Bud abortion in tulip bulbs studied by magnetic resonance imaging

After storage and subsequent planting of flower bulbs, the flower bud frequently appears to be aborted. This physiological aberration is probably caused by a change in the water status of the bulb and may be initiated during storage. The development of bud abortion in tulip bulbs was studied during long-term dry storage of the bulbs at 5 degrees C. The anatomy of individual tulip bulbs was followed non-invasively with T2-weighted NMR imaging, which allowed the monitoring of the growth of the shoot and daughter bulbs. Quantitative maps of T1 and T2 relaxation times of individual bulbs were used to assess regional changes in the water status of different tissues. Parallel to the NMR measurements, bulbs were planted to assess the ultimate flower quality. Moreover, water content, osmolality of tissue sap and ion leakage of excised shoot and scale tissues were determined to obtain information about the water status and viability of the bulbs. Significant decreases during long-term storage were found in T1 and T2 relaxation times in the shoot and particularly in the stamens. An increase in the osmolality of tissue sap and the decrease in relaxation times in the shoot below a certain threshold value attained after 24 weeks of storage, could be indicative for the emergence of bud abortion in tulips.     

Temperature dependent redistribution of organic nitrogen during 'dry' storage of tulip bulbs cv. Apeldoorn

Tulip bulbs cv. Apeldoorn are dry stored at 5°C for 12 weeks to ensure subsequent optimal flowering when planted in the greenhouse at higher temperatures of 17–20°C. Both temperature and duration of the cold treatment determine the subsequent rate of the shoot elongation, the time until anthesis and the flower size, pigmentation and water content. In search for cold-specific physiological changes, possibly related to the development of the potential of proper flowering (flowering preparation), we studied the redistribution of organic nitrogen in both cooled (5°C) and non-cooled (17°C) bulbs.
During 12 weeks of dry storage, the total protein- and free amino acid-nitrogen content decreased in the scales, whereas the opposite was found in the basal plate (with root primordia) and the shoot. In the shoot, this occurred significantly more at 17°C than at 5°C. At the same time, there was a tissue-specific change in the free amino acid composition in both cooled and non-cooled bulbs. Changes specific for the 5°C treatment were only found for the alanine content, in both the basal plate (with root primordia) and the shoot, and for the proline, asparagine, threonine, glycine and isoleucine content, in the shoot only. These changes are, for the greater part, completed within the first 6–8 weeks of dry storage. Bulbs stored for such a short period of time at 5°C still show flowering disorders. Thus, flowering preparation is only partly accompanied by changes in free amino acid contents.     

Exogenous GA<Subscript>3</Subscript> overcomes bud deterioration in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) bulbs during dry storage by promoting endogenous IAA activity in the internodes

Endogenous free IAA was examined with an immunohistochemical method for its involvement in the reduction of bud deterioration after GA₃ was injected into the bulbs. We found that tulip bulbs stored at 20°C constantly developed severe bud deterioration, whereas the symptoms of deterioration was lighter in the bulbs with GA₃ injection and not observed in the bulbs with 4°C treatment. 73% success in overcoming bud deterioration was achieved in 20°C with GA₃ treatment after 8 weeks of bulb storage, and the success rate was 7% after 12 weeks of storage. IAA was detected in the parenchyma cells in the internodes of the shoot after the bulbs were stored at 4°C or at 20°C with GA₃ injection for 4 weeks, but little was detected in the bulbs stored at 20°C constantly. Moreover, a weak IAA signal was present in between the cells of the internodes irrespective of bulb treatment. After planting, the bulbs that had been treated differently exhibited different flowering ability. The bulbs stored at 4°C for 4, 8 and 12 weeks attained high flowering percentage, which was lower in the 20°C with GA₃ treatment and lowest in the 20°C treatment. It may be concluded that GA₃ injection decreases bud deterioration of tulip bulbs during dry storage at 20°C by promoting the endogenous IAA in the internodes.     

Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of <Emphasis Type="Italic">Fritillaria imperialis</Emphasis> L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with higher level of cytokinin (1 mg l⁻¹ BAP). In contrast, auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with lower level of cytokinin (0.1 mg l⁻¹ BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.     

