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.     

Changes in Parameters of the Plasmalemma ATPase during Peach Vegetative Bud Dormancy

Plant dormancy and dormancy breaking depend, at least partially, on close relationships between buds and tissues underlying bud (bud stands). In Prunus persica, the dormancy was related to high nutrient absorption in bud stands linked to high plasmalemma ATPase (EC 3.6.1.3) activity. Two plasmalemma fractions was isolated from peach vegetative buds and bud stands using aqueous phase partitioning and ultracentrifugation. Results of markers enzyme assays indicated that both plasmalemma enriched fractions obtained were highly purified. During the dormancy period plasma membrane ATPase amount and activity were higher in bud stands than in buds. Moreover, assays performed at different temperatures (4, 18, 30 °C) indicated modifications of kinetic parameters (K_m, V_m) in both tissues during dormancy release. In buds, from November to February, K_m declined at 4°C and increased at 30 °C whereas no changes was measured at 18 °C and V_m increased at all temperature. In bud stands, no changes of K_m was measured at 4 °C and 18 °C whereas an increase occurred at 30 °C and V_m decreased at all temperature. According to the results, it can be postulated that dormancy release in peach-tree could be related to modifications of plasma membrane ATPase properties, in buds and bud stands, during winter time.     

Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid, IAA-indole-3-acetic acid     

Dormancy in Peach (Prunus persica L.) Flower Buds : I. Floral Morphogenesis and Endogenous Gibberellins at the End of the Dormancy Period

Flower buds of peach (Prunus persica L.) trees, cv Novedad de Cordoba (Argentina), were collected near the end of the dormant period and immediately before anthesis. After removal of scale leaves, morphological observations of representative buds, made on transverse and longitudinal microtome sections, showed that all verticils making up the flower are present in an undifferentiated form during the dormant period (June). Flower buds collected at the end of dormant period (August) showed additional growth and differentiation, at which time formation of two ovules was beginning in the unicarpelar gynoecium. Dehiscence of anthers had not yet occurred 10 days before full bloom, and the ovules were still developing. Free endogenous gibberellin (GA)-like substances were quantified by bioassay (Tan-ginbozu dwarf rice microdrop) after SiO₂ partition column chromatography, reversed phase C18-high performance liquid chromatography, and finally Nucleosil [N(CH₃)₂]high performance liquid chromatography. Bioactive fractions were then subjected to capillary gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). Gibberellins A₁, A₃, and A₈ were tentatively identified in peach flower buds using GC-SIM and Kovat's retention indices, and relative amounts approximated by GC-SIM (2:8:6 for GA₁, GA₃, and GA₈, respectively). The highest concentration (330 nanograms per gram dry weight) of free GA₁/GA₃ was found in dormant buds (June) and diminished thereafter. The concentration free of GA₁/GA₃ did not increase immediately prior to bud break. However, high GA₁/GA₃ concentrations occurred during stages where rate of growth and cellular differentiation of (mainly fertile) verticils can be influenced.     

Gibberellin, daylength and temperature effects on floral and vegetative development in white clover

The effects of gibberellin A₃ (GA₃) application on five white clover populations was assessed in both glasshouse and controlled environments. Daylength, temperature and GA₃ interactions were also examined. Gibberellin A₃ did not induce vegetative plants to flower when daylength and temperature requirements were not met. In flowering plants, GA₃ increased the number of reproductive buds per stolon and peduncle length, but did not affect other floral characters. Gibberellin A₃ depressed total stolon numbers, but increased the number of nodes per stolon, internode length, leaf area and petiole length.     

A comparative study of genotypic and environmental response to androgenesis in Nicotiana rustica

Summary A total of six genotypes of Nicotiana rustica comprising the two F₁'s (V₂ × V₁₂ and V₁ × V₅) and their parents were evaluated for their efficiency in haploid production. Excised immature flower buds with pollen at late uninucleate to early binucleate stage were pretreated for 21 days at 5 ° or 7 °C, or for 15 days at 9 °C before culturing on Nitsch's medium+ 0.1 mg/l NAA. The effects of genotype, pretreatment and their interaction were tested on anther response, anther productivity and days to first plantlet formation. Highly significant genotype X pretreatment interaction and differences between genotypes were observed for all three characters. Significant differences between pretreatments were observed for anther productivity only. The performance of V₁₂ both in respect of anther productivity and response was highest whereas that of V₅ was the lowest. Analysis of variance showed that a simple additive genetic model was not adequate to explain the above variation due to significant additive genetic and dominance interactions with the pretreatment.     

