Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat     

Role of ABA in Maturation of Rapeseed Embryos

Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investigate the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.     

Gene expression patterns, and uptake and fate of fed ABA in white spruce somatic embryo tissues2

Changes in cellular protein accumulation and in in vivo andin vitro protein synthesis, in somatic embryo tissues of whitespruce during a 42 d maturation period were followed by two-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-DSDS-PAGE). These investigations were complemented by an analysisof uptake and fate of fed abscisic acid (ABA) in somatic embryotissues grown on maturation medium. When Stage 1 somatic embryoswere cultured on ABA-containing maturation medium, many changeswere observed in patterns of gene expression and in proteinsynthesis and accumulation which could be associated with embryodevelopment. The polypeptides observed could be categorizedas constitutive, embryo-abundant, embryo maturation-relatedand embryo stage-related, as well as those with non-specificchanges. Accumulation of label from fed ³H-(+)-ABA in embryotissues reached a plateau 3 d after Stage 1 somatic embryoswere placed on maturation medium. ABA taken into tissues wasrapidly metabolized, and 40% of radioactivity in tissues after1 d of culture resulted from ABA metabolites. This value increasedto 90% after 3 weeks culture. Conjugated ABA and oxidized ABA(phaseic acid and dihydrophaseic acid) were major forms of ABAmetabolites in spruce embryo tissues. Using a single 42 d cultureperiod following transfer to medium with ABA, the conditionsthat stimulate the sequence of developmental changes of somaticembryo maturation during the first 21 d do not reoccur duringthe second 21 d. Unless greater synchronization of Stage 1 culturescan be achieved, it is therefore unlikely that yields of maturesomatic embryos will be increased by this method. Key words: Abscisic acid, gene expression, Picea glauca (Moench) Voss, protein synthesis, somatic embryo maturation     

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     

Temporal and nutritional factors modulate responses to abscisic acid and osmoticum in their regulation of storage protein synthesis in developing seeds of alfalfa (Medicago sativa L.)

Storage protein synthesis during alfalfa (Medicago sativa L.)seed development displays a temporal pattern and is regulatedboth by ABA and low osmotic potentials. Deposition of the 2S,7S and 11S storage proteins occurs maximally at the mid–to late stages of development. Osmoticum, but not ABA alonecould effectively induce storage protein synthesis at earlystages of development in isolated alfalfa embryos placed onwater for several days. Neither ABA nor osmoticum alone couldmaintain storage protein synthesis in isolated late-stage embryosplaced on water. On the other hand, isolated developing embryoscultured on a nutrient (Murashige and Skoog) medium could maintainstorage protein synthesis in the presence of ABA and osmoticum,alone or in combination, for up to 14 d after being dissectedfrom the pod. Of the components of this medium, the inorganicsalts appeared to be the most important. The response to ABAand osmoticum varied with the time of isolation during developmentwith the greatest enhancement of storage protein synthesis inisolated embryos coinciding with the time of maximum synthesiswithin the seed when in planta. Addition of ABA and osmoticumafter the time of maximum storage protein synthesis did notelevate the amount of synthesis, but rather prolonged the timeover which it could take place. Thus, while the amplitude ofstorage protein synthesis could be modified by ABA or osmoticum,the inherent temporal pattern, with maximum synthesis occurringonly at mid– to late stages of embryo development, couldnot. Key words: Embryogenesis, Medicago sativa L., alfalfa, abscisic acid, osmotic potential, storage protein synthesis, nutrient supply     

Seed Dormancy in Acer: The Role of Testa-imposed and Embryo Dormancy in Acer velutinum

The embryo dormancy shown in freshly harvested samples of Acervelutinum seeds is weakly established and very short-lived.Loss of this embryo dormancy occurred during post-harvest fruitstorage at either 5 or 17 C. In contrast, the dormancy of intactfruits and seeds was overcome only during storage at the lowertemperature. Removal of the cotyledons from embryos of freshlyharvested fruits allowed more rapid germination of the embryonicaxes, indicating that the cotyledons exert an inhibitory effect,although the axes still retained a measure of innate dormancy.The inhibitory effect of the cotyledons became less marked withincreasing duration of fruit storage, this loss of inhibitoryeffect occurring at both storage temperatures. Applied ABA stronglysuppressed germinative capacity in intact embryos and isolatedembryonic axes from freshly harvested fruits, but when ABA wasapplied to embryos of fruits that had been stored for variousperiods at 5 or 17 C, the inhibitory effect was first weakenedand then lost with increased storage. Although dormancy in the seeds of A. velutinum may be describedas intermediate between testa-imposed dormancy and true dormancy,it is perhaps more properly included in the former category. Acer velutinum Boiss. var. vanvolxemii, abscisic acid, embryo dormancy, germination, seed storage, testa-imposed dormancy, tissue sensitivity     

An Analysis of Seed Development in Pisum sativum: VIII. DOES ABSCISIC ACID PREVENT PRECOCIOUS GERMINATION AND CONTROL STORAGE PROTEIN SYNTHESIS?

