In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD analysis of sporting and chimerism in chrysanthemum

Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM amiprophos methyl - DMSO dimethyl sulfoxide     

Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture

In vitro chromosome doubling during ovule culture of sugar and fodder beets (Beta vulgaris L.) was studied with four anti-microtubule herbicides: amiprophos-methyl (APM), oryzalin, pronamide, and trifluralin at concentrations of 0–300 μM. Best chromosome doubling results were obtained by treatment of the ovules with 100 μM APM which produced 4.7 diploid plants per 100 ovules. Highest chromosome doubling was found with oryzalin using 1 μM, with trifluralin at 10 μM, and with pronamide at 10 μM producing 2.8, 2.0, and 2.0 diploid plants per 100 ovules, respectively. The APM treatments showed relatively low toxicity on embryo formation which in combination with a high chromosome doubling effect, resulted in up to 89 diploids per 100 plants regenerated. Oryzalin and trifluralin had more severe toxic effects, which reduced embryo formation, thereby lower percentages of chromosome doubled plants were obtained from these treatments. Pronamide had no significant toxic effect but it induced chromosome doubling at lower frequencies. Compared to colchicine, APM seems to be as efficient for chromosome doubling during beet ovule culture, but at molar concentrations 100 times lower than those used for chromosome doubling with colchicine. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of mitotic inhibitors and genotype on chromosome doubling of <Emphasis Type="Italic">Rosa</Emphasis>

Dinitroanilines represent a class of compounds that are widely used in herbicide formulations as they depolymerise plant microtubles, causing chromosome doubling. The potential of microtubule depolymerising herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling of Rosa was studied. Five concentrations (0, 3, 6, 12 and 24 μM) and three exposure periods (12, 24 and 48 h) for each of the compounds were compared. Oryzalin, trifluralin and APM were not significantly different in their ability to induce chromosome doubling of R. hybrida cv Iceberg. At concentration of 6 μM and exposure period of 24 h, chromosome doubling of R. hybrida cv Iceberg was not significantly different with each of the polyplodising agents. At higher concentration (24 μM) and longer exposure period (48 h), 66.7% and 62.5% chromosome doubling was achieved with APM and trifluralin, respectively. However, the application of 6 μM oryzalin to R. persica (2n = 2x), R. hybrida cv Iceberg (2n = 3x) and R. hybrida cv Akito (2n = 4x), resulted in 60.0%, 6.3% and 0% chromosome doubling, respectively, which suggest that chromosome doubling is genotype dependent and plants with lower ploidy level have a higher propensity for chromosome doubling. Flow cytometry results at 18 and 24 weeks after herbicide treatment, indicated that the best time to test the treated plants was after 24 weeks.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

不同除草剂加倍玉米单倍体的效率

通过比较3种除草剂加倍玉米单倍体的效率,提出了利用除草剂加倍玉米单倍体的新方法。以先玉335、中农大4号和8607×8609三个基因型诱导的单倍体籽粒为材料,利用20、40、80和160 μmol L^-1浓度的甲基胺草磷、炔苯酰草胺和氟乐灵作为加倍药剂,在单倍体植株生长到三叶期和五叶期时,用滴心法处理幼苗,选择有花粉的单株自交,收获后调查果穗加倍率;采用细胞学方法观察单倍体的染色体数目和花粉的活性。结果表明,20~160 μmol L^-1的3种除草剂对玉米单倍体加倍均有效果,加倍率在3.42%~26.32%之间。甲基胺草磷、炔苯酰草胺和氟乐灵的加倍率分别为4.29%~26.32%、3.85%~20.81%和3.42%~17.61%;其中80 μmol L^-1甲基胺草磷的加倍效果最佳,使用80 μmol L^-1甲基胺草磷处理3个杂交种的单倍体,平均加倍率分别为25.02%、20.13%和14.99%。方差分析表明,3个基因型间的单倍体加倍率均呈极显著差异,可见使用甲基胺草磷、炔苯酰草胺、氟乐灵可以提高玉米单倍体的加倍频率,但不同基因型单倍体对除草剂的敏感性存在差异。     

Effect of chromosome-doubling agents on somaclonal variation in the progeny of doubled haploids of maize

This study was carried out to determine whether the treatment of anther-culture-derived haploid callus of maize (Zea mays) with chromosome-doubling agents, such as colchicine or the herbicides pronamide and amiprophos-methyl (APM), induces higher than normal levels of somaclonal variation. A total of 79 R₁ families produced by diploid regenerated plants resulting from chromosome-doubling treatments were evaluated in the field in comparison with the three parental inbreds. Four qualitative variant phenotypes — male sterility, chlorophyll deficiency, earless plants, and short plants with narrow leaves and thin stalks —– were observed. The last phenotype (narrow leaves and thin stalks) was also found in the inbreds FR16 and H99 grown from seed, so it may not be directly related to the tissue-culture conditions or the anti-microtubule-agent treatments. The frequency of R₁ families segregating for the other three mutations was 3.8%, which is no higher than the somaclonal variation frequencies observed previously in tissue-culture-derived maize plants. Observations of three quantitative traits—–days to anthesis, days to silk emergence, and plant height—– also failed to detect any extra variation that could be related to the treatments with anti-microtubule agents. These studies indicate that the anti-microtubule agents APM, pronamide and colchicine can be used to induce chromosome doubling of anther-culture-derived callus to produce a high proportion of doubled haploid plants without causing increased rates of mutation (somaclonal variation).     