Carbohydrate Status of Tulip Bulbs during Cold-Induced Flower Stalk Elongation and Flowering

The effect of a cold treatment on the carbohydrate status of the scales and flower stalk of Tulipa gesneriana L. cv Apeldoorn bulbs during growth after planting was studied and compared with bulbs not given cold treatment. Bulbs were stored dry for 12 weeks at 5[deg]C (precooled) or 17[deg]C (noncooled). Only the 5[deg]C treatment led to rapid flower stalk elongation and flowering following planting at higher temperatures. Precooling enhanced mobilization of starch, fructans, and sucrose in the scales. The cold-stimulated starch breakdown was initially accompanied by increased [alpha]-amylase activity per scale. In noncooled bulbs, [alpha]-amylase activity slightly decreased or remained more or less constant. Cold-induced flower stalk elongation was partially accompanied by a decrease in the sucrose content and an increase in the glucose content and invertase activity per g dry weight. The starch content in internodes initially decreased and subsequently increased; [alpha]-amylase activity per g dry weight of the lowermost internode showed a peak pattern during starch breakdown and increased thereafter. The internodes of noncooled bulbs, on the contrary, accumulated sucrose. Their glucose content and invertase activity per g dry weight remained low. Starch breakdown was not found and [alpha]-amylase activity per g dry weight of the lowermost internode remained at a low level. Precooling of tulip bulbs thus favors reserve mobilization in the scales and flower stalk and glucose accumulation in the elongating internodes.     

The accumulation and metabolism of plant growth regulators during organogenesis in cultures of thin cell layers of Nicotiana tabacum

The accumulation and metabolism of exogenously applied and endogenously produced auxins and cytokinins were studied in relation to organogenesis in thin cell layers of Nicotiana tabacum L. It was shown that, in order to obtain maximal flower bud formation, both exogenous auxin and cytokinin needed to be present during the first 4 days of culture (to the formation of a subepidermal meristematic zone) whereas cytokinins needed to be present for at least 4 days more (until formation of organogenic centres). Explants taken from floral branches have higher endogenous indole-3-acetic acid (IAA) levels compared with explants from the basal part of the stem which form only vegetative buds. This might be related to a different IAA metabolism in these two types of explants as was shown by the different accumulation of exogenously applied IAA. Both 'floral' and 'vegetative' cells layers contained comparable amounts of zeatin riboside (ZR) as their major cytokinin. Free bases, zeatin (Z) and dihydrozeatin [(diH)Z], given exogenously, were largely metabolised to their respective ribosides. The observation that Z was less effective than (diH)Z in the induction of flower buds could be related to (diH)ZR apparently not being a substrate for cytokinin oxidase.     

Effects of Temperature on Cold Acclimation and Deacclimation in Flower Buds of Evergreen Azaleas

The effects of various storage temperature/duration combinations(5, 10 and 17°/4, 8, 12 and 16 weeks) on cold acclimationand deacclimation of flower buds were studied in four speciesof evergreen azaleas having different natural distribution andcold hardiness. The freezing process and the exotherm temperaturedistribution of florets in excised whole buds determined bydifferential thermal analysis were used as the diagnostics todetermine the degree of bud acclimation and deacclimation. Theacclimation in buds lasted for as long as 12 to 16 weeks at5°C storage, and from 8 to 12 weeks at 10°C, and itappeared to be maintained after the chilling requirement forbreaking bud dormancy had been satisfied. Therefore, bud acclimationseems to be maintained independently from bud dormancy. Thedehardening effect on acclimated buds occurred as a result ofshort exposures to higher temperatures or long exposures tolower temperatures, and there was no relation between the rateof deacclimation and the degree of hardiness in each species.Among three storage temperatures examined, 5°C was the mosteffective for the maintenance of cold acclimation in flowerbuds and the small difference of floret water contents at 5and 10°C storage is not significant. (Received August 28, 1982; Accepted February 4, 1983)     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Developmental changes and water status in tulip bulbs during storage: visualization by NMR imaging

Magnetic Resonance Imaging (MRI) and light and scanning electron microscopy (SEM) were used to follow time-dependent morphological changes and changes in water status of tulip bulbs (Tulipa gesneriana L., cv. 'Apeldoorn') during bulb storage for 12 weeks at 20 degrees C (non-chilled) or 4 degrees C (chilled) and after planting. MR images reflecting the water content, the relaxation times T1 and T2 (or their reciprocal values, the relaxation rates R1 and R2), and the apparent self-diffusion coefficient of water molecules (ADC), were obtained for intact bulbs. After planting, scape elongation and flowering occurred only in chilled bulbs, while elongation in non-chilled bulbs was retarded. Microscopic observations showed different structural components and high heterogeneity of the bulb tissues. MRI revealed the elongation of the flower bud during storage, which was significantly faster in the chilled bulbs. In addition, MRI demonstrated a redistribution of water between different bulb organs, as well as significant differences in the pattern of this redistribution between the chilled and non-chilled bulbs. Generally, R2 relaxation rates became faster in all bulb organs during storage. At the same time, ADC values remained constant in the chilled bulbs, while exhibiting a significant increase in the non-chilled bulbs.     

