A comparison of several published methods for barley anther culture

A number of methods have been published for barley (Hordeum vulgare L.) anther culture and have gained acceptance in different laboratories. The breeder's requirement is for a compromise method that gives good, repeatable results for a wide range of genotypes. Yet the routine production of spontaneously doubled haploid green regenerants remains difficult. Despite attempts to formulate a widely-applicable anther culture method, the 4 main published methods, compared here with one modified procedure, are quite distinct for a number of important characteristics. The methods interacted strongly with the 3 genotypes, and response ranged from zero to 28 green regenerants per 100 anthers plated. The current methods still require often substantial modification to suit local situations in order that the technology may be exploited by barley breeders.Abbreviations BAP benzylaminopurine - DH doubled haploid - FV final volume - IAA indoleacetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - PABA para-aminobenzoic acid     

Enhancement of embryogenesis and plant regeneration from barley anther culture by low doses of gamma irradiation

Summary The effects of 0,5 and 10 Gy doses of gamma irradiation on the enhancement of embryogenesis and plant regeneration efficiency of three barley (Hordeum vulgare L.) genotypes, Igri, Arabi Abiad and AECS 76, were evaluated. Embryo yields at 5 and 10 Gy doses were significantly higher than those of the control (OGy). This effect was genotype-dependent. The most responsive genotype was Igri, with 592.8 embryos 32 anthers exposed to 10 Gy. However, despite a high embryo induction rate, the green plant regeneration rate was low. Arbi Abiad had a higher ability to generate green plants produced from, with 28. 13 plantlets obtained from 32 anthers at 10 Gy; irradiation had no significant effect on regeneration of Igri and AECS 76 genotypes. In general, the 10 Gy dose produced a much higher embryo yield than the 5 Gy dose. The root-tip chromosome number and the fertility of 298 regenerating green plants of cv. Igri revealed that 64% of the tested plants were spontaneously doubled haploids (DHs) and fertile.     

The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.)

The influence of temperature stress pre-treatment on anther culture response has been examined in eight commercially desirable barley cultivars. Spikes were pre-treated in darkness at 4°C for periods of 0, 7, 14, 21 and 28 days. Overall, the optimum pre-treatment period was 21 days, although there were large genotype by pre-treatment interactions. The most responsive cultivar was Igri, with a mean of 38% anthers responding, and relatively little effect of pre-treatment. The greatest effect of pre-treatment was in cv. Heriot, which had 3% response with no pre-treatment and 52% response from 14 days pre-treatment.     

Barley anther culture: assessment of carbohydrate effects on embryo yield, green plant production and differential plastid development in relation with albinism

Sugars and polyols were tested at different steps of anther culture in barley (Hordeum vulgare L.) to elucidate their influence on both the overall yield of androgenesis and the structure of plastids in relation to albinism. During the pretreatment period, the osmotic regulation in the medium was beneficial to microspore embryogenesis regardless of the type and concentration of the tested osmoticum. The use of mannitol (300 mOsm/kg), sorbitol (180 mOsm/kg), PEG (240 mOsm/kg) and sucrose (180 mOsm/kg) gave the best results in terms of green plant production, although the influence of each substance differed according to the studied parameter. Similarly, during anther culture the regulation of the osmotic pressure in the medium had various effects, according to the osmoticum used. The best results were obtained using mannitol (364 mOsm/kg), providing 139.7 green plants per 100 plated anthers. Plastids were examined by electron microscopy following both pretreatment and culture. In the presence of mannitol and PEG, plastids did not accumulate starch at any stage of the protocol but they started to differentiate into chloroplasts in the microspore-derived embryos. Using sorbitol and sucrose, plastids differentiated poorly but accumulated large amounts of starch, suggesting that these sugars are metabolized by micropores and microspore derived structures. However, the accumulation of starch was not correlated with the occurrence of albinism. These results indicated that, in barley, the osmotic regulation was favourable to switch the microspore gametophytic program toward a sporophytic program regardless of the nature of the osmoticum. In addition, during the pretreatment period, mannito was found to be the most suitable osmoticum for subsequent embryo development.     

