Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

Effects of Cytokinins on In Vitro Seed Germination and Early Seedling Morphogenesis in Lotus corniculatus L.

We determined the effects of zeatin (ZEA), isopentenyl adenine (2iP), kinetin (KIN), benzyladenine (BA), and thidiazuron (TDZ) on seed germination, elongation of seedling shoots and roots, frequency of regeneration, and the number of regenerants per seedling in Lotus corniculatus L. Sterilized seeds were cultured in vitro on Murashige and Skoog (1962) medium containing 3% sucrose, 0.7% agar, and various cytokinins (0, 0.08, 0.22, 0.35, 0.80, 2.20, and 3.50 μM). After 30 days, seedlings were transferred to cytokinin-free medium for another 60 days. All cytokinins stimulated the rate and percentage of seed germination at least twofold in optimum concentrations; TDZ and ZEA were the most active, followed closely by BA, whereas KIN and 2iP stimulated germination in higher concentrations only. Elongation of shoots and roots was strongly inhibited at the lowest TDZ and BA concentrations, whereas ZEA, KIN, and 2iP exerted moderate, dose-dependent inhibition. The frequency of regenerant-producing seeds was highest on ZEA and BA, whereas the greatest number of regenerants per seedling was found on TDZ. It is concluded that the culture of seeds on cytokinin-containing media, followed by transfer to cytokinin-free medium, is a suitable procedure for rapid production of a large number of uniform regenerants. The presumed role of particular cytokinins is discussed.     

Phloridzin transformation and accumulation during the stratification of apple seeds and the culture of isolated embryos

Phloridzin level and phloridzin β-glucosidase activity were estimated during apple seed cold stratification and during the culture of apple embryos isolated from seeds after different times of stratification. Both these factors were found to increase parallel to the progress of stratification as well as to the increasing ability of seeds/embryos to germinate. However, in the seed coats phloridzin dissappears during the progress of stratification despite the increasing β-glucosidase activity. Phloridzin formation in the embryos is postulated to be under light and gibberellin control, when isolated embryos germinate, and under temperature and gibberellin influence during the cold stratification of the seeds. The same factors control β-glucosidase activity development, which is also stimulated by the substrate itself. It is postulated that phloridzin accumulation and its glucosidase activity are indirectly related to the after-ripening process and directly to the germination of the embryos.     

Enhancement of ethylene biosynthesis and germination by cytokinins and 1-aminocyclopropane-l-carboxylic acid in Striga asiatica seeds

Germination of witchweed ( Striga asiatica [L.] Kuntze), an important parasitic weed on several poaceous crops, is stimulated by several synthetic and natural compounds. We investigated the role of ethylene biosynthesis and action in cytokinin-induced germination. Conditioned Striga seeds treated with distilled water, 1-aminocyclopro-pane-1-carboxylic acid (ACC) or the cytokinins thidiazuron (TDZ), trans zeatin (TZ), benzyladenine (BA) and kinetin (KIN) produced little ethylene. Treatments with cytokinin-ACC combinations enhanced ethylene production. The relative order of activity of the cytokinins in elicitation of the phytohormone was TDZ > TZ > BA > KIN. Germination in response to distilled water and ACC treatments was negligible. Induction of germination by cytokinins varied from low (0%) to moderate (52%). Seeds treated with cytokinin-ACC combinations displayed high rates of germination. The observed germination was positively correlated (γ= 0. 8 and 0. 9) with ethylene production. Germination was reduced by silver thiosulphate (STS) and CoCl₂, inhibitors of ethylene action and ACC oxidase, respectively. Aminoethoxyvi-nylglycine (AVG), an ACC-synthase inhibitor, reduced TDZ-induced Striga germination. However, the inhibitory effect of AVG was overcome by addition of ACC. The results are consistent with a model in which Striga germination and embryo growth are limited by low capacity of the seeds to oxidize ACC. The cytokinins promote ACC conversion into ethylene and consequent Striga germination by enhancing ACC oxidase activity and/or synthesis.     

The beneficial effect of small toxic molecules on dormancy alleviation and germination of apple embryos is due to NO formation

Deep dormancy of apple (Malus domestica Borkh.) seeds is terminated by a 3-month-long cold stratification. It is expressed by rapid germination of seeds and undisturbed growth of seedlings. However, stimulation of germination of isolated apple embryos is also observed after applying inhibitors of cytochrome c oxidase: nitric oxide (NO) or hydrogen cyanide (HCN) during the first 3–6 h of imbibition of dormant embryos. The aim of this work was to compare the effect of yet another toxic gaseous molecule carbon monoxide (CO) with the effects of HCN and NO on germination of apple embryos and growth and development of young seedlings. We demonstrated that stimulation of germination after short-term pre-treatment with HCN, NO or CO was accompanied by enhanced NO emission from the embryo axes during their elongation. Moreover, similarly high NO production from non-dormant embryos, after cold stratification, was detected. Therefore, we propose that NO may act as signaling molecule in apple embryo dormancy break.     