Seasonal variation in the competence of the buds of three cultivars from different Citrus species to flower

The role of bud competence in the determination of flowering seasonality was studied in three Citrus cultivars, Bearss lime (Citrus latifolia Tan.), Fino lemon (C. limon [L.] Burm. f.) and Owari satsuma (C. unshiu (Mak.) Marc.), which differ in their adaptation to hot climates and their propensity to produce off-season blooms. Potted plants were kept in a greenhouse under non-inductive conditions (minimum temperature higher than 20°C), and periodically the flowering response was determined of a group of trees exposed for 30 days to an inductive temperature regime (15/8°C). A seasonal change in bud competence was demonstrated, and both bud sprouting and flower formation were highest when the low temperature regime was imposed during February and March. During the summer months, the low temperature regime resulted in a small increase in bud sprouting as compared to non-chilled trees, but only vegetative buds developed and no flowers were formed. The influence of environmental factors on the determination of bud competence was further studied. No effect of photoperiod was found, but raising the minimum air temperature above 25°C during 60 days, eliminated bud competence in Owari satsuma. In Bearss lime trees, the buds reacquired the competence after 4 months at 25/20°C, a temperature regime that does not induce flower formation. The reacquisition of competence was much faster at a lower temperature (15/8°C). A consistent relationship between the flowering response and DNA methylation in buds could not be demonstrated in all cultivars.     

Dormancy in peach (Prunus persica L.) flower buds

Summary Peach buds (floral and vegetative) were periodically collected from midsummer until the spring flowering and sprouted under continuous light, 100% relative humidity and 20–25°C. Treatments with 200 ppm gibberellin A₃ (GA₃) or chilling (2–4°C for 30 days before planting) were applied. Vegetative buds showed well-defined phenological stages: pre-dormancy, true dormancy, and end of dormancy. Both GA₃ and chilling treatments shortened the sprouting times of vegetative dormant buds close to those in predormancy. Isolated floral buds were irresponsive under all conditions and did not sprout even with the GA₃ or chilling treatments. In a comparative study with buds immediately after collection anatomical analysis demonstrated that vegetative buds were almost completely developed by midsummer/early automn and remained in a resting state until the end of winter. Floral buds developed continuously over the same period. Both types of verticils began to differentiate in midsummer. Sepals and petals developed mainly in late summer, androecious floral parts developed throughout the resting period, while gynoecious floral parts showed differentiation in late winter. The flower was completely formed a few days prior to blossoming. Thus, in isolated peach buds fertile verticils are not sufficiently developed during the resting time to allow sprouting.     

Gibberellin A(4/7) and the Promotion of Flowering in Pinus radiata: Effects on Partitioning of Photoassimilate within the Bud during Primordia Diferentiation

Gibberellin A_4/7 mixture (GA_4/7), a highly effective promoter of early and enhanced flowering in the Pinaceae, caused a significant reallocation of dry matter and ¹⁴C-photosynthate within terminal buds of Pinus radiata D. Don within 8 days of hormone treatment. Treatment with GA_4/7 to terminal shoots of vigorous, potentially flowering mature grafted propagules reduced the flow of photoassimilated ¹⁴C and dry matter into the terminal bud as a whole, but significantly increased the dry matter and ¹⁴C allocated within the bud to developing long-shoot primordia (potential seed-cone buds). This was accomplished at the expense of the structural tissues, the apical dome region, and the vegetative branch buds. Although GA₃ caused a similar reallocation of dry matter within the terminal bud, it was significantly less effective than GA_4/7 thus appears to have, in addition to any nutrient diversion abilities, a distinct morphogenic function in sexual differentiation.     

‘圣诞快乐’朱顶红花芽分化研究

采用解剖观测和石蜡切片技术,对朱顶红品种‘圣诞快乐’花芽生长情况、花器官分化和性细胞分化过程进行了研究,以明确朱顶红花芽分化特征,为其花发育、花期调控、杂交育种和系统分类研究提供理论依据。结果表明：‘圣诞快乐’朱顶红每年产生2个花序芽,在第2年完成其内花芽花器官分化,经过低温作用后于第3年盛开,其中第2个花序偶有败育发生;花器官分化过程包括花原基分化期、外花被原基分化期、内花被原基分化期、雄蕊原基分化期、心皮原基分化期,对应的花芽大小分别约为0.02、0.05、0.1、0.2、0.3 cm,所有花器官均为螺旋状向心式发生;朱顶红花药4室,花药壁从外至内由表皮、药室内壁、中层和绒毡层组成,绒毡层类型为分泌型,小孢子减数分裂类型为连续型,四分体排列方式为十字交叉型,成熟花粉粒为2-细胞型;朱顶红雌蕊3心皮,下位子房,中轴胎座,3心室,每室两列倒生胚珠,胚珠为双珠被,厚珠心,具葱型胚囊。     

