A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.     

Somatic embryogenesis and plant regeneration in avocado (Persea americana Mill.): influence of embryogenic culture type

Embryogenic cell lines of Persea americana were classified as SE- or PEM-type based on their morphology in maintenance medium. PEM-type cultures consisted of proembryonic masses (PEMs) with occasional development of proembryos and early-stage somatic embryos, while SE-type cultures consisted of somatic embryos from globular to cotyledonary stages with only a low frequency of proembryos and PEMs. Moreover, histological analysis revealed signs of cellular organisation, with a higher proportion of small cells in peripheral regions of the callus mass in the SE-type, but not in PEM-type cell lines. The effects of time in suspension, inoculum density and maintenance medium on the ability of avocado cell lines to produce mature somatic embryos were evaluated. Morphological differences observed during proliferation were correlated to the subsequent capacity of cultures to develop mature somatic embryos. The period of time in suspension and inoculum density were critical factors that influenced the ability of cultures to undergo maturation. Optimal conditions differed between SE-type cultures and PEM-type cultures. In SE-type cultures, the highest number of somatic embryos was observed in cultures grown in suspension for 9?days and 0.4?g inoculum; in PEM-type cultures a slight increase in mature somatic embryos production could be observed with 4?g inoculum size and 14?days in suspension. The presence of agar in the maintenance medium of SE-type cultures was essential for the maturation of somatic embryos.     

Production of Norway spruce embryos in a temporary immersion system (TIS)

Somatic embryogenesis has already been used for Norway spruce (Picea abies (L.) Karst) embling production on a laboratory scale, but automation is needed to increase efficiency and reduce costs. One option to scale up production is mass production in bioreactors. In a series of experiments, a pro-embryogenic mass was propagated using Plantform temporary immersion system bioreactors, and the effect of different aeration cycles, support pad materials, and post-maturation treatments (rinsing and desiccation) on the embryo yield and embling survival after 4 to 6 mo in a greenhouse was tested. Three genotypes were used to test each treatment. The best aeration frequency was 20 min every 4 h, while a lower or higher frequency did not generally improve embryo production. Filter paper on plastic netting was the best support pad material in terms of usability and embryo production (varying from 177 ± 20 to 696 ± 109 per g pro-embryogenic mass). The separation of the embryos from the undeveloped cell mass by rinsing with sterile water resulted in reduced survival of the emblings. Desiccation treatment on nested plates with the embryos on the inner plate with or without filter paper improved their survival. Bioreactors were laborious to prepare, load, and clean. Improvements in embryo production can be achieved by optimizing the process, but bioreactors based on the requirements of somatic embryogenesis are needed to enable their use in the mass production of Norway spruce emblings.

     

Possible effect from shear stress on maturation of somatic embryos of Norway spruce (Picea abies)

Somatic embryogenesis is the only method with the potential for industrial scale clonal propagation of conifers. Implementation of the method has so far been hampered by the extensive manual labor required for development of the somatic embryos into plants. The utilization of bioreactors is limited since the somatic embryos will not mature and germinate under liquid culture conditions. The negative effect on mature embryo yields from liquid culture conditions has been previously described. We have described the negative effects of shear stress on the development of early stage somatic embryos (proembryogenic masses; PEMs) at shear stresses of 0.086 and 0.14 N/m². In the present study, additional flow rates were studied to determine the effects of shear stress at lower rates resembling shear stress in a suspension culture flask. The results showed that shear stress at 0.009, 0.014, and 0.029 N/m² inhibited the PEM expansions comparing with the control group without shear stress. This study also provides validation for the cross‐correlation method previously developed to show the effect of shear stress on early stage embryo suspensor cell formation and polarization. Furthermore, shear stress was shown to positively affect the uptake of water into the cells. The results indicate that the plasmolyzing effect from macromolecules added to liquid culture medium to stimulate maturation of the embryos are affected by liquid culture conditions and thus can affect the conversion of PEMs into mature somatic embryos. Bioeng. 2011; 108:1089–1099. © 2010 Wiley Periodicals, Inc.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

The influence of culture vessel head-space volatiles on somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.]

