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.     

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.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols     

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.     

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.     

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.     

Abscisic acid is involved in the water stress-induced betaine accumulation in pear leaves

ABA exogenously applied to the leaves of the whole plants of pear (Pyrus bretschneideri Redh. cv. Suly grafted on Pyrus betulaefolia Rehd.) significantly increased the betaine concentrations in the leaves when the plants were well watered. The plants subjected to 'drought plus ABA' treatment had significantly higher betaine concentrations in their leaves than those given drought treatment alone. The 'drought plus ABA' treatment increased the amount of betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) and its activity in the leaves more than did the drought treatment alone. The experiments with detached leaves showed that ABA treatment significantly increased the concentration of betaine, activity of BADH and apparent amount of BADH in non-dehydrated leaves, and enhanced the accumulation of betaine, activity of BADH and apparent amount of BADH in dehydrated leaves. These effects of ABA were both time- and dose-dependent. Two ABA isomers, (-)-cis, trans-ABA and 2-trans, 4-trans-ABA, had no effect on the betaine accumulation in the leaves, showing that the ABA-induced effects are specific. These data demonstrate that ABA is involved in the drought-induced betaine accumulation in the pear leaves.     

Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression

During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.     

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.     

Long-term somatic embryogenesis and maturation of somatic embryos in Hevea brasiliensis

A high frequency of secondary embryogenesis was induced from isolated early cotyledonary-stage somatic embryos of Hevea brasiliensis. A long-term embryogenic line was established by the use of recurrent embryogenesis and maintained for 3 years on hormone-free medium by the transfer of selected proembryogenic masses every 10 days.

The addition of 234 mM sucrose as stress with sucrose and 10⁻⁵ M abscisic acid (ABA) to the culture medium enhanced the maturation of somatic embryos. Under these culture conditions, the embryo population was composed of 45% globular, 18% oblong and 37% torpedo-stage embryos. These somatic embryos had well-formed tissue structure, a well-defined epidermis, protein storage bodies, and a high accumulation of starch. The triglyceride content was five times as high in the torpedo-stage embryos that developed on medium supplemented with 234 mM sucrose and 10⁻⁵ M ABA as in embryos obtained on basal medium with 58 mM sucrose.     

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     

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.     

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.     

Regeneration of plant by somatic embryogenesis in <Emphasis Type="Italic">Pinus rigida</Emphasis>×<Emphasis Type="Italic">P. taeda</Emphasis>

Zygotic embryos at different developmental stages were tested for their potential in the initiation of embryogenic suspensor mass (ESM) lines using immature seeds of Pinus rigida × P. taeda. The highest frequency (1.1%) of ESM was obtained with explants from cones collected on July 1. All excised embryos of the July 1 collection were at the early proembryo stage. Two different culture media were compared. Forty-eight ESM lines were initiated on Pinus taeda basal medium (P6) (0.97%) with 13.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA). However, only four ESM were obtained on a modified Murashige and Skoog medium (MSG; 0.55%). Most of the ESM arose from the seeds that were at the stages ranging from late cleavage polyembryony to the early staged proembryo. Out of 52 lines (0.46%) that were produced from 11,388 explants, only two viable lines (0.018%) (PRT11 and PRT28) survived. As for somatic embryo maturation, the highest number (224/g⁻¹ FW) of matured cotyledonary somatic embryos (line PRT 28) was obtained on a medium containing 100 μM abscisic acid (ABA), 0.2 M maltose, and 1.2% gellan gum. For germination of the somatic embryos, the cotyledonary somatic embryos derived from maturation medium were transferred on half-strength Litvay medium (LM) plus 0.4% gellan gum. The germination rates were high (71.4–96.3%) regardless of the concentrations of either ABA or gellan gum in the maturation medium. Approximately 500 somatic plants were recovered from the germination medium and transferred to the green house; finally most of them were transplanted successfully to the experimental field.     

Treatments affecting maturation and germination of American chestnut somatic embryos

The effects of amino acids, abscisic acid (ABA), polyethylene glycol (PEG), and elevated sucrose were tested on the maturation and germination of American chestnut (Castanea dentata) somatic embryos. Somatic embryos from three lines were matured over an eight week period through a two-stage process. After maturation, somatic embryos were randomly divided into three groups to measure dry weight/ fresh weight ratios, starch levels, and germination rates. Prior to transfer to germination medium, somatic embryos received a four week cold treatment. While some treatments with amino acids, elevated sucrose, PEG or ABA increased either dry weight/fresh weight ratios, starch content or both, only addition of 25mM L-asparagine significantly increased germination rate and taproot length, and this response was only obtained with one of the three lines tested. Six plants survived the transfer to potting mix, acclimatization to greenhouse conditions and field planting.     

Endogenous ABA content in relation to maturation of somatic embryos in <Emphasis Type="Italic">Tulip</Emphasis>a (L.) ‘Apeldoorn’ cultures

The aim of the present study was to estimate the endogenous abscisic acid (ABA) content in tulip ‘Apeldoorn’ torpedo and mature somatic embryos. Moreover, the effect of exogenous ABA and/or its inhibitor fluridone on somatic embryo maturation and conversion into plantlets was investigated. Torpedo-stage somatic embryos were subcultured on media containing 5 μM of picloram and 1 μM of 6-benzyl-aminopurine (BAP)—control, and combinations of ABA (0 or 10 μM) and/or fluridone (0 or 30 μM) for 1 week. Then, the torpedo embryos were transferred to a maturation medium containing 0.25 μM of α-naphthaleneacetic acid (NAA) and 2.5 μM of BAP, without ABA and fluridone treatment, and cultivated under darkness or light for ten weeks. Endogenous ABA content (first time measured in tulip somatic embryos) was evaluated by ELISA test. The obtained results revealed that the highest level of endogenous ABA, at 17.45 nmol g^?1 dry weight (DW), was recorded in torpedo-stage of tulip embryo development, only after 1 week of ABA treatment, and was nearly 10 times higher in comparison with the control. Simultaneous addition of ABA and fluridone to the medium resulted in the lowering of the ABA concentration to 9.58 nmol g^?1 DW. During ten weeks of maturation of the embryos, the endogenous ABA content in mature tissue of tulip somatic embryo considerably decreased to an amount 0.87–1.33 nmol g^?1 DW (irrespective of ABA and fluridone treatment) and did not differ significantly from control (0.59 nmol g^?1 DW). Exogenous ABA and fluridone significantly decreased the growth value of fresh weight (FW) of the tulip torpedo-shaped and mature embryos under light conditions. Percentage of the DW of the torpedo embryos treated with exogenous ABA was significantly higher (15.43–17.02) in comparison with the control (10.87). Three to three and a half times more malformed mature embryos were noted under light conditions than in darkness, irrespective of ABA and fluridone treatment. The highest percentage of mature embryos forming shoots (conversion) was observed under light conditions in the control and after fluridone treatment (26 and 20%, respectively).