Kinetics of ethylene biosynthesis and its effects during maturation of white spruce somatic embryos

The objective of the current investigation was to study the role of ethylene in the maturation of white spruce ( Picea glauca [Moench.] Voss) somatic embryos. This was carried out by examining the effects of (1) 1-aminocyclopropane-1-carboxylic acid (ACC), a direct precursor of ethylene in plant tissue, (2) silver nitrate (AgNO₃), an inhibitor of ethylene action, (3) α -aminooxyamino acid (AOA), a potent inhibitor of ethylene biosynthesis, and (4) enrichment with ethylene. Ethylene biosynthesis was biphasic and gradually increased during embryo development, whereas endogenous ACC and N-malonylaminocyclopropane-1-carboxylic acid (mACC) decreased. Addition of ACC or AOA to the culture medium increased or decreased, respectively, ethylene biosynthesis by altering endogenous ACC levels during the culture period. In contrast to AOA and AgNO₃, ACC and ethylene enrichment significantly decreased the production of mature somatic embryos and increased the browning of the cultures. However, the structure of the shoot apex in mature cotyledonary stage embryos formed under ethylene enrichment was similar to that in control systems. This shows that a reduction in ethylene is beneficial to maturation of white spruce somatic embryos. This is further substantiated by the finding that the inhibitory effects of AOA were partially reversed by the addition of ethylene. The possible effects of the interaction between ethylene and polyamines on somatic embryo development are also discussed.     

Effects of sealed and vented gaseous microenvironments on the maturation of somatic embryos of black spruce with a special emphasis on ethylene

Maturation of black spruce somatic embryos in sealed and vented microenvironments was investigated. The sealed microenvironment induced a larger number of well-formed mature embryos and less precocious germination than the vented microenvironment. Maturation rate of somatic embryos was not changed either by injection of ethylene into the culture vessel or by its removal by potassium permanganate traps. Increased as well as decreased ethylene concentrations, by the addition of either 1-aminocyclopropane-1-carboxylic acid (precursor of ethylene) or cobalt chloride (inhibitor of ethylene biosynthesis), resulted in a decreased number of embryos produced. However, inhibition of ethylene action by the addition of silver nitrate to the maturation medium did not affect either ethylene concentration or somatic embryo production. It was concluded that ethylene accumulation during maturation has no effect on somatic embryo production. Neither the microenvironment nor the modification of the ethylene metabolism affected conversion rate of somatic embryos into plantlets growing in soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.P.

Summary The usefulness of random amplified polymorphic DNA (RAPD) in assessing the genetic stability of somatic embryogenesis-derived populations of black spruce [Picea mariana (Mill.) B.S.P.] was evaluated. Three arbitrary 11-mer primers were successfully used to amplify DNA from both in-vivo and in-vitro material. Twenty-five embryogenic cell lines, additional zygotic embryos and megagametophytes from three controlled crosses involving four selected genotypes of black spruce were used for the segregation analysis of RAPD variants. Ten markers were genetically characterized and used to evaluate the genetic stability of somatic embryos derived from three embryogenic cell lines (one cell line per cross, 30 somatic embryos per cell line). No variation was detected within clones. The utilization of RAPD markers both for the assessment of genetic stability of clonal materials and to certify genetic stability throughout the process of somatic embryogenesis is discussed.     

Stable genetic transformation ofPicea mariana (black spruce) via particle bombardment

Stable genetic transformation ofPicea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell line were obtained from the mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014). The suspension culture from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they have a normal phenotype.     

Somatic embryogenesis of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.     

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.     

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.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

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.     

Abscisic acid and methyl jasmonate as regulators of ethylene biosynthesis during somatic embryogenesis of Medicago sativa L.

Effects of abscisic acid (ABA) and methyl jasmonate (MeJA) on ethylene production, ACC oxidase (ACO) activity, and content of 1-aminocyclopropane-1-carboxylic acid (ACC) during indirect somatic embryogenesis (SE) of Medicago sativa L. were studied. ABA and MeJA, at 50 μM, were applied during the induction, proliferation, or differentiation phase. ABA decreased ethylene production at the beginning of callus and SE induction and during the differentiation of somatic embryos. The hormone inhibited ACO activity in explants with overgrowing callus during the first two weeks of induction, in embryogenic suspension and also in differentiating embryos. The ACC content was reduced by ABA in callus at the end of SE induction, in embryogenic suspension and in globular embryos, but elevated in cotyledonary embryos. MeJA had no significant effect on ethylene production during M. sativa SE, despite the fact, that it inhibited ACO activity during the first two weeks of induction and in torpedo and cotyledonary embryos. The ACC content was increased by MeJA in 14-day-old callus and embryogenic suspension but was inhibited in globular embryos. Both ABA and MeJA seem to be involved in the regulation of ethylene biosynthesis during distinct phases of SE in M. sativa. It might be considered that exogenous ABA, more probably than MeJA, exerts its inhibitory effect on M. sativa somatic embryo formation by modifying ethylene production.     