Microspore culture of white cabbage, Brassica oleracea var. capitata L.: Genetic improvement of non-responsive cultivars and effect of genome doubling agents

For the efficient application of haploid induction procedures in cabbage breeding, a sufficient number of regenerants should be achieved in a broad spectrum of genotypes. However, the majority of genotypes are somewhat recalcitrant. The efficiency of microspore culture was tested by crossing a responsive (28.7 embryos per Petri dish) and a non- responsive (0.1 embryo) cabbage cultivar. The embryo yield of one progeny was intermediate (18.9) while two were superior to the best parent cultivar (52.9 and 64.0 embryos). Thus, genes for haploid embryogenesis, present in responsive lines, can be effectively transmitted to responsive × non-responsive hybrids. Abscisic acid-induced desiccation of embryos was used for the efficient regeneration of plants. High germination percentages (54.7-70.6%) followed by normal plantlet development were achieved. Spontaneous genome doubling measured at the plantlet stage differed markedly in untreated genotypes. The percentage of diploids ranged from 21 to 67%. The effects of two antimitotic drugs applied to freshly isolated microspores were determined in two experiments. In the first experiment, trifluralin (0.5 and 1.0 mg:l) had no effect on embryo induction while oryzalin partly (0.125-0.25mg/l) or completely (0.5.mg/l) inhibited the formation of embryos. In the second experiment, higher concentrations of trifluralin increased the proportion of diploidized plants. Application of anti-mitotic drugs to microspores did generally not improve the overall production of haploid plants, which was higher in an untreated control.     

Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture

The effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.     

Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores

Summary The effect of colchicine on isolated microspore cultures of Brassica napus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chromosome doubling at an early developmental stage, thus avoiding the production of haploid or chimeric plants. Colchicine was added to the culture medium immediately after isolation of B. napus microspores. The cultures were incubated from 6 to 72 h with various concentrations of colchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry.The highest diploidization rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80–90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a lower diploidization rate (76–80%). Treatment durations of 6 h were not long enough to induce a high rate of diploidization, whereas the application of 10 mg/l for 72 h was also very effective.A sample of the plants regenerated from the colchicine treated microspores was transferred to the greenhouse. The plants looked similar to normal diploid rapeseed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessary. The advantage of applying a minimum volume of colchicine under controlled in vitro conditions means a considerable saving of time and labour in DH-breeding programs.     

Functional genomics of microspore embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for breeding purposes. Protocols for the induction of microspore embryogenesis and the subsequent regeneration of doubled haploid (DH) plants have been successfully developed for more than 200 species. These practical advances stand in stark contrast to our knowledge of the underlying molecular genetic mechanism controlling this process. The majority of information regarding the genetic and molecular control of the developmental switch from gametophytic to sporophytic development has been garnered from four intensely studied (crop) plants comprising two dicotyledonous species, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum), and two monocotyledonous species, wheat (Triticum aestivum) and barley (Hordeum vulgare). In these species the efficiency of microspore embryogenesis is very high and reproducible, making them suitable models for molecular studies. In the past, molecular studies on microspore embryogenesis have focussed mainly on the identification of genes that are differentially expressed during this developmental transition and/or early in embryo development, and have identified a number of genes whose expression marks or predicts the developmental fate of stressed microspores. More recently, functional genomics approaches have been used to obtain a broad overview of the molecular processes that take place during the establishment of microspore embryogenesis. In this review we summarise accumulated molecular data obtained in rapeseed, tobacco, wheat and barley on embryogenic induction of microspores and define common aspects involved in the androgenic switch.     