The Influence of Different Cytokinins and Auxins on Flower Neoformation in Thin Cell Layers of Nicotiana tabacum L.

Neoformation of flower buds was studied in thin cell layersderived from floral branches of Nicotiana tabacum L. Organogenesiswas found to be expressed in different ways according to thekind of auxin and cytokinin present in the culture medium. Inthe presence of synthetic auxins (NAA, CTA), more flower budswere generated than with the natural auxins (IAA, IBA), if theculture medium was also supplemented with a cytokinin. Theseconditions also stimulated the development of the buds to flowersor inflorescences. The most active cytokinin was BA. Naturalcytokinins (zeatin, IPA) appeared much less active but led toremarkable development of roots together with flower buds. Anorganogenetic effect of 2,4-D was observed in this experimentalsystem, leading to the conclusion that organogenetic activityof cytokinins and auxins in thin cell layer cultures stronglydepends on the molecular structure of the growth regulators. (Received September 30, 1987; Accepted March 23, 1988)     

In vitro flower bud formation in tobacco: interaction of hormones

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate.     

Changes in free polyamines in Tulipa gesneriana flower stalks during dry-storage and after planting of bulbs

Tulip bulbs cv. Apeldoorn are dry-stored at 5°C for 12 weeks to ensure sufficient elongation of the flower stalk, when subsequently planted at higher temperatures (17–20°C). To investigate whether free polyamines are involved in this process, flower stalk internodes were analyzed during dry-storage and after planting of the bulbs.During dry-storage for 12 weeks at 5°C (cooled) and 17°C (non-cooled), the free putrescine, spermidine and spermine amounts per flower stalk increased. The putrescine amount increased at 5°C significantly more than at 17°C, whereas the opposite was found for the spermine amount. These differences developed early during dry-storage and disappeared rapidly at subsequent higher temperatures.After planting, the lower- and uppermost flower stalk internodes of the pre-cooled bulbs elongated much faster than those of the non-cooled ones. In the pre-cooled bulbs, the free polyamine amounts per internode increased with time after planting, but the time course of these changes was different. In the non-cooled bulbs, the free polyamine amounts increased to a much lesser extent or remained more or less constant.It is argued that the observed changes in the free polyamine contents are probably not required for the cold-induced extension growth of tulips cv. Apeldoorn.Abbreviations PA polyamine - Put putrescine - Spd spermidine - Spm spermine     

Regeneration of calabrian pine from juvenile needles

Cytokinins applied in an agar medium induced adventitious buds on cultured needles from seedling of Pinus brutia Ten. Cytokinins applied as pulses to the explants prior to culture were less effective. Irrespective of the mode of cytokinin application, 8 weeks was the time required to bring about bud formation. Organogenetic potential of the cultured needles decreased with chronological age of the explanted seedlings. The induced buds grew into elongated shoots on culture medium without cytokinins, but the inclusion of activated charcoal (1%) doubled the elongation rate. There was an indication that mixtures of cytokinins were more effective than separate cytokinins in producing buds on explants, but the difference between treatments did not achieve statistical significance. Parenchyma cells in mesophyll layers were evidently the target cells responding to the culture conditions. After a period of activity and division in these cells, meristematic zones developed which later led to formation of bud primordia, and subsequently these primordia developed into well-formed adventitious buds. Subsequent rooting (64%) of shoots was achieved using a combination of two auxins and a low level of cytokinin.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

Development of flower buds in thin-layer cultures of floral stalk tissue from tobacco: Role of hormones in different stages

The in vitro development of flower buds was studied on tissue explants of epidermis and subepidermal cortex from the flower stalks of Nicotiana tabacum L. cv. Samsun. The number of flower buds formed depended mainly on cytokinin concentration. Auxin acted as a modifier in a complex way. In early development, NAA at 1 μ M decreased the number of buds initiated and delayed bud emergence. At a later stage, auxin promoted bud outgrowth at the same concentration. Optimal results were obtained when explants were first incubated at low auxin concentration for 3–5 days and subsequently transferred to an elevated auxin level. Physiological processes that lead to flower bud initiation start very soon after the onset of incubation. This was inferred from experiments whereby explants were first cultured at an inductive cytokinin concentration and then transferred to a non-inductive hormone level.     

