A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Non-freezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4–8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.     

Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana)

The present study was conducted to improve the transition from proliferation to maturation in embryogenic cultures of Nordmanns fir. For that reason, chemicals reported to affect endogenous levels or activity of auxin were included in the growth media during maturation. The auxin antagonist PCIB reduced proliferation and promoted the development of numerous high-quality mature embryos in the tested cell lines. PCIB could not substitute for exogenously supplied ABA and the positive effect was only found when PCIB and ABA were used in combination. The effect of PCIB was dependent on the concentration and the application period. The auxin transport inhibitor TIBA also reduced proliferation, but had no positive effect on maturation. The auxin synergist phloroglucinol had the opposite effect of PCIB; proliferation was increased and no maturation was initiated. A lowered concentration of boron had no effect on proliferation but had some positive effect on maturation. The optimum protocol for PCIB application was strongly genotype dependent, and a general scheme that covered the tested cell lines could not be found. Overexposure to PCIB during maturation caused abnormal development of the mature embryos, which was revealed by a reduced number of cotyledons. These results suggest that endogenously produced auxin may be one reason for low or failing maturation of embryogenic cultures of Nordmanns fir, but also imply that auxin may play a critical role for proper development of cotyledons during the later stages of embryo maturation.     

A method for quantification of the level of somatic embryogenesis among Norway spruce callus lines

A method for quantitative determination of the level of somatic embryogenesis in Norway spruce embryogenic callus is described. Embryogenic callus was dispersed in liquid by agitation and plated in a thin layer of medium containing 0.6% low melting point agarose. The number of embedded somatic embryos per mg of callus ranged from 0.2 to 1.5 among 11 embryogenic callus lines surveyed. Each callus line was derived from an individual immature embryo explant. Further development occurred as somatic embryos grew out of the agarose layer. This method was useful for identifying highly embryogenic callus lines among phenotypically similar lines, and should be useful for quantitatively determining the effect of medium and growth regulator modifications on somatic embryo density and developmental capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid - ABA abscisic acid     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Increase in permeability to oxygen and in oxygen uptake of soybean nodules under limiting phosphorus nutrition

In vitro cultures and endogenous abscisic acid (ABA) analyses with gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) were used to study the effects of AgNO₃ and polyethylene glycol (PEG) on white spruce ( Picea glauca ) somatic embryo maturation and endogenous ABA contents, Normally, in the absence of ABA, white spruce somatic embryos cannot mature. However, AgNO₃ and PEG stimulated somatic embryo maturation by increasing the number of cotyledonary embryos. A combination of 100 μ M AgNO₃ and 40 g l⁻¹ PEG was the treatment that was most effective in enhancing cotyledonary embryo formation without exogenous ABA. Either AgNO₃ or PEG was able to increase endogenous ABA levels of the embryogenic culture but a combination of AgNO₃ and PEG was the most effective treatment. Stimulation of cotyledonary embryo formation by AgNO₃ occurred only at low ABA levels, while PEG promoted embryo formation at all exogenous ABA concentrations. Germination tests indicated that AgNO₃ had no negative effects on embryo germination and conversion while the PEG-treated embryos failed to germinate.     

Improvement of somatic embryogenesis in wild cherry (Prunus avium). Effect of maltose and ABA supplements

Three different types of morphogenesis were identified in embryogenic cultures of Prunus avium grown on a proliferation medium containing 0.54 μM NAA, 0.46 μM kinetin and 0.44 μM BA: a friable hyperhydric callus, repetitive embryogenesis and an embryogenic tissue. Translucent and white somatic embryos were produced from the three types of morphogenesis but mainly from the embryogenic tissue. These somatic embryos showed histological and cytological teratological features such as highly differentiated cells with shrunken cytoplasm and destructured nuclei. For the four lines studied, somatic embryo production was improved by transferring the embryogenic tissue to developmental media without auxin and cytokinin but supplemented with maltose alone or maltose and 10 μM ABA. Three weeks after transfer, the line showing the most embryogenesis produced 1404 somatic embryos per gram of embryogenic tissue. A concentration of 263 mM maltose significantly increased the number of white somatic embryos for L 10 line, while translucent somatic embryo production was improved by 88 mM maltose for L 16 line. The combination of maltose and ABA produced different effects with each line. When used with 88 mM maltose, 10 μM ABA significantly increased white somatic embryo production for two lines but decreased the production for one line. When combined with 263 mM maltose, ABA had no effect on white somatic embryo production but significantly decreased the number of translucent somatic embryos. Cells of white somatic embryos contained protein storage reserves and numerous lipid bodies, while those of translucent embryos did not contain storage reserves or lipid bodies. After a two-month cold treatment conversion rate of white and translucent somatic embryos reached 8.5% and 35.2% respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Involvement of ethylene in the maturation of black spruce embryogenic cell lines with different maturation capacities.

