Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Regeneration of doubled haploid plants by androgenesis of cucumber (<Emphasis Type="Italic">Cucumis sativus </Emphasis>L.)

Six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted to develop an efficient cucumber (Cucumis sativus L., 2n = 2x = 14) anther culture protocol. Pretreatment and embryo induction were key factors for successful anther culture. Suitable temperature stress depended on the ecotype, i.e., cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment. The best medium for embryonic callus induction was MS medium supplemented with 4.44 μM BA, 2.26 μM 2, 4-D, 4.64 μM KIN, 3% sucrose and 0.8% agar. For embryo induction, MS medium supplemented with 0.54 μM NAA, 13.32 μM BA, 3% sucrose and 0.8% agar was optimal, and for embryo germination MS medium containing 2.22 μM BA, 6% sucrose and 1.2% agar was best. Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No. 1, which was an improved result over a previous report. The origin of regenerants from microspores was determined by cytological, morphological and AFLP analyses.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Effect of genotype,induction medium,carbohydrate source,and polyethylene glycol on embryogenesis in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) anther culture

This study conducted two experiments involving in vitro anther culture of Zea mays L. The first experiment tested 46 maize genotypes, including inbred lines, single and three-way cross hybrids, and line A188 as control, in three different induction basal media (IMSS, N6 and YPm) for their androgenic responses. The results showed that the embryos were established 2–3 weeks after the anthers of the few responsive genotypes were cultured. Most responsive genotypes produced embryos in at least one of the three basal media; therefore, genotype is more important than the type of medium for androgenesis in maize. The mean number of anthers that developed to embryo ranged from 19 embryos per Petri dish in YPm medium for the cross (DH5 × DH7) genotype to 0 for some maize genotypes. In the second experiment, this research reports for the first time the effect of carbohydrates and polyethylene glycol (PEG) as a non-metabolized osmoticum on the embryogenesis anther culture of maize. The genotype DH5 × DH7 was used for this experiment, and the media were varied by altering sucrose, maltose, and PEG concentrations. Results showed that the maximum embryogenesis (32 embryos per Petri dish) was obtained by YPm basal medium supplemented with 60 gl^?1 sucrose + 0.0125 M PEG and 30 gl^?1 sucrose + 30 gl^?1 maltose + 0.0125 M PEG. The lowest rate of embryogenesis was observed in YPm basal medium with 60 gl^?1 maltose and 0.0125 or 0.025 M PEG. Sucrose or a high concentration of maltose was found to be necessary for embryogenesis in anther culture of maize. Therefore, the addition of low levels of PEG and/or different sugars in the experimental design appeared to improve the protocol currently available in the world, especially for anther embryo yield and haploid plant regeneration in maize.     

Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine-supplemented media

Plating rice anthers on a semisolid induction medium containing 250 or 500 mg/l colchicine for 24 or 48 h-incubations followed by transfer to colchicine-free medium and standard anther culture procedures resulted in overall 1.5- to 2.5- fold increases in doubled haploid green plant productions compared to control anther cultures. The addition of colchicine had no detrimental effects on the different anther culture efficiency parameters, but in some treatments led to significant enhancement of anther callusing frequency or callus green plant regenerating ability. The most efficient treatment raised doubled haploid plant recovery from 31% to 65.5%. These results suggest that post-plating colchicine treatment of anthers, since it was found to improve both anther culture efficiency and doubled haploid plant recovery frequency, could be integrated into rice doubled haploid plant production programmes.Abbreviations DH doubled haploid - NAA naphthalenacetic acid - PAS periodic acid Schiff     

Identification and validation of QTLs for green plant percentage in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) anther culture

In cereals, albinism is a major obstacle to produce doubled haploids (DH) for breeding programs. In order to identify QTLs for green plant percentage in barley anther culture, a specific population was developed. This population, consisting of 100 DH lines, was generated by crossing the model cultivar for anther culture “Igri” with an albino-producing DH line (DH46) selected from Igri × Dobla, in search of a maximum segregation for the trait and minimum for the other anther culture variables. A combination of bulked segregant analysis and AFLP methodology was used to identify markers linked to the trait. A linkage map was constructed using these AFLPs, together with RAPD, STS and SSR markers. This study identified a new QTL for green plant percentage on chromosome 3H and confirmed the previously reported one on chromosome 5H. Up to 65.2% of the phenotypic variance for this trait was explained by the additive effects of these two QTLs. Thirty elite cultivars of barley from different origin, row type, growth habit and end use, were selected to validate these QTLs. Since two of the markers linked to the QTLs were AFLPs, we successfully converted them into simple PCR-based SCAR markers. Only the SSR HVM60, on chromosome 3H, was significantly associated with the trait, explaining near 20% of the phenotypic variance. Among the allelic variants identified for this marker, HVM60-120bp was associated with the highest values of green plant percentage.     

Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants

Three interspecific diploid potato hybrids between selections of Solanum phureja Juz. & Buk. and S. chacoense Bitt. were used in anther culture experiments to construct a monoploid family. Different aspects of the anther culture process were affected by the treatments, such as: growing conditions of donor plants, ways of preparing the anther culture medium, and conditions of anthers in culture. Genotype and date of culture initiation were among the most significant sources of variation. Significant improvements in anther culture response were achieved by growing plants at 30°C and by a heat shock of 35°C for 12 h given to anthers in culture, which gave an increase of up to 40% in embryo yield. However, the heat shock reduced the plant regeneration rate. The majority of regenerated plants was diploid, suggesting that there were several recessive lethal alleles in heterozygous status in the anther-donor. Among the regenerants, the homozygotes could be successfully identified by simple sequence repeat analysis, using eight polymorphic primer pairs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Factors affecting somatic embryogenesis in anther cultures of Chinese pink <Emphasis Type="Italic">(Dianthus chinensis</Emphasis> L<Emphasis Type="Italic">.)</Emphasis>

In this study, we aimed to maximize the rates of somatic embryogenesis achievable in anther cultures of Chinese pink (Dianthus chinensis L.) (2n = 2x = 30). The genotype of the donor plant was found to be a major factor in determining the success rate. Conditions imposed during anther culture (notably medium composition and light conditions) and pretreatments (namely, cold, heat, and mannitol incubations) were also found to influence somatic embryo induction. For example, the highest levels of embryogenic callus induction were achieved when the donor buds had been cold pretreated and the subsequent anther culture was maintained in darkness. Furthermore, there appeared to be an interaction of genotype with culture conditions. Thus, in cultures of the cultivar (cv.) ‘Carpet’, the highest rates of embryogenesis were obtained when the anthers had received a 5-d heat-shock, but such a thermal treatment did not generally produce a significant effect. Likewise, a 3-d mannitol pretreatment was optimal only for the cross-hybrid line ‘HC’. Assessment of the ploidy of the plants regenerated from the anther cultures revealed both diploid and tetraploid plants. Histological and cytological observations showed that all of these (both from n-pollen and 2n-pollen lines) derived from anther wall cells. Spontaneous chromosome doubling was inferred to have occurred during the embryogenic callus culture period.     

Gametic and somatic embryogenesis through in vitro anther culture of different Citrus genotypes

In vitro tissue culture represents a useful technique for advancing Citrus breeding and propagation. Among in vitro regeneration systems, anther culture is commonly used to produce haploids and doubled haploids for a fast-track producing homozygous lines, in comparison with the traditional self-pollination approach, which involves several generations of selfing. In addition, anthers culture can produce somatic embryos that can also be used for clonal propagation. In this study, two thermal shocks were applied to the anthers of six Citrus genotypes (two clementine and four sweet oranges), just after they were put in culture. The response obtained was different depending on the genotype: both clementines, namely Hernandina and Corsica, produced homozygous and triploid regenerants (microspore-derived embryos), whereas all of the analyzed regenerants from sweet oranges, three cultivars of Tarocco and Moro, produced heterozygous and diploid regenerants similar to the parental genotypes (somatic embryos).     

Reflexions sur nos cultures d'antheres de plantes cultivées

Abstract

Considerations about our anther cultures of cultivated plants. – One of the main activities performed at the Casaccia Nuclear Centre, in the framework of a contract between CNEN and the European Communities, centers on the induction of haploid plants by anther culture and the subsequent chromosome doubling in order to obtain completely homozygous diploid plants. In tobacco, it is now possible to obtain haploid plants from any cultivar; we perform in vitro culture of internodes from which homozygous diploid plants are regenerated, taking advantage of natural phenomenon of endopolyploidy. In order to try to generalize this method of producing haploid plants in other plant species, we are studying the mechanism involved in haploid embryogenesis which occurs in vitro in the microspores. Datura, Nicotiana and Atropa are among the genera in which a direct embryogenesis from the microspore is observed; it is interesting to note that all three genera belong to the family Solanaceae and are very rich in alkaloids. In almost all the other cases of in vitro induction of haploids, microspores produce calli from which plantlets can be differentiated, but this way of plant regeneration is less interesting because only few plantlets are obtained and it is not sure that each haploid comes from a single microspore. We examined the factors which could influence the transformation of microspores into embryoids in tobacco, namely: the developmental stage of microspore, the degeneration of tapaetal cells, the genotype of microspore, the composition of cultural media, the physiological conditions of the plant from which the anthers were taken. From a practical point of view, it would be desirable to have informations on methods giving a maximum number of haploid plants from one embryogenic anther and the greatest number of embryogenic anthers from the cultured anthers. Our recent experiments on anther culture in liquid shaken medium have yielded good results (about 7,000 embryoids from 25 embryogenic anthers). Further, we are conducting several experiments in order to synchronize the development of the microspores in the anthers; to this end, we analyse the effect of cold treatment, ionizing radiation and gravity force. Experiments are being performed with other cultivated species, beside tobacco, in order to solve some problems of plant breeding more easily and quickly through haploidy. With the aim of introducing, in cultivated tomato, some desirable characters from the wild species, Lycopersicum peruvianum, (self-incompatibility, disease resistance, simultaneous flowering), we have obtained the interspecific hybrid through in vitro culture of young embryos. Haploid production from this hybrid could allow to quickly obtain various genetic recombinations from these two species. For this purpose we are carrying out anther cultures as well as single microspore cultures. In rice, strawberry and L. peruvianum, several diploid and tetraploid plantlets were obtained from our anther cultures. Work is in progress to ascertain the mode of their origin.     

Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

Anther Culture in Nicotiana tabacum: The Role of the Culture Vessel Atmosphere in Pollen Embryo Induction and Growth

A comparison of four volumes of culture vessel atmosphere revealedthat this variable greatly influenced the induction and growthof pollen embryos from anthers of Nicotiana tabacum. The optimumfrequency of anthers producing embryos and plantlets was foundwith a culture atmosphere of 15 ml per anther, whereas the optimumnumber of plantlets was found with 5.5 ml per anther. A smallvolume (0.5 ml per anther) almost completely suppressed embryoinduction. Removal of specific components of the culture atmosphere(ethylene, carbon dioxide, oxygen) influenced the response ofthe anthers but did not produce a satisfactory explanation ofthe inhibition of pollen embryogenesis by the small cultureatmosphere volume. In particular, the influence of ethyleneabsorption on embryo induction and growth depended both on theculture atmosphere volume and on the stage of development ofthe pollen at the start of culture. Using anthers containingpollen at a stage after the first pollen grain mitosis. ethyleneabsorption was found to increase the survival of induced embryos.Treatment of anthers for 3 d with silver nitrate, a known antagonistof ethylene action, was not an efficient means of increasingthe yield of pollen plantlets.     

Assay for doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 1. In vitro anther culture

Sunflower anthers placed on solid medium developed calli and embryos after 12 days. Embryogenesis was improved by the addition of 0.1% polyvinylpyrrolidone (PVP) that alleviated anther and medium browning. As in other species, genotypic variability was an important parameter in the anther response and a medium genotype interaction was suggested with a different PVP effect depending on the genotype. Embryo germination was largely increased by the successive use of germination media with decreasing sucrose concentrations (10%6%3%). Histological examination of the anthers during the first ten days of culture showed that, under our conditions, the embryos were of somatic origin, arising directly from the anther wall on the outside or inside of the anther loculus, or indirectly from proliferating anther wall- or connective tissue-derived callus. Finally, the ploidy status of 78 embryo-derived plants was determined by Feulgen stain or flow cytometry: all plants were diploid (2n=34).Abbreviations PVP polyvinylpyrrolidone     

Micrografting and <Emphasis Type="Italic">ex vitro</Emphasis> grafting for somatic embryo rescue and plant recovery in avocado (<Emphasis Type="Italic">Persea americana</Emphasis>)

To improve plant regeneration from oat (Avena sativa L.) anther culture, the effects of induction medium supplements and culture conditions were studied. Significantly better plant regeneration rates were obtained with cultivars Lisbeth (naked type) and Aslak when a medium containing W₁₄ salts and vitamins, supplemented with 2,4-d, BAP, Ethephon, l-cysteine and myo-inositol, was used for induction in the dark compared with a medium containing only 2,4-d and kinetin. Genotypes reacted differently on the light during the induction phase. Use of dim light significantly decreased the green plant regeneration rates in cv. Lisbeth, while in cv. Aslak the difference was not so clear. Up to 30 green plants per 100 anthers were recovered from Aslak × Lisbeth progeny and in total, over 500 oat regenerants were produced. With these numbers, acceptable rates of DH-production for cultivar breeding and genetic study purposes are approached. The agronomic performance of some DH lines was compared with that of the plants derived from commercial seeds of the same cultivars in the field experiment. A few differences were found, but generally DH lines yielded the same or more as the commercial cultivars.     

The influence of several factors on the efficiency of androgenesis in carrot

The influence of cultivar, donor plant and culture procedure on the efficiency of androgenesis was studied in carrot anther culture. Experiments were carried out on five carrot cultivars: CxC 9900 F1, Lucky B F1, HCM, Beta III and Perfekcja, which were chosen because of their high carotene contents. Two procedures of anther culture were compared: (1) incubation in darkness for two weeks, followed by exposure to continuous light and transfer onto a fresh medium of the same composition; and (2) incubation in darkness until embryos appeared, without transfer onto a fresh medium. Temperature was +27 degrees C all the time. Genotype played an important role in the process of androgenesis in carrot anther culture.The efficiency was the highest in cv. HCM - 5.6 embryos per 100 anthers. Considerable differences in the capacity for androgenesis were observed between individual donor plants. The ratio of embryos obtained per 100 anthers for cv. HCM varied from 0.0 to 48.9. The second procedure of anther culture proved to be more efficient, cheaper and less complicated.