Flower bud differentiation in Salix pentandra as affected by photoperiod, temperature and growth regulators

Flower bud initiation in seedlings and vegetatively propagated plants of Salix pentandra from different locations has been studied under controlled conditions. In mature plants flower bud formation was induced by 2-chloroethyltrimethylammoniumchloride (CCC) and by short day treatment. The effect of CCC was antagonized by GA₃. The critical photoperiod for flower bud formation was about 18 h for a southern clone (from 49°48'N), but cuttings of a northern ecotype (from 69°39'N) formed flower buds even at 24 h photoperiod. Generally, flower bud formation occurred simultaneously with apical growth cessation. However, apical growth cessation was not a prerequisite for floral initiation and flower buds were also found in elongating plants. Seedlings up to 60 days old did not form flower buds in growth chamber studies. The length of the juvenile phase has not been studied in detail, but cuttings taken from seedlings approximately 20 cm high and 60 days old were able to develop flower buds when treated with CCC. A gradual transition from the juvenile to the mature phase was obtained by repeated pruning of seedlings grown at 18°C and 24 h photoperiod.     

Induction of Continuous Tepal Differentiation from in Vitro Regenerated Flower Buds of Hyacinthus orientalis

Continuous differentiation of tepals was successively induced from regenerated flower buds in Hyacinthus orientalis L. cv. White Pearl by controlling the exogenous hormones and explant ages. In 250 days of subculture, each flower bud differentiated an average of more than 70 tepals, with a maximum of over 140 tepals. Studies on the morphogenesis and characteristics of growth and development of the flower buds indicate that the first whorled organ of the flower bud was perianth which consisted of perianth tube and tepals grown at the top of the perianth tube, which is the same as the flower bud of the wild type in H. orentalis. The second and third whorls of the flower bud, which should be stamen and pistil in the wild type, but remained as the tepals in the regenerated flower bud. Growth of the regenerated flower bud was faster in the first several months of culture, then slowed down gradually with time. After 150 days in culture the flower bud growth and organ differentiation became very slow. Other than the tepal differentiation the regenerated flower buds also differentiated at random positions some small flower buds that also differentiated the tepals only. Histological observation revealed that the origin of the regenerated flower buds was jointly participated by some cells in the epidermal and subepidermal layers at the inner surface of the perianth explant, and the inner small flower buds were originated from the meristem which was formed by the transformation of the parenchyma at the base of the very young tepal. The authors also compared and discussed the similarities and differences of the phenotypes between the regenerated flower bud in Hyacinthus and agamous flower in Arabidopsis, from which, they have hypothesized on the role of the hormones in the promotion and termination of the gene expressions by an order of development in plant.     

Elucidating the unusual floral features of Swartzia dipetala (Fabaceae)

In this study, we evaluated the floral ontogeny of Swartzia dipetala, which has peculiar floral features compared with other legumes, such as an entire calyx in the floral bud, a corolla with one or two petals, a dimorphic and polyandrous androecium and a bicarpellate gynoecium. We provide new information on the function of pollen in both stamen morphs and whether both carpels of a flower are able to form fruit. Floral buds, flowers and fruits were processed for observation under light, scanning and transmission electron microscopy and for quantitative analyses. The entire calyx results from the initiation, elongation and fusion of three sepal primordia. A unique petal primordium (or rarely two) is produced on the adaxial side of a ring meristem, which is formed after the initiation of the calyx. The polyandrous and dimorphic androecium also originates from the activity of the ring meristem. It produces three larger stamen primordia on the abaxial side and numerous smaller stamen primordia on the adaxial side. These two types of stamens bear morphologically similar ripening pollen grains. However, prior to the dehiscence of thecae and presentation of pollen in the anther, only the pollen grains of the larger stamens contain amyloplasts. Two carpel primordia are initiated as distinct protuberances, alternating with the larger stamens, in a slightly inner position in the floral meristem, constituting the bicarpellate gynoecium. Both carpels are able to form fruit, although only one fruit is generally produced in a flower. The increase in gynoecium merism probably results in an increase in the surface deposition of pollen grains and consequently in the chance of pollination. This is the first study to thoroughly investigate organogenesis and the ability of the carpel to form fruit in a bicarpellate flower from a member of Fabaceae, in addition to the pollen ultrastructure in the heteromorphic stamens associated with the ‘division of labour’ sensu Darwin. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 303–320.     

