Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) genotypes

This is the first report on isolated microspore culture—derived spelt wheat. The efficiency of anther- and isolated microspore was compared using four genotypes (‘Franckenkorn’, ‘GK Fehér’, ‘Mv Martongold’, ‘Oberkulmer Rotkorn’). In anther culture, genotype dependency was observed, and cold pre-treatment enhanced the efficiency of the method. In isolated microspore culture, the ovary co-culture supported the development of embryo-like structures. The presence of growth regulators (0.5 mg/l 2,4-D and 0.5 mg/l kinetin) were not essential for the induction of androgenesis, but these increased the production of embryo-like structures, green and albino plantlets. The low plant regeneration rate and high number of albinos hinder the practical application of isolated microspore culture while anther culture was efficient for in vitro green plantlets production in spelt wheat. The mean of green plantlets production was 41.45/100 anthers (from 20.93 to 83.07 depending on genotype). The phenomenon of albinism was mitigated in anther culture (3.48 albinos/100 anthers). Altogether, 1720 anther culture—derived green plantlets were produced from the four genotypes.     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999     

Inheritance of anther culture derived green plantlet regeneration in wheat (Triticum aestivum L.)

A study was set up to determine the inheritance and combining ability of the factors anther culture response and green plant regeneration. Reciprocal crosses were made between cultivar Ringo Sztar, showing high anther culture response and the cultivars Ciano 067 and Benoist H77022, showing a high level of green plant regeneration. Averaged over all genotypes, 23.0% of the anthers responded and a callus induction frequency of 77.8% was observed. Of all the embryos, 43.0% developed into plantlets, 25.6% of the regenerants being green, the result being that 3.3 green plants per 100 anthers were formed. Genotypic effects accounted for 57.7%, 86.3% and 77.5% of the total variance of anther culture response, callus induction frequency and embryo induction frequency, respectively. Additive and dominant gene action was detected for all characteristics, including green plant regeneration. No reciprocal differences were found for anther culture response, embryo induction frequency and green plant regeneration, indicating no cytoplasmic effects. A small but significant reciprocal difference was found for callus induction frequency. Embryo production was primarily correlated with anther culture response and not with the number of embryos produced per plated anther or per responding anther. Possible mechanisms for the inheritance of green plant regeneration are discussed.Abbreviations CIRA callus induction frequency per responding anther - ERA embryo induction frequency per responding anther - FHB fusarium head blight - MS-medium Murashige & Skoog (1962) medium - REML residual maximum likelihood     

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%.     

Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.     

Improved plant regeneration from shed microspore culture in barley (Hordeum vulgare L.) cv. igri

This report describes rapid regeneration of green plants from microspores of the barley cultivar Igri. Use of 0.3 M mannitol during maceration and isolation was essential for response from mechanically isolated microspores of barley cv. Igri grown under our conditions. A shed microspore culture system proved to be simple and gave a fast response; plants were obtained as early as 25 days after the material was taken from the donor plant. A 28-day cold-pretreatment of spikes can also be replaced with a 3–4 day pretreatment of anthers in mannitol. Shed microspores from 100 anthers produced an average of 292 plants with 91% of them green. Approximately 80% of the regenerated plants were spontaneously doubled-haploids.Abbreviations IAA Indole-3-acetic acid - FHG Hunter's media (1988) - MS Murashige and Skoog     

Osmotic potential of media affecting green plant percentage in wheat anther culture

The percentage of green plants in anther culture is known to be controlled by the genetics of anther donor materials. The objective of this study was to determine whether components in the culture media also would have a significant influence on the percentage of green plants from wheat anther culture. Anthers of a spring wheat cultivar, Pavon 76, were cultured on potato 4 (P4) induction media with various modifications. Addition of 200 g/l ficoll to the liquid P4 medium significantly increased the percentage of green plants even though the final yield of green plants per 100 anthers was lower than the liquid medium. A higher concentration of maltose (135 g/l) produced significantly higher percentage of green plants than the medium containing 90 g/l maltose or sucrose. These results demonstrate culture medium effects on albinism, indicating that the percentage of green plants in wheat anther culture can be increased by optimizing medium osmotic potential.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Culture conditions for induction of green plants from barley microspores by anther culture methods