The influence of abscisic acid (ABA) on the precocious germinationand storage protein production of pea seeds has been examinedusing embryo and pod culture. The precocious germination ofembryos in culture could not be inhibited fully by ABA on apermissive medium (2% sucrose) even at 0.1 mol m^–3. However,increasing the sucrose concentration to 5% caused near completeinhibition when ABA was added to the medium. Embryos of differentweights cultured on a high osmoticum (mannitol-containing medium),equivalent to 10% sucrose, did not show any consistent differencein ABA content. When fluridone was added to a non-permissiveculture medium, no decrease in ABA content of the embryos couldbe observed and no precocious germination was induced. In contrast,fluridone was able to prevent the accumulation of ABA in seedspresent in pods cultured in its presence from an early stageof development. These seeds, however, grew normally and reachedmaturity, did not germinate precociously in vivo, were desiccationtolerant and still produced storage protein message whetheror not ABA was included in the culture medium. It does not appear,therefore, that ABA regulates normal development or storageprotein synthesis in pea embryos. Key words: Abscisic acid, peas, Pisum sativum, seed development     

The role of abscisic acid in germination, storage protein synthesis and desiccation tolerance in alfalfa (Medicago sativa L.) seeds, as shown by inhibition of its synthesis by fluridone during development

Abscisic acid and osmoticum maintain maturation and proteinsynthesis of developing alfalfa embryos, individually and incombination. The in situ environment of developing alfalfa zygoticembryos is rich in ABA and low in osmotic potential. When ABAsynthesis was inhibited by treating the pods with fluridoneat an early stage of development, the seeds which subsequentlydeveloped contained low amounts of ABA, but had a similar osmoticpotential as untreated control seeds. The reduced ABA in seedsfrom fluridone-treated pods did not change the morphology exceptthe colour of seeds, nor did it induce viviparous germinationor affect storage protein synthesis. However, two nonstorageproteins which were synthesized in control seeds during earlyto mid-development were absent from fluridone-treated seeds.Control seeds containing these two proteins were desiccation-tolerant,whereas the fluridone-treated seeds which lacked them were desiccation-intolerant,at least until the deposition of storage proteins was nearlycomplete. Culture of isolated embryos on nutrient medium inducedgermination and curtailed storage protein synthesis in the embryos.Addition of either ABA or osmoticum to the nutrient medium preventedgermination and maintained storage protein synthesis. When fluridonewas added along with osmoticum, germination occurred, but storageprotein synthesis was maintained. Key words: Embryogenesis, Medicago sativa L., alfalfa, ABA, osmotic potential, fluridone, desiccation, storage protein synthesis     

Effects of Desiccation, Medium Osmolarity and Abscisic acid on the Maturation of Hevea brasiliensis Somatic Embryos

The effects of desiccation of Hevea somatic embryos and of sucroseand ABA concentrations in the maturation medium on their germinabilitywere investigated. Conversion into plant, water and histochemicalstatus of somatic embryos were compared systematically to thoseof the zygotic embryos used as reference. Slow desiccation ormaturation on 351 mol m^–3 sucrose supplemented with 1mmol m^–3 ABA strongly improved germinability and conversionof embryos into plants. The combination of the two treatmentswas the most effective, increasing the germination frequencyby 3·7 and plant conversion by 6·6 in clone PR107. Each of these two treatments increased the vigour of somaticembryos, stimulated the formation of root and shoot meristemsand the synthesis and accumulation of starch and protein reserves.At the end of maturation, the Hevea somatic embryos bore ananatomical and histochemical resemblance to mature zygotic embryos.Likewise, the two treatments brought the water status of somaticembryos closer to that of the mature zygotic embryos, but withoutachieving a perfect match. Optimization of the successful conversioninto plants may require full acquisition of this water status. Key words: ABA, embryo maturation, Hevea, somatic embryogenesis, water status     

Changes in Abscisic Acid Content in Axis and Cotyledons of Developing Phaseolus vulgaris Embryos and their Physiological Consequences

Prevost, I. and Le Page–Degivry, M. Th. 1985. Changesin absicisic acid content in axis and cotyledons of developingPhaseolus vulgaris embryos and their physiological consequences.—J.exp. Bot. 36: 1900–1905.Changes in abscisic acid (ABA)content with time were measured in embryonic axes and in cotyledonsof Phaseolus vulgaris embryos using a radio–immunoassay.During embryogenesis, a similar pattern was observed in bothtissues: ABA increased to a maximum 29 d after an thesis, followedby a decrease as the seed matured. The level of ABA in the cotyledonswas always much higher than that in the axes. In in vitro cultures,the duration of the lag phase before germination of isolatedembryonic axes increased with ABA content. The presence of cotyledonsalways lengthened the lag phase; longer lag phases were associatedwith greater concentrations of ABA in the cotyledons. Moreoverthe presence of cotyledons stimulated the growth of seedlings. Key words: ABA distribution, embryo maturation, axis and embryo germinability     

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.     

