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.     

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

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.     

From callus to embryo: a proteomic view on the development and maturation of somatic embryos in <Emphasis Type="Italic">Cyclamen persicum</Emphasis>

In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.     

Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis

In vitro somatic embryogenesis of Glehnia littoralis Fr. schm. was observed when zygotic embryos were cultured on a medium containing 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (0.01-10 μM), with 1 μM being the optimum. Microscopic observations revealed globular, heart-shaped and torpedo-shaped embryo formations and plantlet regeneration. These somatic embryos seemed to be produced directly from cells of the zygotic embryos used as explants. Of seven types of media tested, Nitsch's medium showed the highest rate of somatic embryogenesis. Somatic embryos developed into normal plantlets and were able to be potted. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of <Emphasis Type="Italic">Hippeastrum</Emphasis> × <Emphasis Type="Italic">chmielii</Emphasis> Chm. – influence of flurprimidol and the culture in solid or liquid medium and in temporary immersion systems

At Weyerhaeuser Company, somatic embryo production in liquid medium and manufactured seed delivery has been developed to reduce labor costs and increase efficiency of mass clonal propagation. We have scaled-up embryonal suspensor masses (ESM) of Douglas-fir in 1-l liquid medium flasks. Over 10,000 somatic embryos have been produced from a single flask. We have cryostored ESM of over 700 genotypes in liquid nitrogen. Somatic embryos of Douglas-fir have also been produced from liquid medium in a bioreactor. Different types of bioreactors are required for embryo multiplication and for cotyledonary embryo development. Over 250,000 Douglas-fir somatic seedlings from a large number of genotypes have been produced for clonal field tests.     

Somatic embryo productions by liquid shake culture of embryogenic calluses in <Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper

The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed into plants.     

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.     

Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

Tissue-specific localization of mRNA for carrot homeobox genes, CHBs, in carrot somatic embryos

We examined spatial and temporal expression patterns of four carrot HD-Zip I homeobox genes in somatic embryos. The mRNAs for CHB3, CHB4 and CHB5 were accumulated preferentially in the innermost cortical cell layers of the embryo axis in the torpedo-shaped embryo. In contrast, the accumulation of CHB6 mRNA was restricted to procambial cells of the heart- and torpedo-shaped embryos. In the embryonic cotyledons and the hypocotyl of the seedlings, all of the mRNAs for the four genes were located in the vascular tissues. These findings indicate that different HD-Zip I homeobox genes may be involved in the differentiation of specific tissues during somatic embryogenesis.     

Effect of desiccation to low moisture content on germination,synchronization of root emergence,and plantlet regeneration of black spruce somatic embryos

A desiccation protocol was developed to evaluate the effect of different levels of desiccation on germination and plantlet regeneration of black spruce somatic embryos. Large desiccation chambers (80 l) with four liters of saturated salt solutions provided constant relative humidities (RH) of 63, 79, 88, and 97% (± 2%). Under these conditions, an embryo mass of 10 mg always dried fast even at 97% RH. In contrast, an embryo mass of 80 mg generated different kinetics of water loss, from fast drying at 63% RH to slow drying at 97% RH. Drying rates similar to those obtained with 80 mg embryos were also generated by combining 40 mg embryos with 40 mg water. The effects of drying rate and embryo MC on germination rate, root elongation, and plantlet regeneration were examined. A fast drying rate to 4–5% embryo MC, obtained under 63% RH, was detrimental to germination and plantlet development. However slower drying rates, obtained under 79–97% RH and generating 7–19% MC in the embryos, gave developmental responses similar to the control. Synchronization of root emergence was improved only for embryos desiccated to approx. 16% MC under 97% RH. The optimal desiccation protocol using large desiccation chamber at 97% RH and a constant embryo mass of 40 mg embryos plus 40 mg water was applied to five genotypes of black spruce. For all genotypes, desiccated embryos gave plantlet regeneration rates similar to the control undesiccated embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of immersion cycles on growth,phenolics content,and antioxidant properties of Castilleja tenuiflora shoots