To examine the possible relationship between ethylene and the capacity of embryogenic cell lines to produce mature somatic embryos of black spruce (Picea mariana (Mill.) B.S.P.), two embryogenic cell lines which exhibit different maturation capacities were used to analyse ethylene biosynthesis and that of its immediate precursor, 1-aminocyclopropane-1- carboxylic acid (ACC). Several compounds known to alter ethylene metabolism were also evaluated for their effect on the number of mature somatic embryos produced. The results showed that in the high capacity cell line, ethylene production and endogenous ACC pools were less than in the low capacity cell line. It was also demonstrated that limiting ethylene biosynthesis by adding inhibitors of ethylene biosynthesis or its physiological action to the maturation medium promoted somatic embryo production for the low capacity cell line. Conversely, lowering ethylene biosynthesis reduced the number of somatic embryos in the high capacity cell line. These results were further substantiated by the finding that the effects of amino-oxyacetic acid (AOA), an inhibitor of ethylene biosynthesis, were partially reversed by adding ethylene to both embryogenic cell lines. It is concluded that ethylene is implicated in somatic embryogenesis of black spruce and that the low capacity cell line had excess, i.e. supraoptimal, ethylene production, whereas the high capacity cell line had nearly optimal ethylene production. The relationship between ethylene and other phytohormones, and the possible effects of the interaction between ethylene and polyamines on the maturation of the somatic embryos are discussed.     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

Genotype effects on ABA consumption and somatic embryo maturation in interior spruce (Picea glaucaxengelmanni)

Abscisic acid (ABA) plays an important role during somatic embryo development and maturation in coniferous species. The purpose of this research was to study ABA utilization by genotypes with different embryo maturation capabilities in interior spruce. Cell lines ISP11 and ISP48 were of high embryo maturation capability. By contrast, the tissue of line ISP16 contained numerous immature embryos, but only a few mature embryos developed. Exogenous ABA, i.e. S-ABA [(+)-cis, trans-ABA], racemic ABA, or ABA isomers were added into suspension cultures at a final concentration of 30 microM. In comparison to racemic ABA and ABA isomers, S-ABA reduced tissue proliferation the most. In all cell lines, about half of the racemic ABA was used within 2 weeks; the remaining ABA was (-)-cis, trans-ABA. The concentration of ABA showed little change thereafter. In the cultures supplied with ABA isomers, about half of (+/-)-cis, trans-ABA was utilized during 22 d. By contrast, (+/-)-trans, trans-ABA was hardly used, especially in line ISP16. S-ABA was almost completely metabolized by line ISP11. However, approximately 28% and 22% of the S-ABA remained in the culture of cell lines ISP16 and ISP48, respectively. Cell line ISP16 grew the fastest in culture. By 3 weeks, S-ABA consumption by ISP11 and ISP48 on the basis of tissue growth was, respectively, 2.2-fold and 3.4-fold greater than that of ISP16. A higher ratio of dihydrophaseic acid to phaseic acid existed with cell lines of higher embryo maturation capability, especially when the exogenously supplied ABA was chemically synthesized.     

Applications of DL-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of dl-buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg⁻¹ tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell–cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.     

Ascorbic acid metabolism during white spruce somatic embryo maturation and germination

Changes in ascorbic acid (AA) metabolism were investigated in 3 different cell lines of white spruce; a non-embryogenic line (NE)WS and two embryogenic lines with low (E)WS1 and high (E)WS2 percentage of embryo germination. During embryo maturation, a high AA/dehydroascorbate (DHA) ratio was observed in the two embryogenic cultures. The increment of this ratio was mainly ascribed to the high ability of the (E)WS1 and (E)WS2 lines to generate AA from its last precursor, l -galactono- γ -lactone, since no increase in the activities of the AA-recycling enzymes, i.e., AA free-radical reductase (AFRR) and DHA reductase (DHAR), was observed as the embryos developed. During the partial drying treatment needed for successful embryo germination, the ability to de novo generate AA continued to increase in the embryos of the two embryogenic lines, although the total ascorbate pool (AA+DHA) sharply decreased. A reactivation of the overall AA metabolism was observed at the inception of germination, especially in the (E)WS2 embryos. These results suggest that (1) the ability to metabolically alter the AA/DHA ratio may be a fundamental requirement for somatic embryo maturation and (2) the restoration of the AA metabolism during the first days of germination may be necessary for successful embryo germination.     

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers.     

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst)

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.     

Effect of sugars,amino acids,and culture technique on maturation of somatic embryos of Pinus strobus on medium with two gellan gum concentrations

Maturation of five embryogenic lines of Pinus strobus L. was tested on media with various sugars and sources of organic nitrogen, and solidified with two gellan gum concentrations (0.6 and 1.0%). Mature somatic embryo production was more abundant at 1.0% gellan gum than at 0.6%. Complex combinations of amino acids had little effect on mature embryo production of most tested embryogenic lines. Increasing glutamine concentration of the maturation medium from 1.7 to 7.3 g l⁻¹ was beneficial to one embryogenic line. Increasing sucrose concentration or substituting part of the sucrose with mannitol or sorbitol had variable effects on somatic embryo maturation depending on the embryogenic line. A medium with 88 mM sucrose plus 175 mM sorbitol solidified with 1.0% gellan gum produced high numbers of somatic embryos in four out of five embryogenic lines tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.