Anther Culture Technique Research Articles

Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.

Comparison of fertility between the F1, F2 and anther derived lines in the crosses of indica/japonica and japonica/indica in rice (Oryza sativa L.)

Response of anthers in in vitro culture was examined in the indica-japonica hybrids of rice (Oryza sativa L.). Significant genotypic differences were observed for callus induction and regeneration among the different interracial hybrids of indica-japonica races. Induction frequency of haploids ranged from 57.7 to 72.9 per cent and doubled haploid androgenic lines ranged from 27.1 to 42.3 per cent in the anther culture of the different hybrids. The indica-japonica hybrids recorded partial pollen grain and spikelet fertility in F1 (29.9 to 41.5% and 19.4 to 48.7% respectively) as well as in F2 (42.7 to 50.6% and 37.1 to 54.4% respectively). In contrast, the androgenic doubled haploid lines recorded significant increase and the pollen grain and spikelet fertility was 76.3 and 78.6 per cent respecitively. The results suggested that the sterility barriers for realising genetic recombinants and fixation of fertile homozygous lines in indica-japonica hybridization programme could be overcome through F1 anther culture technique.

The effect of polyamines on protein kinase activities of wheat (Triticum aestivum) L. anthers

Protein kinase activities were extracted from anther tissue of wheat (Triticum aestivum L. cv. Zaragoza) and characterized with regard to the effects of polyamines, second messengers and plant growth hormones. The protein kinases were inhibited by polyamines and cyclic nucleotides, but were stimulated by the addition of auxins, gibberellic acid and kinetin. The dominant polyamine-sensitive kinase activity was partially purified and characterized. The optimal pH of the reaction was 7.5 to 8.0 and casein was the preferred exogenous substrate. Polyamines were inhibiting in the decreasing order of putrecine > spermidine > spermine. The results are discussed against the context of the anther culture technique.

Comparative Morphological Studies of Microspore Derived and F2 Plants Obtained from an Interspecific Rice Hybrid Oryza sativa Linn. ×O. rufipogon Griff.)*

AbstractAnther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F2 plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F2 segregation. (3) It was possible to recover indica type recombinants among the anther‐derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice.

Microspore development in the anther culture of wheat(Triticum aestivum L.).

A cultivar (Chris) of hard red spring wheat (Triticum aestivum L.) and a one-step haploid generation medium were used to study microspore development and callus formation by using an anther culture technique. Cytological observations on microspore development were made 7, 10, 13 and 16 days after the anther was placed on the medium. The first mitotic division of the uninucleate microspore formed two nuclei, which were similar or different in size. Further divisions of the vegetative nucleus produced multicellular pollen grains that continued to grow and then were released from the exine. This extensive division of the vegetative nucleus could be the source of embryoid or callus generation.

Agronomic Performance of Wheat Doubled‐Haploid Lines Derived from Cultivars by Anther Culture

AbstractTo determine the level of gametoclonal variation among doubled‐haploid lines (DHLs) of Triticum aestivum L. developed using anther culture techniques and its effect on agronomic performance, 70 anther culture‐derived DHLs of ‘Kitt’ were compared for 7 agronomic traits to 50 single‐seed descent‐derived lines (SSDLs) of ‘Kitt’ and to the cultivar ‘Kitt’. In a second experiment, 26 DHLs of ‘Chris’ were compared for 7 agronomic traits to 29 SSDLs of ‘Chris’ and to the cultivar ‘Chris’. Each experiment was grown as a randomized complete block design with three replications in three environments. For ‘Kitt’, the DHLs averaged significantly lower grain yields than the comparable SSDLs. For ‘Chris’, the DHLs averaged lower, but not significantly lower, grain yield than the SSDLs. In both ‘Kitt’ and ‘Chris’, the genetic component of variance for yield of the DHLs was significantly larger than that of the SSDLs indicating the presence of gametoclonal variation. The lower average grain yield of the DHLs was explained by a larger group of low‐yielding DHLs than was present in the SSDLs. Six ‘Kitt’ DHLs and 3 ‘Chris’ DHLs were lower yielding than the lowest yielding SSDL, respectively. Elite DHLs and SSDLs were similar for mean grain yield performance. Though the DHLs and SSDLs were significantly different for some yield components, the affected yield component changed with the cultivar and there was no consistent effect. Significant genotype × environment interactions were detected for some traits which were caused by changes in the magnitude of differences, rather than reversals in ranking, indicating that low yielding DHLs could be culled on the basis of visual selection or single‐environment testing. Hence, gametoclonal variation was induced by the anther culture techniques used in this study, tended to be deleterious for grain yield, and was sensitive to the growing environment. However, as the DHLs and SSDLs had similar expected population means based upon expected gains from selection, this gametoclonal variation should not be a major hindrance to wheat breeding.