Improved Cold and Drought Tolerance of Doubled Haploid Maize Plants Selected for Resistance to Prooxidant tert‐Butyl Hydroperoxide

To improve the abiotic stress tolerance of maize (Zea mays L.), doubled haploid (DH) plants were produced by in vitro selection of microspores exposed to tert‐butyl hydroperoxide (t‐BuOOH) as a powerful prooxidant This study investigated the tolerance of the progenies of t‐BuOOH‐selected DH lines to oxidative stress, cold and drought in controlled environment pot experiments by analyses of photosynthetic electron transport and CO₂ assimilation processes, chlorophyll bleaching and lipid peroxidation of leaves. Our results demonstrated that the t‐BuOOH‐selected DH plants exhibited enhanced tolerance not only to oxidative stress‐induced by t‐BuOOH but also to cold and drought stresses. In addition, they showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase and glutathione S‐transferase when compared with the DH lines derived from microspores that were not exposed to t‐BuOOH and to the original hybrid plants. The results suggest that the simultaneous up‐regulation of several antioxidant enzymes may contribute to the oxidative and cold stress tolerance of the t‐BuOOH‐selected DH lines, and that the in vitro microspore selection represents a potential way to improve abiotic stress tolerance in maize.     

Doubling the chromosome number of bahiagrass via tissue culture

Crop improvement in bahiagrass (Paspalum notatum Flüggé) is limited by apomixis in most natural tetraploids, however, diploid sexual types occur. Production of sexual tetraploids by chromosome doubling will allow hybridization with apomictic tetraploids. Diploid bahiagrass (Paspalum notatum Flüggé) embryogenic callus tissue was exposed to three concentrations of three antimitotic chemical agents, colchicine, oryzalin and trifluralin. Callus was generated to plants and ploidy was evaluated by stomata size, mitotic chromosome counts, and flow cytometry. A total of 310 plants were verified as tetraploid of 1,432 plants that reached transplanting size. All treatments yielded 4x plants. The mean percentage success over all treatments was 22%, with means of 31% for oryzalin, 24% for colchicine and 16% for trifluralin. The high rates of success indicate that all agents can be successfully used to double chromosome numbers in bahiagrass. The percentage of 4x plants ranged from 9% (20 μM trifluralin) to 43% (20 μM oryzalin). Several treatments adversely affected regeneration. Mitotic chromosome counts are difficult and labor intensive in bahiagrass. Therefore, leaf stomata measurements were used as a preliminary screen. Data gave a bimodal distribution with overlapping tails and based on chromosome counts would have given an error rate of 12%. Flow cytometry analysis of regenerated plants resulted in mean nucleus fluorescence distributions consistent with control diploid or tetraploid values. These values agreed with chromosome counts, and this method is recommended for determining bahiagrass ploidy level. Research goals and available resources should be taken into consideration when selecting a treatment for chromosome doubling in bahiagrass.     

Doubled haploid production of sunflower (Helianthus annuus L.) through irradiated pollen-induced parthenogenesis

Induced parthenogenesis as a possible method of haploidization in sunflower (H. annuus L.) was tested in previous investigations (Todorova et al., 1994) and conditions for reproducible regeneration of gynogenic doubled haploids were established by the present work. Forty eight treatments were studied, involving four pollen donors and four recipient hybrids. Pollen was irradiated with doses of 300 Gy, 600 Gy and 900 Gy. In total, 2279 embryos were cultivated in vitro of which 1107 plants were obtained and 582 of them produced seeds after selfing. The ploidy level of the regenerants was evaluated at the two – three leaf stage and 296 of the plantlets obtained were haploids. Some of them underwent spontaneous diploidization, the others were treated with colchicine solution for chromosome doubling. The diploid plantlets were checked for their gynogenic origin by genetical and biochemical methods. The effectiveness of the method expressed as the number of agronomically useful DH lines to the number obtained that were fertile and resistant to downy mildew, branched and unbranched plants was 8,6% on average. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transgene Homozygosity Following Particle Bombardment of Haploid Barley Microspores

Information from previously published studies that are basic to this study is： 1） Following isolated barley microspore culture, around 80% of the resulting barley plants are completely fertile and genetically homozygous doubled haploids （DH） （Kasha et al., 2003）. 2） Chromosome doubling occurred during early stages of microspore culture, mainly by nuclear fusion （Kasha et al., 2001）. 3） A fairly synchronous population of microspores was selected to determine the cell cycle stages （G1, S, G2） by relative DNA density measurements （Shim and Kasha, 2003a）. 4） The pretreatments of spikes used to induce microspore embryogenesis influenced the cell cycle progression during pretreatment. （i） Using a combination of cold （4℃） and 0.3 mol/L mannitol for 4 days, the microspores were held at the G1 uninucleate stage. （ii） The often used pretreatment of cold （4℃ for 21 to 28 d） permitted slow microspore stage progression so that most cells were at the G2 stage or had become binucleate by the end of the pretreatment. （iii） Pretreatment with mannitol for 4 days at 25 ℃ did not block cell cycle progression but prevented cell wall formation after the 1^st mitotic division, leading to nuclear fusion and chromosome doubling.     