The role of gibberellic acid (GA3) in the removal of dormancy inFraxinus excelsior L. seeds

The seeds ofFraxinus excelsior L. were stratified at 17-20 °C (warm stratification), at 4-6 °C (cold stratification) and at alternating temperature (warm — cold stratification). The seeds subjected to warm stratification only, remained dormant. The seeds stratified only at 4-6 °C germinated gradually during a long period of time. The seeds subjected to warm — cold stratification, however, germinated with great intensity within a relatively short period of time. GA₃ was shown to stimulate the growth of embryos markedly, and its effect on the germination of seeds depended on the temperature of stratification. GA₃ applied during the cold stratification accelerated the removal of dormancy by shortening the period of stratification and by influencing the germination of seeds. The results obtained indicate a similarity between the effect of temperature 17-20 °C during the warm stratification and that of gibberellic acid. Both those factors applied separately affect favourably after-ripening of the embryos and accelerate the germination of seeds.     

Dormancy Release,Germination, and Electrolyte Leakage from Apple Embryos during Stratification in the Presence of Sucrose

The dynamics of dormancy release during the stratification of apple (Malus domestica Borkh.) seeds was quantitatively described by three characteristics of seeds germination: the percentage of seeds that germinated by the tenth day, mean germination time, and the sum of seeds germinated in each of ten days (Timson's parameter), which allowed the assessment of the viability, the rate of dormancy release, and seed heterogeneity. We showed that apple seeds were characterized by a combined (physical and physiological) type of dormancy, with the seed coat and the embryo envelope being involved in the maintenance of physical dormancy. The addition of sucrose to the stratification medium accelerated the release of seed dormancy and improved all characteristics that determine seed germinability. Electrolyte leakage from embryos hardly changed during stratification, which agrees with the fact that all seeds remained viable throughout the entire period of dormancy. We assume that the release of seed dormancy is not a single-stage process.     

The role of lipases in the removal of dormancy in apple seeds

Summary It was found that the temperature optimum for apple (Malus domestica Borb.) seed acid lipase is the same as that for seed after-ripening process. The activity of the enzyme occurs between the 40th and 70th days of stratification, whereas the activity of alkaline lipase very low at that time appears about 20 days later. The changes of both enzyme activities were also studied during dark and light culture of embryos isolated from seeds after different times of stratification. Only the alkaline enzyme activity is under the control of light. It was concluded that essentially the same process, i.e. the hydrolysis of reserve fats is catalysed by two different enzymes: acid lipase acting during the cold-mediated breaking of embryo dormancy and alkaline lipase acting during the germination of dormant embryos, thus being under light control.     

Dormancy removal of apple seeds by cold stratification is associated with fluctuation in H2O2, NO production and protein carbonylation level

Reactive oxygen (ROS) and nitrogen (RNS) species play a signaling role in seed dormancy alleviation and germination. Their action may be described by the oxidative/nitrosative “window/door”. ROS accumulation in embryos could lead to oxidative modification of protein through carbonylation. Mature apple (Malus domestica Borkh.) seeds are dormant and do not germinate. Their dormancy may be overcome by 70–90 days long cold stratification. The aim of this work was to analyze the relationship between germinability of embryos isolated from cold (5 °C) or warm (25 °C) stratified apple seeds and ROS or nitric oxide (NO) production and accumulation of protein carbonyl groups. A biphasic pattern of variation in H₂O₂ concentration in the embryos during cold stratification was detected. H₂O₂ content increased markedly after 7 days of seeds imbibition at 5 °C. After an additional two months of cold stratification, the H₂O₂ concentration in embryos reached the maximum. NO production by the embryos was low during entire period of stratification, but increased significantly in germination sensu stricto (i.e. phase II of the germination process). The highest content of protein carbonyl groups was detected after 6 weeks of cold stratification treatment. Fluctuation of H₂O₂ and protein carbonylation seems to play a pivotal role in seed dormancy alleviation by cold stratification, while NO appears to be necessary for seed germination.     