The maturation of somatic embryos of Sitka spruce [Picea sitchensis (Bong.) Carr.] was found to be highly dependent on the method used to seal plastic Petri dishes. Large numbers of well-formed mature embryos developed if dishes were sealed with PVC cling-film (CF) whilst sealing with Parafilm M (PF) greatly reduced the numbers of embryos forming. Inclusion of potassium permanganate oxidation traps, normally used to deplete the atmospheric ethylene, greatly stimulated somatic embryo maturation under PF sealing. Similarly, traps of adsorption agents (Tenax, activated charcoal or soft white paraffin), capable of removing volatiles from the culture vessel head-space, stimulated somatic embryo maturation under PF sealing although to a lesser extent than the oxidation traps. Incorporation of silver nitrate or 2-chloroethylphosphonic acid (ethephon) in the culture medium indicated that ethylene was not the agent supressing somatic embryo maturation under PF sealing.Abbreviations ABA abscisic acid - CF PVC cling-film - PF Parafilm M     

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) 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 (PGRs) 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 Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 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.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Single-cell origin and development of somatic embryos in Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce)

The origin and development of somatic embryos in calli initiated from immature zygotic embryos of Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce) was studied. Immature zygotic embryos cultured on callus induction medium produced two types of white calli that were phenotypically different from one another. The callus that proliferated from the hypocotyl region was white to translucent, glossy, mucilaginous and embryogenic. The callus mass which originated from the radicle end was reddish-white, nonmucilaginous and nonembryogenic. Whole mount preparations of the entire explant with two different types of calli showed the presence of embryogenic cells in the mucilaginous callus mass derived from the hypocotyl region of the zygotic embryo. The origin of somatic embryos in both Norway and white spruce could be traced to single cells of the hypocotyl callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

The Influence of Auxin and Cytokinin on Proliferation and Morphology of Somatic Embryos of Picea abies (L.) Karst

Embryogenic cell-lines of Picea abies were initiated from maturezygotic embryos and cultured on medium containing 2,4-D andBA The cell-lines were categorized into two main groups (solar/polarand undeveloped embryos), based on the morphology of the somaticembryos and their ability to go through a maturation processwhen treated with ABA The cell-lines were transferred to mediacontaining (1) 2,4-D and BA, (2) only BA, (3) only 2,4-D or(4) no growth regulators When cultured on a medium containingboth 2,4-D and BA new somatic embryos were continually formedIn contrast, when they were cultured without one or both ofthe growth regulators no new somatic embryos were formed Solar/polarand undeveloped embryos responded in the same way On a mediumcontaining only BA the somatic embryos already present increasedin size and developed an extremely large embryonic region Ona medium containing only 2,4-D the embryos already present becamedisorganized into loose aggregates When transferred from a mediumcontaining both 2,4-D and BA to one containing ABA, mature somaticembryos developed from the solar/polar type but not from theundeveloped type The ability of the solar/polar somatic embryosto go through a maturation process decreased when they wereprecultured on a medium lacking auxin and was lost when theywere precultured on medium lacking cytokinin The cell-linescontaining undeveloped somatic embryos produced mature somaticembryos in one cell-line out of three that had been culturedon the medium containing only BA Auxin, cytokinin, embryogenic cultures, Norway spruce, somatic embryos, Picea abies (L.) Karst     

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.)

Summary Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.     

Maturation,germination, and conversion of norway spruce (Picea abies L.) somatic embryos to plants