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.     

The effect of AOA on ethylene and polyamine metabolism during early phases of somatic embryogenesis in Medicago sativa

Changes in the levels of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and polyamines were simultaneously investigated during the early phases of alfalfa somatic embryogenesis. These included the period of induction and subculture of callus, and 3- and 7-day suspension cultures for the induction of somatic embryogenesis. The polyamines contained in the embryogenic callus were found to include putrescine (Put), spermidine (Spd) and spermine (Spm), but the level of Spm was much less than that of Put and Spd. There was a dramatic increase in MACC after induction of embryogenesis, and ACC levels were lower in somatic embryos than in embryogenic callus. Induction of embryogenesis for 3 days increased the levels of ACC and polyamines to a maximum level, and these then reduced as the embryogenesis proceeded. The ratios of Put/Spd and ACC/MACC were decreased during the induction. This indicated that both high levels of ACC and polyamines might be a prerequisite for early differentiation during the induction of the embryogenesis. Thus, there appears not to be competition between polyamine biosynthesis and ethylene biosynthesis at least during the induction of somatic embryogenesis, because both the polyamines and ACC were simultaneously increased during the induction period. Conversion of ACC into MACC and the maintenance of a relatively high level of polyamines, especially Spd, appear to be important for further development of the embryos.
When aminooxylvinylglycine (AOA) was added at the initiation of the callus subculture, it had no significant effect on the callus growth, the ethylene production and ACC level of the callus. However, AOA increased the numbers of the embryos accompanying an increase in Spd level and S-adenosylmethionine decarboxylase (SAMDC) activity. Thus, the AOA effect could be associated with Spd increase rather than with the effect of ethylene biosynthesis.     

Biochemical differences between carrot inbreds differing in plant regeneration potential

Cultures derived from domestic carrot (Daucus carota L.) inbreds were found to vary with respect to regeneration potential as measured by the production of somatic embryos in suspension cultures. A number of biochemical parameters previously reported to distinguish embryogenic from non-embryogenic cultures of other species were measured in these carrot cell lines. Ethylene production was found to be inversely related to regeneration potential. The cell line producing the greatest number of somatic embryos exhibited the lowest rate of ethylene biosynthesis, even when grown on 2, 4-D-containing maintenance medium. A specific isozyme of acid phosphatase was associated with embryogenic calli. Proteins visualized by SDS-PAGE did not discriminate between embryo-forming and proliferating calli in all inbreds.     

Maturation of somatic embryos in two embryogenic cultures of <Emphasis Type="Italic">Picea morrisonicola</Emphasis> Hayata as affected by alternation of endogenous IAA content

Embryogenic culture lines T4 and T2 were initiated from two mature zygotic embryos of Picea morrisonicola Hay. Mature somatic embryos (SEs) were produced in culture line T2 but not in line T4 after 8-week abscisic acid (ABA) treatment. High performance liquid chromatography (HPLC) analysis of the endogenous indole-3-acetic acid (IAA) content has shown 7.5 times higher IAA production in T4 line than in T2 line during the proliferation phase. However, after ABA incubation the line T4 produced much less IAA than line T2. The application of an anti-auxin, 2,3,5-triiodobenzoic acid (TIBA) or 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB) induced culture line T4 to produce mature SEs. Both 1 μM TIBA and 5 μM PCIB increased the production of stage 2 SEs in T4 culture line when cultures were treated during the proliferation stage for 8 weeks. Occasionally cotyledonary (stage 3) SEs were even produced from treated T4 culture line. Both chemicals have also been demonstrated to significantly decrease the amount of IAA in the treated T4 and T2 embryogenic lines. However the decrease of the IAA level was not beneficial for SE production in the T2 embryogenic line. These results indicated the importance of endogenous IAA level in manipulating the process of SE maturation in spruce embryogenic cultures.     

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     

Clonal plant production from self- and cross-pollinated seed families of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.     

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.     

Genetic variation in somatic embryogenic response in open-pollinated families of black spruce

Summary Zygotic embryos from open-pollinated seeds of 20 black spruce (Picea mariana) families were used to investigate the proportion of genotypes that would give rise to embryogenic tissue (ET) and mature somatic embryos. Eighty-five percent of the maternal genotypes gave rise to embryogenic tissue. Within-family rates of ET induction ranged from 0 to 17%, with an average of 8%. The largest proportion of variation was among families, indicating the additive nature of the genetic variation. On a medium with 6% sucrose and 3.7 M ABA, 90% of the embryogenic lines gave rise to abundant (>100/100 mg of ET), well-formed, mature somatic embryos. A medium with 2% sucrose, without 2,4-D, was used to germinate the mature somatic embryos. These were grown in the greenhouse and have now been established in field trials.