Castilleja tenuiflora, a species highly valued for its medicinal properties, is threatened in the wild. We evaluated the effects of six different immersion cycles in a temporary immersion bioreactor on C. tenuiflora shoot growth, proliferation rate, phenolics content, flavonoid content, and antioxidant activity. We also evaluated the regeneration capacity of the shoots. The highest proliferation rate (nine shoots per explant) was obtained using an immersion cycle of 5 min every 12 h, and the longest shoots (38.8?±?1.9 mm) were obtained using an immersion cycle of 5 min every 24 h. Shoots obtained from immersion cycles of 30 min every 24 h or 5 min every 24 h showed 100% rooting efficiency. Shoots obtained from immersion cycles of 30 min every 3 h or 30 min every 12 h accumulated H₂O₂, developed abnormal stomata, and showed symptoms of hyperhydricity. These characteristics were associated with a low survival rate (16–80%) when the plants were transferred to potting mix. The shoots from an immersion cycle of 30 min every 24 h showed the highest total phenolics content, which coincided with the highest antioxidant activity in the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) free-radical scavenging assay (161.74?±?10.06 μmol Trolox/g dry weight (DW)). The shoots from an immersion cycle of 5 min every 24 h showed the highest activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay, and those from an immersion cycle of 5 min every 3 h showed the strongest reducing power. These results show that temporary immersion culture represents a reliable and efficient method for in vitro micropropagation of C. tenuiflora.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

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.     

Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr.

Summary Suspension cultures initiated from calluses derived from seedling leaf explants of Acacia sinuata (Lour.) Merr. produced somatic embryos. Embryogenic callus was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.22 μM 6-benzylaminopurine. A high frequency of somatic embryos was induced in MS liquid medium supplemented with 4.52 μM 2,4-D and 10% coconut water. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent development led to the formation of globular, heart, torpedo-shaped and cotyledonary-stage embryos. The conversion of somatic embryos occurred on auxin-free MS medium. Effects of various auxins, cytokinins, carbohydrates and amino acids in enhancing productin, of somatic embryos were studied. MS medium supplemented with 87.64 mM sucrose and 342.46 μM glutamine promoted higher somatic embryo production whereas cytokinin had no effect and led to recallusing of embryos. About 8–10% of embryos converted into plants.     

Temporary immersion systems (RITA®) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production

Somatic embryogenesis in cork oak (Quercus suber L.) is an efficient tool that allows the production of large number of embryos from selected quality and productive trees. Temporary immersion systems (TIS) are an alternative to semi-solid or liquid culture that combine the advantages of liquid culture and avoid the associated problems. Parameters that affect the TIS multiplication efficiency of Q. suber L. embryogenic cultures were evaluated. Immersion frequencies of 1 min every 6 or 4 h increased the fresh weight 3.7 or 7.5-fold compared with an immersion frequency of 1 min every 12 h or cultures on semi-solid medium, respectively. The cellular fate of embryogenic cultures was also affected by the immersion frequency, 1 min every 6 h was the best for mass propagation of proliferative developmental stages (embryogenic calli and embryo clusters) while 1 min every 4 h promoted the formation of single, fully developed cotyledonary embryos. An initial amount of 1.5 g fresh weight of proliferative tissues produced the best results in RITA^® containers while 0.5 g of embryogenic callus was the best for semi-solid cultures.     

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

Summary A 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.     