Influence of Different Cytokinins on the Germination of Lettuce (Lactuca sativa) and Celery (Apium graveolens) Seeds

The naturally occurring cytokinins, zeatin, zeatin riboside and dihydrozeatin did not promote the germination of celery (Apium graveolens L.) seeds and 6-Δ²-isopentenyladenine (2iPA) and its riboside were only moderately active. Of the synthetic cytokinins, kinetin, kinetin riboside, and the disubstituted urea, N-phenyl-N′-pyridyl urea (NC5392) were moderately active, and 6-benzyl-aminopurine (BA) and its derivatives BA riboside and 6-benzyl-amino-9(tetrahydropyran-2yl)purine (SD8339) were the most active cytokinins tested. 6-(o-hydroxybenzyl)aminopurine (hyd-BA) and its naturally occurring riboside inhibited germination under normally inductive conditions. All the cytokinins examined were more active in promoting germination of lettuce (Lactuca sativa L.) than celery seeds. BA, BA riboside and SD8339 were again the most active cytokinins. In contrast to the results with celery, zeatin and zeatin riboside were highly active. The other cytokinins also showed high activity with the exception of dihydrozeatin, hyd-BA and hyd-BA riboside which were less active. Cytokinin ribosides were less active than the corresponding free bases during the early period of the lettuce seed incubation but total germination after 90 h was similar.     

Cold stratification and growth of radicles of loblolly pine (Pinus taeda) embryos

The mechanism of germination enhancement by cold stratification was examined in seeds of loblolly pine ( Pinus taeda L. ), Removal of the seed coat permitted elongation of radicles from unstratified embryos, but both rates of germination and radicle elongation were increased by stratification. Radicles of both stratified and unstratified embryos excised from the megagamethophyte elongated only when in contact with solid incubation media supplemented with sucrose. Stratification of embryos either in the presence or absence of the megagametophyte resulted in similar enhancement of radicle elongation. Elongation rates of radicles were increased after stratification independent of sucrose concentration, and changes in sucrose content in the megagamethophyte during stratification or incubations subsequent to stratification were insufficient to regulate radicle growth. Our results support the hypothesis that the embryos of pine seeds perceive the low temperature stimulus directly and this stimulus results in a growth potential increase in the embryonic axes. We propose that this growth potential increase enables the embryos to overcome the mechanical restraint of the seeds coats and to germinate.     

Jasmonic acid promotes germination and lipase activity in non‐stratified apple embryos

The effects of (−)jasmonic acid (JA) on germination of embryos isolated from dormant seeds of apple ( Malus domestica Borb. cv. Antonówka) cultured in darkness or at 12‐h photoperiod were studied, as well as its effects on the activity of alkaline lipase (AlkL, EC 3.1.1.3) in these embryos. The maximum sensitivity of germination to JA occurred on days 3 and 4 of embryo culture. Both germination and enzyme activity were stimulated by JA, its effect being additive to that of light. Inhibitors of lipoxygenase inhibited embryo germination and AlkL activity, both effects being partially reversed by JA treatment. We suggest that (1) JA is implicated in an endogenous complex controlling apple seed germination, and that (2) it acts independently of the mechanism triggered by light.     

The interaction of abscisic acid with growth stimulators in germination of partially after-ripened apple embryos

The effect of abscisic acid (ABA), gibberellin A₄₊₇ and benzyladenine on germination of apple embryos which had been stratified for different periods of time was investigated. The growth stimulators accelerated the germination of non-stratified embryos or those stratified for a short time. After a longer period of stratification, the effect of growth stimulators was visible only when germination of the embryos was artificially inhibited by simultaneous application of synthetic ABA. The sensitivity of stratified apple embryos to the exogenous ABA diminished with the number of hours elapsed between the imbibiting by the embryos of the water or gibberellin and the application of the ABA. This was similar to the change in the sensitivity of apple embryos during stratification due to the increasing concentrations of ABA.     

Free and conjugated gibberellins in dormancy and germination of apple seeds

Halińska, A. and Lewak, St. 1987. Free and conjugated gibberellins in dormancy and germination of apple seeds.
The presence of gibberellin A₄ (GA₄) was confirmed in partly stratified seeds of apple ( Malus domestica Borb., cv. Antonówka) by mass spectrometry of the methyl ester. Levels of free and conjugated gibberellins A₄₊₇ and A₉ changed during drying of mature seeds, during cold and warm stratification, as well as during germination of dormant and non-dormant embryos. The temporary rise in GA₄₊₇ during cold stratification and during the culture of dormant embryos as well as the lack of it under conditions of warm stratification, allowed us to postulate a role for GA₄₊₇ in the removal of dormancy. In addition, GA₉ was absent in dormant embryos and increased during cold stratification and during the culture of non-dormant embryos. This suggests the involvement of GA₉, in induction of normal development of the seedling. The equivalence between changes in free and conjugated GAs suggests that formation and hydrolysis of conjugates are involved in the control of the physiologically active levels of free GA₄₊₇ and GA₉.     