Rapid production of recombinant barley yellow mosaic virus resistant Hordeum vulgare lines by anther culture

Summary In a winter barley breeding program for barley yellow mosaic virus (BaYMV) resistance, the resistant six-rowed cv. Franka was crossed to 17 susceptible and two resistant cultivars, three of which were tworowed. A total of 233,445 anthers of the 19 hybrids and their parents were cultured and 831 green plants regenerated. Anther culture responsiveness varied greatly between genotypes, and the responsiveness of F₁hybrids was generally related to that of the more responsive (high) parent. On average, 3.6 green plants were recovered from 1,000 cultured anthers, almost twice as many as in comparable spring barley experiments. Androgenetic green plants were tested for their reaction to mechanical inoculation of BaYMV. In crosses of resistant parents, all the cross progeny proved to be resistant, which indicates that both parents carry identical gene(s). In the crosses of the resistant cv. Franka to susceptible parents, an average of 62% of the androgenetic progenies were resistant, which indicates that probably more than one gene is responsible for Franka's BaYMV-resistance. From the crosses of Franka to two-rowed cultivars, 282 androgenetic plants were produced. When 132 of these were tested for their reaction to BaYMV, 79 (59.8%) were resistant, and 30 of the latter were shown to be two-rowed recombinant lines. Doubled haploid lines are field-tested for other agronomic characters including grain yield and its components.     

Tests for the presence of gametoclonal variation in barley and wheat doubled haploids produced using the Hordeum bulbosum system

Summary To investigate whether the Hordeum bulbosum system of doubled haploid production generates gametoclonal variation, populations of second generation doubled haploid lines were developed from first generation doubled haploid lines of two barley varieties and three wheat genotypes. In barley, no variation between doubled haploids from doubled haploids was detected for a range of quantitative characters, suggesting the absence of any gametoclonal effects. However, the original selfed-seed stocks were shown to contain cryptic allelic variation for some of the characters investigated. In wheat, gametoclonal variation was detected for ear emergence time, plant height and yield, and its components for two out of the three genotypes investigated. The type and range of variation was similar to that reported from studies of somaclonal variation from immature embryos and gametoclonal variation from anther culture. Generally, the effects appeared to reduce the yield performance of individual lines. The difference in response between the two species and the consequences for the use of the doubled haploid system in breeding programmes are discussed.     

Effects of donor plant genotype and growth environment on anther culture of soft-red winter wheat (Triticum aestivum L.)

The effects of donor plant growth temperature and photoperiod on embryo formation and plant regeneration from cultured anthers in five genotypes of soft-red winter wheat (Triticum aestivum L.) were examined. There were no significant differences between the three environments studied (15°C - 16/8 h light/dark, 20°C - 16/8 h light/dark, and 20°C - 12/12 h light/dark) when frequencies were averaged over genotypes; however, significant genotype and genotype x environment interactions were observed for embryo formation. When averaged over environments, highest embryo and plant production frequencies were exhibited by a line derived from the cross IL 72-2219-1/Amigo. A mean of 8.6 embryos per 100 anthers plated was observed for this genotype grown in the 20°C - 16/8 h light/dark environment. The cultivar Scotty averaged 4.2 plants produced per 100 anthers plated when grown in the 15°C - 16/8 h light/dark environment. The results from this study suggest a potential for increasing embryo and plant production in this material and point toward the need to optimize donor plant growth environmental conditions to maximize response frequencies for specific genotypes of interest.     

Regeneration of Immature Inflorescences of Barley In Vitro

Two spring barley cultivars, Golden Promise and Galan, were screened for callus induction and shoot regeneration from cultured immature inflorescences. Genotype Galan have better regeneration capacity in in vitro conditions than Golden Promise.     

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (flat). When anthers were plated with only one loculus in contact with the medium (up), a high proportion of the anthers produced calluses. The top loculus of the up anthers was most productive. Flat anthers, when compared with up anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in flat anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.     

Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) anther cultures

The generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative to the classical inbreeding and selection programs. However, this technology is poorly developed in tomato, where doubled haploid tomato plants have only been obtained through anther culture. Despite the fact that anther culture is routinely used in a number of economically interesting crops, there are still many drawbacks that prevent tomato breeders from adopting this technique, and improvements in methodology are required. One key issue is the correct identification of the optimal stage for anther excision and culture. In this paper we characterise in vivo microsporogenesis in tomato, defining the different microspore stages and relating them to the length of the donor flower bud. In parallel, we cultured anthers of these stages to obtain embryogenic callus, and followed the microscopic development of the callus contained within the anther. Our data suggest that the stage with the highest response, in terms of callus generation, is meiosis. In particular, we propose the window from metaphase I to telophase II, including tetrad cellularisation, as the timeframe where induction can be accomplished in tomato anther cultures.     