Improved and synchronized maturation of Norway spruce (<Emphasis Type="Italic">Picea abies</Emphasis> (L.) H.Karst.) somatic embryos in temporary immersion bioreactors

Somatic embryogenesis offers many benefits for clonal propagation in large-scale plant production of conifers. A key rate-limiting step is the conversion from early-stage somatic embryos in pro-embryogenic masses (PEMs) to the maturation stage. Immature embryos in PEMs are present at different developmental stages, where some are unable to respond to the maturation treatment, thus limiting yields of mature embryos. Synchronization of early somatic embryo development in PEMs could greatly improve subsequent yields of mature embryos. A temporary immersion bioreactor designed for Norway spruce (Picea abies (L.) H.Karst.) was used in this study. Through a specific system for dispersion, connected tissue of PEMs, composed of immature embryos grown in liquid medium in the temporary immersion bioreactors or on solid medium as a control, was dispersed and redistributed in a more uniform spatial arrangement. It was demonstrated that development of mature embryos could be significantly stimulated by dispersion, compared to controls, in both medium types. Synchronization of maturation was evaluated by a statistical approach. The present study shows that the yield of mature embryos from dispersed PEMs was three to five times higher than that from non-dispersed controls in three of four cell lines of Norway spruce tested, both in bioreactors and on solid medium.     

Organization of initial stages of somatic embryogenesis in tissue culture of Citrus sinensis cv. Tarocco at the organismal level

Four-step protocol was established for the in vitro regeneration of Citrus sinensis cv. Tarocco somatic embryos that were morphologically similar to small somatic embryos in vivo. The regeneration procedure comprises a mechanical destruction of embryogenic culture to obtain proembryogenic cell masses (PEMs) (step 1) followed by culturing on three different media (steps 2–4). The approach developed allows in vitro simulating somatic embryogenesis by dividing this process into three partially independent steps: PEM → globular somatic embryo → heart-shaped somatic embryo → somatic embryo with developed cotyledons. The highest frequency of morphogenetic stage transition was 64, 40, and 26%, respectively. It was shown that the first step (PEM → globular embryo) was associated with the formation of heterogeneous population of spherical bodies 50–500 μm in diameter, among which about 40% were somatic embryos at globular stage. The scheme is offered of alternative pathways for the development of spherical bodies in vitro, and interrelations between their sizes and ability to direct morphogenesis are discussed.     

Efficient induction of microspore embryogenesis using abscisic acid,jasmonic acid and salicylic acid in Brassica napus L

The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l^?1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l^?1 for 24 h. Highest embryo yield (286.0 embryos Petri dish^?1) was achieved using 1.0 mg l^?1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l^?1 JA for 12 h. JA (5.0 mg l^?1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l^?1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish^?1) relative to the control (136.2 embryos Petri dish^?1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.     

Analysis of the effects of maturation treatments on the probabilities of somatic embryo germination and plantlet regeneration in pistachio using a linear logistic method

Embryogenic masses (EMSes) of pistachio (Pistacia vera L.) were proliferated in liquid Murashige and Skoog (MS) medium without growth regulators. To determine the effects of benzylaminopurine (BAP), racemic (±) abscisic acid (ABA) and sucrose treatments during maturation on the subsequent germination and plantlet regeneration, clusters of mature somatic embryos were transferred from maturation medium onto the surface of 0.7% agar-solidified Murashige and Skoog medium. Neither germination nor plantlet development medium contained BAP or ABA. Germination studies were carried out using 80 somatic embryos at every combination of four sucrose concentrations, three maturation periods and either five concentrations of BAP or four of ABA, and the numbers germinating were recorded after four durations of culture. A similar experimental plan was used to study plantlet regeneration. The number of germinated somatic embryos increased markedly with duration of the culture on germination medium, and was influenced by the concentrations of BAP or ABA in the maturation medium; the concentration of sucrose in this medium had little influence. Plantlet regeneration also increased with culture duration and was reduced at the highest levels of BAP or ABA; with ABA, the probability of plantlet regeneration was lower for longer maturation periods. ABA and BAP have similar effects on somatic embryo germination (except at the highest levels used), but BAP is superior to ABA for promoting subsequent plantlet regeneration. Linear logistic models were used to investigate the significance of the treatments, and to estimate the optimum conditions for germination and plantlet regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.