %) and highest root number (3.7 roots per shoot) were achieved in 1.0 mg l IAA — containing medium after exposure to six immersions per day, ten minutes each. The mean survival rate of plants rooted in TIS was 94 % under standard greenhouse conditions.

Temporary immersion, solid and liquid culture methods were compared to evaluate propagation of three species and a rootstock. The growth of plants cultured at 30 and 60 minutes of immersion per day was compared to that in solid and stationary liquid conditions. After 60 days, multiplication rate, water, chlorophyll, carotenoid and fructose contents were evaluated. Stationary liquid culture negatively affected plant growth by reducing multiplication rate, chlorophyll and fructose contents and by inducing hyperhydricity and necrosis. The multiplication rate did not differ on solid medium and in temporary immersion, but hyperhydricity was present to a certain degree on solid medium but never in temporary immersion. Moreover chlorophylls, carotenoids and fructose, in the form of sucrose, increased in temporary immersion, particularly at the optimal immersion time. The accumulation of sucrose and the increase of photosynthetic pigment content could be due to a partial restoration of autotrophic activity.

The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 growth in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentrations of cytokinins during both shoot multiplication and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4 µmol BAP and 0.5 µmol IBA during the shoot multiplication phase and 1.1 µmol BAP and 0.25 µmol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher multiplication rate and longer shoots than 8 times per day. The explant size of 0.5 cm or 1 cm resulted in a significantly higher shoot production rate compared with that of 1.5 cm, but shoot length and hyperhydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90 % rooting and the rooted plantlets acclimatised well in the greenhouse.

A novel temporary immersion system (TIS), designed at the laboratory of VITRO HELLAS S.A., was used for the liquid culture of olive microshoots during their proliferation phase. The results were compared with those obtained in agar solidified medium, which was used as control, and with other bioreactor systems including the LifeReactor ©, liquid culture in Erlenmeyer flasks under agitation and liquid culture on filter paper bridges. After 30 days of culture, results derived from the novel TIS (1.93 new microshoots per explant, 0.95 cm shoot length) were statistically the same as the control (1.75 microshoots per explant, 1.22 cm shoot length respectively). Equally high results were obtained with the agitated Erlenmeyer flasks, while LifeReactor © gave the poorest ones (0.35 new microshoots per explant, 0.40 cm shoot length). Cultures on filter paper bridges developed large masses of callus at the explant bases. However, all the liquid cultures suffered from hyperhydricity, the most serious was observed in the Erlenmeyer flasks and the least in the novel TIS. In order to reduce hyperhydricity, the TIS was combined with cultures on agar solidified medium, which eliminated the problem but shoot proliferation was also reduced. It seems that the novel TIS provides a promising device, although some technical problems need to be overcome for large scale production.

Nodules of maintained in liquid Murashige and Skoog (MS) medium with 20 µmol BA in the dark were subjected to different treatments under continuous light for shoot regeneration. A high regeneration rate without hyperhydration of the shoots was observed on semisolid basal MS medium with 1 % sucrose. The use of liquid MS medium (1 % sucrose, no growth regulators) resulted in a significantly lower amount of shoots per gramme of nodules under both submerged and temporary immersion (TI) conditions. Shoot hyperhydration was lowest in a TI system with one 5 min immersion every 24 hours. When compared on a per container base, large amounts of shoots could be produced in the TI system with less labour input than in the system with semisolid medium.

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce () vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an -like gene () and a homeobox gene (), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of and leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.