Fertile maize lines obtained from isolated microspores

Microspore response of three- way cross maize hybrid genotype 3AL/95 (Zea mays L.) was studied under simplified isolation and culture conditions. Fertile plant production was achieved through abundant plant regeneration. As a total, microspores of 160 tassels were inoculated and five sustainable microspore derived callus cultures (SMC) were obtained. Hybrid seeds (ML SC), which were produced by crossing of regenerates from two SMCs, gave rise to subsequent vigorous and fertile progeny. The response of the 3AL/95 and ML SC microspores was studied in three liquid culture media in order to improve the early viability of microspores. Them N6M medium provided better survival of cultured microspores (p = 5%) than the ppN6M/89 and the YPM-G media. The pH 5.8 in mN6M medium revealed significant increase (p = 1%) in microspore viability as compared to pH 3.0. The ML SC microspores showed higher viability(30%) on the first day of culture in the mN₆M than those of the3AL/95 (19%) but without improved rate of callus formation and plant regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

油菜小孢子培养技术体系研究

小孢子培养在油菜的基础研究和应用研究中均具有十分重要的意义。自1982年Lichter首次在甘蓝型油菜中进行小孢子培养获得成功以来,国内外在油菜小孢子培养技术方面已取得大量研究成果,包括油菜小孢子胚状体发生的影响因素,小孢子植株的再生、成苗、大田移栽、染色体加倍等,近年来又对一些关键技术环节加以了改进,笔者在对这些研究成果进行总结的基础上针对中国国情建立了大田条件下油菜高效小孢子培养技术体系。用该体系对甘蓝型油菜和新疆野生油菜的体细胞杂种后代进行小孢子培养的出胚率达到300枚/皿以上,采用小孢子苗直接移栽大田技术,成活率达到89.0%。此外还成功构建了含127个DH系的黄籽油菜DH群体及含115个DH系的粒重分离群体。     

Pollen production in soybeans with respect to genotype,environment, and stamen position

Summary Our objective was to determine the average numbers of pollen grains from fertile plants (Ms ₁) and the average numbers of coenocytic microspores from genetic male sterile plants (ms ₁ ms ₁) in soybeans, Glycine max L. Merr. Comparisons were made between the average numbers of pollen grains and the average numbers of coenocytic microspores with respect to environment where plants were grown and to stamen position in the flower. Five male sterile lines were used. They included the North Carolina ms ₁ mutant, the cultivar Hark with the ms ₁ gene, and mutants identified as the Urbana, Tonica, and Ames male steriles. Three environments used were the Agronomy Farm, University of Georgia, Athens, Georgia; the Agronomy and Agricultural Engineering Research Center, Iowa State University, Ames, Iowa; and the Agronomy Greenhouse, Iowa State University, Ames, Iowa.Pollen production from fertile plants varied from 374 to 760 pollen grains per anther among genetic lines and environments. This variation may be an important consideration in selecting a male parent to use as a pollinator for hybrid seed production.Among fertile plants, the average numbers of pollen grains per anther of the separate stamen and and of the lower whorl of stamens were significantly different only in greenhouse-grown plants. Among male sterile plants, the average numbers of coenocytic microspores per anther of the separate stamen and of the lower whorl of stamens were significantly different in three genotype x environment combinations. These three exceptions did not conform to any genetic or environmental pattern. Deviations from the expected ratio of 4 pollen grains from fertile plants: 1 coenocytic microspore from sterile plants were attributed to initial differences in the average number of microspore mother cells between the two genotypes.Joint contribution: Agricultural Research Service, USDA, North Central Region, and Journal Paper No. J-8910 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.     

Harnessing mutant donor plants for microspore culture in Indian mustard [Brassica juncea (L.) Czern and Coss]

Haploid mutagenesis offers several advantages over conventional (seed) approach. However, its potential has not been utilised for Brassica juncea, an important oilseed. In this study, mutant donor plants of three Indian B. juncea genotypes, generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU), were used for microspore culture. The response of mutant donor plants was about 100 times lower than non-mutant controls; a total of 9,411 embryos were produced from the EMS treated donor plants, while microspores isolated from ENU treated donors did not yield any embryos. The lethality of induced mutations demonstrated itself mainly as the induction of abnormal embryos (80%), failure of germination (70%) and failure of plantlet development (70%). Nine doubled haploid (DH) mutant lines and three non-mutant DH lines obtained through this approach were tested for agronomic and biochemical variation over two growing seasons. High variability was observed and stable mutants were recovered for reduced height (125 vs. 168 cm for the control), appressed pod character, altered fatty acid composition higher protein proportion in de-oiled meal (48%) and a lower glucosinolate content in de-oiled meal (59.5 μM/g) relative to controls. The approach demonstrates that despite severe reduction in efficiency of the DH line production, valuable mutants can be recovered from mutated donor plants.     

Assessment of different anther culture approaches to produce doubled haploids in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.