Summary Quantitative data are presented on the efficiency of three stages of plant regeneration from somatic embryos of Norway spruce (Picea abies L.): 1) Maturation, the development of immature embryos to the cotyledonary stage; 2) Germination, primary root growth; and 3) Conversion, plantlet survival and continued growth in nonaxenic conditions. Maturation frequency was calculated relative to the number of immature somatic embryos induced to develop on the basal medium of von Arnold and Eriksson (1981). The average number of immature somatic embryos was 700 per gram of embryogenic callus, on medium supplemented with ABA and IBA (1 μM each). Maturation was the least efficient stage of regeneration; an average of 3% of the embryos induced to develop reached the cotyledonary stage. Mean germination frequencies were improved on treatments which avoided immersion of the radicle in medium solidified with agar. Whereas, 27% of the somatic embryos germinated when radicles were immersed in agar medium, 45% germinated when placed on the surface of the medium, and 56% germinated when cotyledons were immersed in agar medium and the culture vessel inverted. Twenty-nine percent of the somatic embryos germinatedin vitro were converted to plants. Under greenhouse conditions these plants set dormant buds, subsequently survived overwintering (to −5°C), and renewed vegetative growth synchronously with seedlings grown under the same conditions. Our results verified long-term (2 year) growth and development potential of conifer somatic embryo plants.     

Overexpression of HBK3, a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos

In order to investigate the effects of HBK3, a spruce gene member of the class I KNOX family, during somatic embryogenesis, sense (HBK3-S) and antisense (HBK3-A) Norway spruce (Picea abies) lines were generated. Somatic embryos produced from these lines were then analysed at morphological and structural levels. Compared with control, differentiation of immature somatic embryos from pro-embryogenic masses (PEMs) was accelerated in lines overexpressing HBK3 (HBK3-S). Such immature embryos showed enlarged embryogenic heads and were able to produce fully developed cotyledonary embryos at higher frequency. Furthermore, HBK3-S embryos had enlarged shoot apical meristems (SAMs) and enlarged expression pattern of PgAGO, a molecular marker gene specific to meristematic cells. Lines in which HBK3 (HBK3-A) was down-regulated had reduced ability to produce immature somatic embryos from PEMs and were not able to complete the maturation processes. To assess the function of HBK3 in comparison with that of angiosperm KNOX genes, this gene was ectopically expressed in Arabidopsis plants. As observed for spruce, Arabidopsis embryos overexpressing HBK3 had enlarged meristems and enlarged expression pattern of SHOOTMERISTEMLESS, a SAM molecular marker gene. In addition, transformed embryos were able to germinate at a higher rate and the resulting plants showed a variety of phenotypic aberrations, including abnormal leaves and reduced apical dominance. Overall, these data confirm the importance of KNOTTED genes during development and reveal the participation of HBK3 in conifer embryogeny. Furthermore, the results show redundant functions of this gene during embryonic growth of spruce and Arabidopsis, but not during post-embryonic growth.     

Development and germination of American chestnut somatic embryos

American chestnut (Castanea dentata (Marsh.) Borkh.) plants were regenerated from developing ovules through somatic embryogenesis. On an initiation medium containing 18.18 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine (BA), 25 out of 1,576 ovules were induced to form proembryogenic masses (PEMs). These PEMs were cultivated on a development medium for 4 weeks. Individual somatic embryos were then grown on a maturation medium for at least one month, until shoot meristems and radicles were developed. Both development and maturation media consisted of Gamborg's B-5 basal medium, 0.5 μM BA, and 0.5 μM α-naphthaleneacetic acid, but the former contained 20 g l⁻¹ sucrose and the later contained 60 g l⁻¹ sucrose. A range of 86 to 586 embryos per gram PEMs was observed beyond the cotyledonary stage. These embryos then germinated, resulting in plantlets with a 3.3% conversion rate. An additional 6.3% of the mature embryos produced shoots, which could also result in plantlets by rooting of microcuttings. Proembryogenic masses that were established in continuous culture and maintained on initiation medium for 17 months retained regenerability, though the embryo yield decreased over time. Twenty plantlets were acclimatized and grown in potting mix in a greenhouse. The largest 6 were transplanted, along with seedling controls, into a nursery bed in 1997. As of July, 1999, 4 out of the 6 were surviving. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genotypic and media factors affecting stabilization of polyembryogenic cultures in norway spruce (Picea abies (L.) Karst.)