Barley tissue as direct template for PCR: a practical breeding tool

A method for using alkali treated intact plant tissue as a DNA source for the polymerase chain reaction (PCR) was applied to barley. This method saves up to two days and more than USD 50 per 40 samples by eliminating the need for DNA extraction to produce template for PCR. The conditions were optimized for various barley tissues. Fresh leaves, freeze-dried leaves, and anthers worked well as templates while root, embryo, and endosperm tissues did not. The method was shown to work with several genotypes and different primers. The resulting PCR product could be cut with restriction enzyme to produce clear polymorphism without any interference. This method can be a practical breeding tool by providing a fast, inexpensive method for screening large populations.     

The improvement in regenerated doubled haploids from anther culture of wheat by anther transfer

This study was conducted to determine the most suitable method of regeneration by comparing two approaches: transfer of anthers (with and without embryo-like structures) to regeneration conditions after a period of two to four weeks on induction medium (= anther-transfer treatment) and transfer of embryo-like structures to regeneration conditions after five to eight weeks on induction medium. The early transfer of anthers brought about a significant reduction in the number of embryos formed, but nevertheless significantly improved the frequency of plant regeneration. Combining an optimal date of anther transfer with the early addition of colchicine to the induction medium (100 mg l⁻¹ for 1 and 3 days) led to an increase in the number of doubled haploid regenerants. The results indicate that transferring the anthers after 28 days and adding 100 mg l⁻¹ colchicine to the induction medium on one day only caused a significant improvement in the ability of green plants to regenerate (7.0 compared to 0.50) as well as in chromosome doubling (success index: 4.0 compared to 0.33). This revised version was published online in August 2006 with corrections to the Cover Date.     

Screening salt-tolerant barley genotypes via F1 anther culture in salt stress media

Summary Anthers of two six-row barley cultivars Diamond (a germination salt sensitive cultivar) and Men Yuan Liang Lan (a germination salt tolerant cultivar), and their F₁ reciprocal crosses were cultured in liquid media containing 0, 0.4, 0.6, and 0.8% Na₂SO₄. A total of 138 green pollen plants were obtained: 7 from Na₂SO₄ media, 128 from Na₂SO₄ free medium. Seeds of two successive generations of 61 pollen plants were germinated in a series of Na₂SO₄ solution (0 to 5.5%). It was found that among 37 progenies from F₁ pollen in Na₂SO₄ free medium, 11 were as sensitive as Diamond, 12 were intermediate to the two parents, 7 were equal to the salt tolerant parent and 7 were more tolerant to Na₂SO₄ than Men Yuan Liang Lan. Whereas, no progeny from F₁ pollen in high salt media was as susceptible as the susceptible parent; 2 were intermediate, 2 were equal to the salt tolerant parent and 2 were more tolerant than the salt tolerant parent. The results indicate that culturing anthers in Na₂SO₄ media effectively eliminated salt susceptible progenies. All 16 microspore-derived lines of Diamond were as susceptible as Diamond to Na₂SO₄. The 5 lines from Men Yuan Liang Lan microspores were as resistant to Na₂SO₄ as Men Yuan Liang Lan. All of the lines breed-true. The results indicate that the lines exhibiting elevated levels of tolerance to salt probably resulted from recombination of genes rather than from spontaneous mutation.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

Interaction of environment and ABA and GA treatments on the maize anther culture response

Treatments designed to influence abscisic acid (ABA) or gibberellin (GA) concentrations were applied to developing tassels of maize (Zea mays L.) plants in different environments or to anthers in culture to determine the effect on formation of embryo-like structures (ELS). Production of ELS was significantly affected in certain environments when ABA, GA₃, ancymidol, or fluridone solutions were pipetted into whorls of field-grown plants approximately 3 days before tassel harvest. In 1996 anthers from 10 M ancymidol-treated plants were most responsive, producing 35 ELS/100 anthers and 50 M GA₃-treated plants were least responsive, producing 12 ELS/100 anthers. In 1997 under hotter, drier conditions, anthers from 50 M GA₃-treated plants were most responsive, producing 20 ELS/100 anthers and those from 50 M ABA-treated plants were least responsive, producing 2.4 ELS/100 anthers. Anthers from growth chamber plants were significantly more responsive when grown in a 16-h than a 12-h photoperiod. With the 16-h photoperiod the response was significantly greater with a 250 M ABA whorl treatment. With the 12-h photoperiod there was no significant effect from whorl treatments. Modification of the culture medium with added ABA, GA₃, ancymidol, or fluridone was generally ineffective, except in 1997 when the response was significantly higher with 1 M ABA added to the culture medium. The results suggest that the maize anther culture response may be influenced by environmental conditions that interact with ABA and GA treatments to donor plants during tassel development.     