The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies

Resting vegetative buds of Picea abies collected from phytotron-grown rooted cuttings of 24-year-old trees or a 12-year-old hedge were tested for their capacity to form adventitious buds after various cytokinin treatments. The most effective method for obtaining a high yield of adventitious buds within 8 weeks was to pulse treat the buds in 250 M BA for 3 h and then culture them on medium containing 5 M each of BA and kinetin for 1 week. The developmental pattern for adventitious bud production, with the formation of 10 to 20 adventitious buds per bud, was similar for all tested genotypes, although the number of buds giving rise to adventitious buds varied significantly. The capability of some clones to form adventitious buds was correlated to endogenous cytokinin content. The clone which contained most endogenous cytokinin in its resting bud had the highest potential for adventitious bud formation.     

Endogenous Cytokinins in Flower Bud Forming Explants of Tobacco

The accumulation of endogenous cytokinins was studied in pedicelexplants of tobacco (Nicotiana tabacumL.) during regenerationof flower buds in vitro. Maximal bud formation was induced onmedia containing 1.0 mmol m^–3 of benzyladenine or dihydrozeatin.No buds were formed in the absence of cytokinin. The levelsof dihydrozeatin, zeatin, and the corresponding ribosides weredetermined in explants cultured in the presence or absence ofcytokinin by means of a competitive ELISA technique. In explantsincubated without a cytokinin, only the dihydrozeatin concentrationincreased significantly during the first day of incubation anddecreased during the second day. No increase was observed inexplants incubated in the presence of benzyladenine. The concentrationof dihydrozeatin in these bud-forming explants was only 10 to15% of the concentration built up in explants cultured on dihydrozeatininstead of benzyladenine. This suggests that the endogenouscytokinins only play a minor role in the regeneration of flowerbuds in vitro. Key words: cytokinin, flower bud development, tissue culture, tobacco     

Changes in the carbohydrate and hormonal status in <Emphasis Type="Italic">Tulipa bifloriformis</Emphasis> bulbs forced into bloom in a greenhouse and in the open ground

Time-course of ABA, cytokinins, monosaccharides (MS), and water-soluble polysaccharides (WSP) contents were followed during cold dormancy period in the bulbs of tulip (Tulipa bifloriformis Vved.) that stayed over winter in the open ground or were cold-forced into bloom in a greenhouse. In both cases, the level of monosaccharides and water-soluble polysaccharides in the storage scale tissues increased, whereas the MS/WSP ratio in the bulbs planted in the open remained essentially the same and in case of forcing treatment decreased almost fivefold. In the apical bud tissues, the level of monosaccharides also rose in both cases, but the MS/WSP ratio in the open was greater throughout the whole experiment. The level of cytokinins in the apical bud tissues in the open was higher than in the forcing case, although the changes in their total content were identical following both treatments. Following the forcing treatment, the contents of free and bound ABA in the apical bud tissues increased reaching their peaks by the end of cold period. In the open, there were two peaks of free ABA: in October (when early frosts occurred) and in March (at the end of the wintering period). The winter forcing treatment resulted in rapid depletion of energy and plastic resources in T. bifloriformis and the death of 20% of embryonic flower buds (in the open, all flower buds survived). Nevertheless, plant adaptation potential ensured the development of generative shoots with 4–5 normal flowers, which makes it possible to use this species of multiflorous tulip for winter forcing in a greenhouse.     

The effect of LAB 173711 and ethephon on time of flowering and cold hardiness of peach flower buds

Peach flowers are often killed during bloom by spring frosts. LAB 173711, a compound with abscisic (ABA)-like activity, and ethephon delayed flowering in peach trees. In greenhouse experiments, LAB 173711, at concentrations of 10^?3–10^?2 M, was most effective in delaying bloom when applied after a 5°C cold storage period, rather than before the dormancy breaking treatment. In contrast, ethephon delayed bloom most effectively when applied before 5°C cold storage; ethephon caused flower bud abscission when treatments were made after the chilling requirement had been satisfied. In field experiments, ethephon delayed flowering by 6–7 days, which reduced bud injury after a spring frost during bloom. No flower bud injury was found on ethephon-treated trees after temperatures of ?4.3°C; whereas without ethephon 25% of the flower buds were frost damaged. LAB 173711 delayed the time to 50% bloom by 2–3 days. However, this was not long enough to avoid low-temperature injury to the flower buds.