The expression of tgas118, encoding a defensin in Lycopersicon esculentum, is regulated by gibberellin.

A flower specific cDNA, tgas118, has been isolated after differential screening of a gib-1 anther cDNA library of Lycopersicon esculentum. The corresponding mRNA was present in all tissues analysed. Northern blot analysis revealed that in wild-type tomato the gene was predominantly expressed throughout flower development, while in the gibberellin (GA)-deficient mutant of tomato (gib-1) the abundance declined. Treatment of the mutant with GA resulted in an accumulation of the tgas118 mRNA within hours in leaf and bud tissues. In the leaf, GA1, GA3 and GA9 were effective in enhancing the expression while GA4 was not. In addition to GA, wounding and dehydration also increased the accumulation of tgas118 mRNA in leaf tissue. In situ hybridization showed that application of 50 ng GA3 bud(-1) induced a similar spatial expression of the tgas118 mRNA in gib-1 buds 24 h post treatment to that found in wild-type flower buds. The deduced TGAS118 protein displays up to 77% similarity with defensins and as its expression is up-regulated by stimuli such as wounding it is proposed that it may play a role in protection against pathogens.     

Early flower development in Arabidopsis.

The early development of the flower of Arabidopsis thaliana is described from initiation until the opening of the bud. The morphogenesis, growth rate, and surface structure of floral organs were recorded in detail using scanning electron microscopy. Flower development has been divided into 12 stages using a series of landmark events. Stage 1 begins with the initiation of a floral buttress on the flank of the apical meristem. Stage 2 commences when the flower primordium becomes separate from the meristem. Sepal primordia then arise (stage 3) and grow to overlie the primordium (stage 4). Petal and stamen primordia appear next (stage 5) and are soon enclosed by the sepals (stage 6). During stage 6, petal primordia grow slowly, whereas stamen primordia enlarge more rapidly. Stage 7 begins when the medial stamens become stalked. These soon develop locules (stage 8). A long stage 9 then commences with the petal primordia becoming stalked. During this stage all organs lengthen rapidly. This includes the gynoecium, which commences growth as an open-ended tube during stage 6. When the petals reach the length of the lateral stamens, stage 10 begins. Stigmatic papillae appear soon after (stage 11), and the petals rapidly reach the height of the medial stamens (stage 12). This final stage ends when the 1-millimeter-long bud opens. Under our growing conditions 1.9 buds were initiated per day on average, and they took 13.25 days to progress through the 12 stages from initiation until opening.     

Flower Morphogenesis in Rosa hybrida 'Mercedes' as Studied by Cryo-scanning Electron and Light Microscopy. Effects on Light and Shoot Position on a Branch

Flower bud development in Rosa hybrida cv. 'Mercedes' was studied in shoots grown at different irradiances and sprouting from axillary buds at different branch positions. Cryo-scanning electron microscopy and light microscopy were used to visualize, characterize and determine flower morphogenesis during early shoot development. Up to the moment of visible flower bud appearance on the plant, flower morphogenesis was divided into nine stages. This classification was based on external and internal characteristics of the primordium. All shoots of the rose 'Mercedes' whether positioned uppermost or second on a branch and whether grown at 300 or 150 μmol m^-2 s^-1 PAR (12 h d^-1) developed equally up to flower stage 7, i.e. the stage just before visible initiation of stamen and pistils. Signs of flower bud abortion were the compactness of the flower bud at developmental stage 7 (height/width < 1·5) and the sprouting of axillary buds positioned just below the flower bud primordium. It was concluded that once a flower bud has reached a height to width ratio larger than 1·5, and once stamen and pistil developmental has started, it has passed the critical developmental stage in which abortion may occur. Flower developmental stage was closely related to shoot length. This relationship was not affected by irradiance level nor by shoot position on a branch. Therefore, cultivation treatments aimed to improve flower production by reducing flower abortion, such as supplementary lighting, will be most effective when applied during the first 2 weeks of shoot growth in which the flower develops up to stage 7.     

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.     