Polyamines and ethylene in the removal of embryonal dormancy in apple seeds

Putrescine (Put), spermidine (Spd) and spermine (Spm) were found in seeds of apple ( Malus domestica Borkh. cv. Antonovka), in amounts that increased in the order given. The levels slowly decreased during 30 days of stratification. Exogenous polyamines (PAs) affected germination of isolated embryos in a way dependent on the type of polyamine, its concentration, and the state of the embryo dormancy. The effect of Put and Spd on germination was stimulatory, while that of Spm was inhibitory. Stimulation of germination was also observed when embryos were treated with arginine, ornithine and methionine. Canavanine inhibited germination, and this effect was reversed by arginine or Put. Ethephon, aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) present during seed stratification had no effect on the levels of endogenous PAs. Put and Spd did not change ethylene production, neither during seed stratification nor during embryo germination, whereas Spm reduced ethylene evolution. The data suggest that Spm plays a role in the maintenance of dormancy by preventing ethylene production, while Put and Spd participate in dormancy removal, independently of ethylene.     

Somatic embryogenesis in Agave tequilana Weber cultivar azul

Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(γ-γ-dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 μM BA) compared to auxin (4.5 μM 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 μM 2,4-D and 1.3 and 4.0 μM BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.     

Relationships among cytokinins, ethylene and poly amines during the stratification-germination process in seeds of Acer saccharum

Relatively high levels of dihydrozeatin and trans-zeatin were detected in unstratified seeds of Acer saccharum Marsh. Both cytokinins increased substantially over the first 20 days of stratification at 5°C and then fell rapidly to values well below original levels by first germination on day 55. In seeds held at 20°C, a non-afterripening temperature, cytokinin levels remained constant for the first 10 days and then declined to their lowest levels by day 20. Levels of putrescine, spermidine and spermine in the radicles and cotyledons did not change during the full course of the afterripening process, but large increments were noted during radicle emergence. A large increase in ethylene production at germination suggests that competition for S-adenosyl-methionine by the ethylene and polyamine biosynthetic pathways did not inhibit synthesis of ethylene or polyamines during seedling emergence and establishment. In seeds stratified at 20°C, ethylene showed an exceptionally large peak early in the stratification period, but polyamine contents remained low throughout the test. The present results are consistent with the hypothesis that cytokinins play a significant role in overcoming the metabolic block present in dormant seeds. This conclusion is supported by data showing that high levels of cytokinins develop concurrently with the start of tissue differentiation and at the time when abscisic acid and phenolic inhibitors decline in stratifying seeds. Changes in ethylene and polyamine contents did not correlate with any events in the afterripening process; however, large increases in levels of these substances were closely associated with the germinative process and, in the case of polyamines, specifically with the start of cell division.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Nitric oxide,hydrogen cyanide and ethylene are required in the control of germination and undisturbed development of young apple seedlings

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are emerging as important regulators of plant development (germination, flowering, senescence), acting as secondary messengers in cooperation with classical phytohormones. Apple seeds are dormant, unless they undergo a 3 month long cold stratification. Deep dormancy of isolated apple embryos can also be broken by short pre-treatment with HCN or NO with the effect associated with enhanced ethylene synthesis. Non-dormant embryos germinate well and young seedlings grown from non-dormant embryos do not exhibit any morphological anomalies, such as asymmetric growth and greening of cotyledons. One of the aims of this work was to investigate the correlation between RNS- mediated (HCN- and NO-dependent) dormancy removal and ROS (H₂O₂ and O₂^−•) accumulation in the embryos. The beneficial effect of NO and HCN on germination of dormant apple embryos has been associated with marked increases in H₂O₂ and O₂^−• concentration in the embryos at early germination stages. We also analyzed growth of young seedlings developed from embryos pre-treatment with HCN or NO or exposed to ethylene (ethephone) and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC). ACC and ethephone removed all morphological anomalies of the seedlings (asymmetric growth and greening of cotyledons) but the radicle growth was rather slight. We propose that accumulation of ROS provoked by HCN and NO pre-treatment is required for embryo germination “sensu stricto”, while ethylene is required for post-germination seedling growth.     

The dynamics of indole-3-acetic acid in Acer platanoides seeds during stratification and germination

During stratification at 5°C indole-3-acetic acid (IAA) levels in embryos of Acer platanoides decreased during the early stages but subsequently increased again throughout the remainder of a 144 day period. The reduction in IAA levels in embryos of fruits stored at 17°C was even more pronounced, and in addition, no increase was observed after longer storage periods at this temperature, the levels of IAA remaining very low. Germination in seeds maintained at 5°C was not observed until after 120 days or longer, but germination potential increased at an earlier stage, as shown by the fact that seeds transferred to 20°C gave appreciable increases in germination after much shorter chilling periods. Endogenous IAA levels in embryos from seeds transferred to 20°C after a chilling period, long enough to break dormancy, increased within 24 h, i.e. before visible germination, to levels similar to those observed in embryos from seeds chilled continuously for 144 days. Embryos from seeds chilled for 120 days, i.e. when the samples already showed visible germination and when the endogenous IAA content was already high, showed no further increase in endogenous IAA during a three day incubation at 20°C. None of the treatments employed was effective in inducing germination of seeds or embryos from fruits stored at 17°C.