Diallel analysis of barley anther culture response

The genetics of barley microspore development in culture was examined by means of diallel analysis. The frequency of microspore derived green and albino plant production was shown to be under genetic control. This genotypic limitation to microspore development will limit the application of anther culture techniques to barley breeding programmes. However, significant additive genetic effects were detected for the characters measured and indicate that the frequency of green plant regeneration may be improved by the hybridization of suitable parents. Significant reciprocal differences were also detected and indicate that the direction of the cross is important in determining microspore development. An embryogenic route to green plantlet formation was observed in a number of genotypes in the diallel experiment. The implications of these findings for barley improvement and genetics are discussed.Key words: doubled haploids, barley, anther culture, microspore, embryoid.

Expression of various gametic types in pollen plants regenerated from hybrids between Triticum-Agropyron and wheat

Studies of the chromosomal composition of pollen plants regenerated from the F1 of hybrids produced from Triticum-Agropyron intermediate type and common wheat demonstrated that various gametic types of the F1 could be fully expressed at the whole plant level via anther culture. The observed frequency of each of the eight types of pollen plants (based on their chromosome numbers) was in good agreement with the theoretical probabilities as shown by X2 analysis. Comparative studies of the chromosome composition of somatic cells and pollen mother cells (PMC's) of selected pollen plants permitted classification of the plants into four distinct classes. The majority of these regenerated pollen plants had identical chromosome numbers in both root tip cells and PMC's. An alien disomic addition line, which was cytologically stable for two generations, was obtained directly from anther culture. Moreover, the addition line exhibits resistance to stripe rust disease, a trait which is conferred by the Agropyron chromosome. We suggest that anther culture techniques provide a unique and expeditious route for the introduction of alien genes or chromosomes into wheat cultivars.

Comparison of haploid production efficiency through anther culture and intergeneric crossing in three wheat varieties and their F1 hybrids.

Frequencies of haploid production in wheat were compared between the two methods of anther culture and intergeneric crossing with Hordeum bulbosum L. using three varieties and their reciprocal F1 hybrids. Average frequencies of embryo and plant production per 100 florets were 5. 02 embryoids and 1.27 plants (including 0.76 albino plants) through the anther culture technique, 55.0 embryos and 39. 4 green plants through the bulbosum technique, indicating that the bulbosum technique was obviously much more efficient. Problems to be solved for applying both methods to all the wheat genotypes were discussed.

Anther culture of Solanum genotypes

Anther culture response was examined in five Solanum genotypes. Large genotypic differences exist in response to culture and liquid culture media were found to be superior to agar solidified media systems. Pre-treatment effects on embryoid induction were also investigated and two of the genotypes displayed differential response to temperature stress pretreatment. In general the beneficial effect of charcoal in the media was confirmed. Successful embryoid production and plantlet regeneration was obtained from the wild potato species, Solanum papita. The influence of sucrose concentration on embryoid production was also investigated. Large genotype by sucrose interactions were detected and this was mainly due to the differential response of the tuberosum clones to increasing sucrose concentration when compared with S. papita. Embryoids were produced from this species in media containing 15% sucrose although the optimum concentration of sucrose for embryoid production was 9%. The possible role of anther culture techniques in gene introgression and potato improvement is discussed.

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). I. Comparison of sexual and doubled-haploid populations

The genetic consequences of anther culture and chromosome-doubling techniques on burley tobacco (Nicotiana tabacum L.) were examined in this study. Three diploid populations, obtained from a burley tobacco inbred by conventional and anther-culture techniques, were compared. The first population consisted of 50 conventionally-selfed lines; the second population was made up of 35 doubled-haploid lines obtained from individual haploid plants by in vitro techniques (IVDH); and the third population consisted of 20 doubled-haploid lines whose chromosome complements had been doubled with colchicine (CDH). Comparisons of doubled-haploid lines with sexually-derived lines revealed significant differences for yield, maturity, leaf length, and alkaloid content. Yield reductions in the doubled-haploid populations averaged 8.5%. Significant differences observed between the IVDH and CDH populations indicate that the reported deleterious effects of colchicine contributed to the vigor reduction of doubled haploids. The anther derived lines in this study exhibited greater variation than did the sexual materials. This variation could provide useful variation for a breeding program. Variation exhibited by the sexual progeny of the highly inbred line, ‘Kentucky 16’, suggests that the differences among anther-derived materials are at least partially due to natural phenomena.