枣花芽分化过程中营养物质和内源激素含量及抗氧化酶活性变化研究

以新疆主栽品种灰枣和骏枣的花芽为材料,测定不同分化时期花芽的可溶性糖、还原糖、淀粉、可溶性蛋白含量,SOD、POD、PPO、CAT活性以及内源GA₃、IAA、ABA、ZT水平的变化,并分析它们与花芽分化的关系,为枣花芽分化调控提供理论参考。结果表明:(1)灰枣和骏枣花芽可溶性糖、还原糖和淀粉含量在花芽分化过程的变化趋势基本相似,于花原基分化期至雌蕊分化期先降低后升高,至雌蕊分化期到达峰值;而可溶性蛋白质含量变化趋势相反,在花原基分化期至雌蕊分化期先上升再降低。(2)在整个花芽分化过程中,其POD、PPO、CAT活性变化趋势基本一致,从花芽开始分化后逐步降低,最低点出现在雌蕊分化期;两个品种花芽SOD活性在花原基分化期至分化初期时显著上升,之后SOD活性在灰枣中不断降低,而在骏枣中则显著上升。(3)两品种花芽IAA、GA₃、ZT含量在分化过程中的变化规律基本相似,它们均在萼片分化期前呈下降趋势,之后GA₃、ZT含量及灰枣中IAA含量逐渐上升,而骏枣IAA含量在萼片分化期至花瓣分化期呈先显著上升后下降再上升;灰枣ABA含量在花原基分化期至萼片分化期显著上升,而同期骏枣则显著降低,随着分化进程的推进,灰枣ABA含量在萼片分化期后逐步降低,而骏枣则逐步上升并在雌蕊分化期达到峰值。(4)花芽分化开始后,骏枣ABA/IAA、ZT/IAA、GA₃/IAA比值快速上升,但GA₃/ABA、ZT/ABA的比值呈下降趋势;灰枣ZT/IAA、GA₃/IAA在花原基分化期至萼片分化期显著上升后降低,分化结束后低于花原基分化期。研究认为,枣花芽开始分化后会消耗大量的营养物质,导致花芽的可溶性糖、淀粉和还原糖含量降低,且整个分化过程中淀粉含量始终高于可溶性糖和还原糖含量;两个品种枣花芽分化过程中POD、CAT、PPO活性下降以及骏枣花芽分化过程中SOD活性的上升均有利于枣营养生长向生殖生长的转变,且枣花芽分化过程中低水平的GA₃和IAA、中等水平的ABA、较高水平的ZT,以及较高的ZT/IAA、ABA/IAA和GA₃/IAA有利于枣花芽分化和花芽形成。     

Persistence of 14C-gibberellin A3 on the surface of Citrus fruit peel and on inert glass surfaces

The persistence of gibberellin A₃ on plant surfaces was examined using fruit of Marsh seedless grapefruit (Citrus paradisi Macf.) and an inert glass model system. ¹⁴C-gibberellin A₃ was applied to surfaces in aqueous treatment solutions or in waxing solutions. Dried-out treatment residues were removed by washing and analyzed for total and GA₃-like radioactivity. Gibberellin A₃ persisted without significant loss for at least 7 d in aqueous treatment solutions (pH 4.0 or 6.2) but was less persistent in the pH 10.4 waxing solution (t1/2=7 d).Loss of total peel surface radioactivity was fast during the first 3 days, slowing down afterwards. After 14 days 73% of the initial radioactivity could still be recovered from fruit peel surface and 70% of the recovered radioactivity was still in the form of gibberellin A₃. Gibberellin A₃ was somewhat more persistent in residues from pH 4 than pH 7 treatment solutions. Light had a slight enhancing effect on gibberellin A₃ decomposition on fruit peel under growth chamber conditions. After 12 d at 100% relative humidity, 88% of the radioactivity on glass surfaces was still in the form of gibberellin A₃, as against 45% at a relative humidity of 50%. Simulated field conditions, combining daily fluctuations in light, temperature and relative humidity, markedly enhanced gibberellin A₃ decomposition on glass surfaces (t1/2=2 d). Gibberellin A₃ was very persistent (90% after 9 d) in the waxing residues on fruit peel surface.Abbreviations GA₃ gibberellin A₃ - RH relative humidity     

Stimulation of androgenesis in white cabbage (Brassica oleracea var. capitata) anthers by low temperature and anther dissection

Anther culture was performed on two local cultivars, Ljubljansko and Varadinsko, and the F₁ cv. Krautman (Bejo-Zaden). The effects on androgenesis of hot and cold temperature treatments and different dissections of anthers were evaluated. In contrast to cv. Krautman, cvs. Ljubljansko and Varadinsko produced more embryos after cold pretreatment of flower buds (4°C, 48 h) than after standard treatment (35°C, 24h). Simultaneous cutting of the anther tip and removal of the filament gave the best results in comparison to other tested dissections. Microscopical observations of sectioned anthers revealed enhanced embryo development near the cut ends of the anthers. Ploidy analysis revealed the presence of haploids among embryos resulting from cold treatment (4°C, 48 h), treatment at elevated temperature (35°C, 24 h), and among embryos resulting from dissections of anther tips.