Embryonal-suspensor mass lines (ESM lines) from mature embryos of three open-pollinated families of Norway spruce [Picea abies (L.) Karst.] were stabilized on several culture media varying in nutrient composition. A pattern of relationship between parameters of polyembryogenic culture performance and medium composition was genetically determined. Stabilized ESM lines could be classified into two phenovariants: (i) lines of homogenous ESM composed entirely of somatic proembryos, and (ii) lines of heterogeneous ESM composed of a mixture of nonembryogenic callus and aggregates of somatic proembryos. The formation of one or the other phenovariant was independent of genotype, but strongly determined by the medium composition.Abbreviations ABA abscisic acid - BA 6-benzyladenine - ESM embryonal-suspensor mass - N nitrogen - NAA -naphthyl-1-acetic acid     

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.     

Regeneration of somatic embryos in Theobroma cacao L. in temporary immersion bioreactor and analyses of free amino acids in different tissues

The present study aimed at developing temporary immersion bioreactor techniques for multiplication of cacao somatic embryos. Temporary Immersion System (TIS), i.e. flooding of plant tissue at regular time intervals provides an efficient way to propagate plants. Somatic embryos were regenerated in twin flask bioreactors. The TIS proved to be suitable for mass regeneration of somatic embryos and for their subsequent direct sowing. The number of embryos after 3 months of culture was significantly higher in TIS cultures than in the solid medium variant. TIS also improved embryo development regarding the conversion to torpedo shaped forms. Matured embryos derived from TIS and pre-treated with 6% sucrose were converted into plants after direct sowing. Additionally to the influence of culture conditions on the development of somatic embryogenesis the content and composition of free amino acids were analysed. The content of free amino acids in somatic embryos rose as immersion frequency increased. The endogenous free GABA content in embryogenic callus was significantly higher than in non-embryogenic callus.     

Robust carbohydrate dynamics based on sucrose resynthesis in developing Norway spruce somatic embryos at variable sugar supply

Growth regulators and carbohydrates are key regulatory factors that affect somatic embryogenesis. Carbohydrates serve as energy and carbon sources, osmotica and osmoprotectants and are important signal molecules. Most information about the role of carbohydrates in somatic embryogenesis in Norway spruce has been obtained with embryos grown on semi-solid media. The aim of the present study was to gain a better understanding of the effects of exogenous carbohydrates through modification of medium components (sugars) and physical state (liquid and semi-solid media). Rafts, floating on liquid medium, were used to allow precise manipulation of carbohydrate availability, though it did not result in the highest embryo yields. Our results indicate the following for Norway spruce somatic embryo development: (1) overall carbohydrate dynamics in somatic embryos cultivated on liquid or semi-solid media were similar; (2) the total carbohydrate content, however, was higher in somatic embryos cultivated on liquid media; (3) sucrose was present in somatic embryos even when they matured on sucrose-free media; (4) sucrose content in liquid sucrose-supplemented maturation media decreased sharply during a 1-wk subculture interval; (5) the accumulation of the raffinose family oligosaccharides during desiccation was determined independently of previous sugar supply; and (6) a decrease of sucrose and an increase of hexoses contents accompanied somatic embryo germination.     

Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies)

The morphology of somatic embryos of Norway spruce ( Picea abies ) varies among different cell lines, from less developed somatic embryos with small embryonic regions (group B) to well developed embryos with large embryonic regions (group A). Only well developed somatic embryos will undergo a maturation process after a treatment with ABA and develop into mature somatic embryos, which is required for plant regeneration. We have previously shown that the presence of specific extracellular proteins can be correlated with the morphology of the somatic embryos. In the present study we show that extracellular proteins concentrated from group A cell lines can stimulate group B embryos to develop further and that seed extract can stably convert B embryos into A embryos. The arabinogalactan protein (AGP) fraction of the extracellular proteins and of the seed extract was shown to be an active component for stimulating B embryos to develop further. Furthermore, the amount and type of extracellular AGPs, as detected with β-glucosyl Yariv reagent and monoclonal antibodies, varied among different types of tissues and cell lines. The data show that development of somatic embryos in Norway spruce is associated with particular extracellular AGPs, which have a regulatory function.     

Improving secondary embryogenesis in <Emphasis Type="Italic">Quercus robur</Emphasis>: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA^® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.