Summary With barley a large variation in frequency of plant formation from microspores of spikes from the same plant has been observed. The highest frequency of plant formation was obtained when culturing anthers in the dark on a high Ficoll medium containing 2,4-D and kinetin to induce proembryo (or callus) formation. Subsequently the proembryos or calli were cultured in dim light on a high Ficoll-high sugar medium containing IBA and kinetin. Finally the embryos were transferred to a starch agar medium. A maximum of 13 green plants were obtained from microspores of a single anther.The ratios of green to albino microspore derived plants varied from 91 to 19 depending on culture conditions. Under anaerobic conditions, lactic acid and other organic acids may have damaged the organelles in the cells resulting in the formation of albino plants. Thus, direct embryogenesis by using a well-buffered, high Ficoll-high sugar medium and proper aeration are essential for obtaining high frequency of green plants from microspores.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA 3 indolylbutyric acid     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

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.     

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).     

Androgenic response to preculture stress in microspore cultures of barley

Summary. Various stresses such as starvation and cold or heat shocks have been identified as triggers in the induction of the microspore embryogenesis. This study attempts to quantify the effects of different pretreatment conditions for successful microspore culture of malting barley (cv. Scarlett). While the sporophytic microspore development could be induced from treated and nontreated microspores, abiotic stress was essential for embryo formation and plant regeneration. The type of stress treatment applied affected the numbers and the ratios of albino and green plants regenerated, as well as their fertility. The highest number of green plants was obtained after the treatment of anthers in 0.3 M mannitol at 32 °C for 24 h before microspore culture. Correspondence and reprints: Department of Plant Biotechnology and Cytogenetics, Institute of Plant Breeding and Acclimatization, Radzików, 05-870 Blonie, Poland.     

Anther culture with different genotypes of opium poppy (Papaver somniferum L.): effect of cold treatment

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 10⁵ microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 10⁵ microspores per ml is recommended for isolated microspore culture.Abbreviations LSmean least square mean - MES 2-N-morpholinoethane sulfonic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphtaleneacetic acid     

Production of doubled haploid lines in frequencies sufficient for barley breeding programs

Winter barley (Hordeum vulgare L.) anthers were cultured on different liquid and on starch-solidified media. The optimal embryo and callus formation with different F₁-lines and the cv. Igri was obtained on a liquid medium with 20% Ficoll, 20 g/l maltose and barley starch. But the influence of the growth conditions of the donor plants and the genotypical differences are still enormous. The procedure has been optimized to such an extent that it can be used economically.     

Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture

Embryogenesis from isolated microspore cultures of wheat was improved by ovary co-culture when compared to a completely defined medium. This indicates that essential factor(s) in addition to PAA or its analogs may be supplied by the ovaries. Isolated microspores cocultured with 20 ovaries of wheat on the top of semi-solid MMS3 induction medium for 21–30 days gave the best response. Both the number and quality of the embryos was significantly increased. The maximum frequencies of dividing microspores and of embryogenesis were 94% and 2.4%, respectively. Up to 2583 embryos were formed per 100 anthers of cv Chris and between 18% and 43% of the larger embryos regenerated into green plants upon transfer. Genotype differences for both induction and embryogenesis phases were reduced using ovary co-culture. However, there was still a strong genotype influence on plant regeneration with cv Chris, with the F1 of Chris × Sinton displaying the highest frequencies. These results are important with respect to enhancing haploidy applications in wheat biotechnology and plant breeding.Abbreviations PAA Phenylacetic acid - MMS modified MS medium - MS Murashige and Skoog's medium 1962 - FHG Hunter's FHG medium 1988     

Influence of different concentrations of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale

In this work, the effect of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale was studied. The study of androgenesis was conducted on two model samples YaTKh-327-11 and YaTKh-18-11. The research revealed that the addition of the antioxidant ascorbic acid at a concentration of 2–8 mg/L into the culture medium to induce embryogenesis enhanced the formation of androgen structures and regeneration of green plants. The addition of gibberellic acid into the culture medium to induce embryogenesis gave negative effects. The average yield of green plants in the anther culture of spring triticale was 2 pcs/100 anthers. According to the research, 277 green plants were received and spontaneous doubling of chromosomes was recorded in 26.7%.