Cold pretreatment to enhance green plant regeneration from rye anther culture

Cold pretreatment of detached tillers of rye, Secale cereale, was tested under two light regimes. The ryes included two spring and two winter cultivars. Significant increases in green plant regeneration were recorded in each experiment when cold pretreatments of two to four weeks were applied. Dim light during the stress period improved green plant regeneration for two of the four cultivars tested. The highest regeneration rate, 30.6 green plants per 100 plated anthers, was reached following three weeks at +4 °C under dim light, for spring rye Jo02. Starvation stress applied to plated anthers in mannitol medium suppressed anther response. This revised version was published online in June 2006 with corrections to the Cover Date.     

The statistical analysis of potato anther culture data

Methods for the production and utilisation of haploids have been developed for a range of crop plants. Genetical and environmental factors are known to influence both the rate of haploid induction and the mode of regeneration. Investigations designed to examine the parameters that influence haploid production from anthers are usually based on the overall percentage of responding anthers. Previous studies have rarely taken into account the frequency or distribution of anther culture response within a potato flower. In this paper the binomial, poisson and multiplicative binomial distributions are used for the first time to describe the distribution of anther culture response within a Solanum tuberosum flower.     

Protein markers for anther culturability in barley

Two-dimensional electrophoresis of proteins from a recombinant population of anther culture-derived doubled haploid lines identified 4 loci or linkage groups showing a deviation from an expected 11 segregation. It was hypothesized that these markers are linked to genes involved in the process of haploid plant production and that the deviation was due to a selection for alleles conferring higher anther culture response. To check this hypothesis, the anther culturability of 50 of the doubled haploid lines and their two inbred parents was assessed. It was found that 2 of the loci which had a distortion of segregation showed a significant effect on anther culture response, the most efficient allele being the most frequent in both loci. In addition, 2 more markers associated with anther culturability were found. One of the first mentioned 2 loci and one of the latter 2 were found to be linked to genes involved in both embryoid production and subsequent green plant regeneration. The remaining two were linked to genes involved only in green plant regeneration. Of the 4 favorable alleles 3 were inherited from one parent.     

Effects of Paclobutrazol on Response of Two Barley Cultivars to Salt Stress

The seeds of two barley (Hordeum vulgare L.) cultivars (a drought resistant cv. Tokak-137/57 and a drought sensitive cv. Erginel-90) were imbibed either in distilled water (control) or in a solution containing 40 mg dm⁻³ paclobutrazol (PBZ) and air dried. Seeds were germinated and grown in a glasshouse for 21 d and seedlings were subjected to salt stress by treating them with 100 and 200 mM NaCl for 12 d. The height of shoots was significantly decreased and root length was increased in PBZ-treated plants prior and after NaCl stress for 12 d leading to an increase in root to shoot ratio. Leaf chlorophyll and carotenoid contents in PBZ treated plants were increased in controls and especially in plants subjected to salt stress. PBZ induced increase in superoxide dismutase (SOD) activities was higher in cv. Tokak-157/37, than in cv. Erginel-90. However, an increase in SOD activity was not accompanied by an increase in peroxidase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of genotype and medium on rye (Secale cereale L.) anther culture

Anthers of three rye inbred lines - L9, L318, Dw28, one synthetic hybrid - F₁(5) and one variety, Dakowskie Zote (DZ), were cultured on two media based on N6, and two based on P2 components. The induction rate significantly depended on genotype - the best results were obtained for line L318 and the lowest percentage of responding anthers was noticed in line L9. There was no universal medium for all tested genotypes. The highest induction rate (IR) for lines L318, L9 and hybrid F₁(5) was obtained on medium CI (11.94, 0.71 and 1.75 respectively). For DZ, the P2I medium was better than the others while for Dw28 CIP turned to be as suitable as CI. A highly significant interaction between genotype and medium was proved. Single anthers of DZ, L318 and L9 produced embryos on CI, CIP and P2I media. They did not develop into plants but after transferring them to CS1.7 medium, a secondary, embryogenic callus was obtained. Such a reaction has not been described in rye until now. In spite of a relatively high IR, plant regeneration was rather poor. An elaboration of this step in haploid production is needed. Most of the analyzed calluses and microspore derived regenerants proved to be haploids, according to flow cytometry analysis. Plants treated with colchicine were doubled haploids.