Abscisic Acid and Precocious Germination in Soybeans

Immature embryos of soybeans (Glycine max L. Merr.) can be inducedto germinate precociously by depleting the endogenous pool ofabscisic acid (ABA). Washing the embryos or allowing the embryosto dry slowly within or out of detached pods causes a gradualdecline in ABA content. The extent of germination is correlatedwith the length of washing or drying treatments, which in turn,affects the level of endogenous ABA. Providing embryos are threeweeks of age or older, maturation and precocious germinationcan be induced by treating embryos in a way that causes a reductionin embryo ABA content. Drying is not an obligatory requirementfor soybean seed maturation or germination. Key words: ABA, Germination, Embryos     

The rea (red embryonic axis) phenotype describes a new mutation affecting the response of maize embryos to abscisic acid and osmotic stress

During a screen for mutants with defective germination, a newphenotype was observed consisting of red pigmentation of theembryonic axis in the dormant seed. Segregation ratios, as determinedin F₂ and back-crossed progeny, indicate that the phenotypeis due to a recessive single gene mutation that has been symbolizedrea to denote red embryonic axis. A closer inspection of therea phenotype revealed that the mutant is occasionally viviparous,indicating a defect in abscisic acid (ABA) metabolism. The mutationprobably affects ABA sensitivity since no difference in ABAcontent was detected in mutant versus normal tissues. Moreover,when immature mutant and wild-type embryos were incubated onmedia containing 10 M ABA, only the mutants germinated. ABA-regulatedgene expression in rea embryos differed from that of embryosof the viviparous mutant vp1 which does not respond to the inhibitoryaction of ABA at the level of immature embryo germination. Theseresults, therefore, indicate that the two genes exert a differentrole in the control of embryogenesis. Key words: Zea mays L, embryo dormancy, ABA     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

Endogenous cis,trans-Abscisic Acid and Pea Seed Development: Evidence for a Role in Seed Growth from Changes Induced by Temperature

Measurements were made using GC/MS SIM¹ of the effects of temperatureon cis,trans-ABA levels in developing ovules and embryos oftwo pea genotypes contrasted in seed size. These effects werethen related to differences in the growth of the pods, seeds,embryos, and testae. In both genotypes high temperatures hastenedthe onset and rate of logarithmic and then linear growth, greatlyshortening the duration of pod and seed development but withoutgreatly altering seed size. Cis,trans-ABA was most concentratedxin the ovules immediately after fertilization. It also accumulatedin the embryo, more rapidly in the larger-seeded line, duringseed maturation. The stage when accumulation in the embryo beganwas the same irrespective of temperature. Accumulation ceasedwhen the pods started to desiccate. The effects of differentconstant temperatures on the maximum levels of embryo cis,trans-ABAwere relatively small and confounded in one genotype by variationin ovule abortion and in the other by differences in the stagewhen cis,trans-ABA accumulation ceased. However, when plantswere transferred from 13 °C to 29 °C at two differentstages during seed maturation, further seed growth was greatlyinhibited coincident with a substantial increase in embryo cis-trans-ABA.The results suggested a role for cis,trans-ABA in the controlof cotyledon enlargement during the linear phase of seed growth.     

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

mRNAs for two ribosomal proteins are preferentially translated in the chloroplast of Chlamydomonas reinhardtii under conditions of reduced protein synthesis

Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.     

Involvement of Endogenous Abscisic Acid in Onset and Release of Helianthus annuus Embryo Dormancy

Mature seeds of Helianthus annuus L. exhibit dormancy that is eliminated during storage in dry conditions. In vitro culture of immature embryos isolated at different times after anthesis showed that the youngest embryos are able to germinate, but within the third week after pollination, dormancy progressively affected most of the embryos. A radioimmunoassay showed that the endogenous abscisic acid (ABA) level, which increased sharply in the first half of the development period, fell at precisely the moment when embryo dormancy became established. An application of fluridone, before the increase of ABA level, prevented both ABA synthesis and development of embryo dormancy. Applied later, after the rise of the ABA level, fluridone could not prevent embryo dormancy development. Dormancy thus appears to be dependent on ABA synthesis but not concomitant with its accumulation; it must therefore be induced by ABA during maturation. Furthermore, a preincubation in water allowed dormant embryos to germinate. This acquisition of germinability could not be directly related to a leaching of free ABA. Possible effects of this treatment are discussed.