Variation for improved protein and yield from rice anther culture

The anther culture technique is useful for the recovery of haploids which when doubled provide homozygosity. Additionally, beneficial as well as deleterious genetic and epigenetic changes are promoted by the in vitro procedures. The majority of plants recovered from anther calli of the cultivar 'Calrose 76' were similar to the starting cultivar but plants regenerated from microspore calli had a wider range of responses than controls for several characteristics. Plants with larger seeds, higher levels of seed protein, shorter stature and more highly tillered than the starting cultivar were obtained from selfed anther-derived plants. The data also support the concept that in vitro procedures including anther culture of specific cultivars of rice promoted the recovery of phenotypes with increased seed storage proteins.

Recovery of Heritable Variability in Anther‐Derived Doubled‐Haploid Rice1

The anther culture technique for the derivation of dihaplolds in rice, Oryza sativa L., has not been applied extensively to commercial cultivars in the United States. The objective of this study was to identify suitable genotypes among U.S. cultivars for the recovery of useful variability from doubled haploid plants regenerated from anther‐derived calli. Six rice cultivars were evaluated for their ability to produce haploids by anther culture. The cultivar ‘Calrose 76’ produced, on the average, anther‐derived calli in more than 2% of the anthers excised. Some haploids recovered from Calrose 76 were shorter than the original cultivar. Second‐generation, colchicine‐doubled and spontaneously doubled dihaploids produced progeny significantly shorter than the original cultivar. The enhanced dwarfness was transmitted to offspring by selfing. Third‐generation plants derived from doubled haploids were 15 to 30% shorter than Calrose 76. Seed and leaf size and seed weight were significantly lower in the third‐generation progeny than in the controls. The work shows that both useful and deleterious variability can be recovered from Calrose 76 anthers, and the technique should be useful to rice breeders for the development of short stature cultivars and other unique characteristics.

Plantlet Formation by Anther Culture of Citrus aurantium L.

An anther culture technique was employed in Citrus aurantiuln L.; 'Sour Orange', 'Bouquet', and two varieties grown in Japan, 'Kabusu (Shu-to)' and 'Choshu-to'. Anthers collected from immature flower buds were cultured on the MURASHIGE and SKOOG's (19692) medium supplemented with or without indole-3-acetic acid (IAA) and kinetin at 28°C in darkness. Embryoid formation vvas generally evident 14 weeks after inoculation of anthers of 'Sour Orange' and 'Choshu-to'. Anthers containing pollen grains at the late uninucleate stage formed embryoids effectively on media with 0.02mg/l of kinetin. Nuclear divisions of pollen grains and multinucleate pollen grains were observed in the 'Sour Orange' an-thers, while embryoids from pollen grains were not observed microscopically so far. After the incubation in light with a 16-hour photoperiod, some of these embryoids differentiated into shoots, developing into plantlets, while some of the other embryoids produced more embryoids mainly from the hypocotyl region. Induction of roots and shoots was stimulated by transfer from the embryoid induction media containing growth regulators to a medium eliminated growth regulators. Root tip cells of some of the differentiated plantlets had the diploid chromosome number.

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium. This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO2 concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum. Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10−6 g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10−6 g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.

Induced mutants from dihaploid potatoes after pollen mother cell treatment

Microspore mother cells of dihaploid Solanum tuberosum plants were mutagenically treated during the stage of meiosis. Mutagenesis was performed either by irradiation with x- or γ-rays or by the application of nitrosomethylurethane or methylnitronitrosoguanidine. Then, by use of the anther culture technique, 913 functional plants and 442 untreated control plants were regenerated. From the exposed plants seven distinct mutants could be isolated, predominantly chlorophyll deficient lines, while from the controls no clear-cut mutants arose. One mutant turned out to be photomorphogenetic in addition to having a chlorophyll defect. In addition to the production of mutants the treatments significantly increased the frequency of multicellular structure formation from microspores.

Induction of haploidy from pollen grains in angiosperms ? the current status

Since the successful induction of haploids from anthers cultured in vitro in 1964, a great deal of attention has been given to this problem by those interested in obtaining pure lines and mutants for crop improvement and biochemical genetics. In the last 16 years the anther culture technique has been refined and extended to over one hundred and fifty different species. More recently, isolated pollen culture - which is a refinement of the original anther culture technique - has also been developed. In this review we have made an effort to critically examine existing reports with the objective of analysing the effects of various factors - e.g. culture medium, the cultural conditions, and the effect of genotype and physiological state of the parent plant on pollen induction - and to speculate on the mechanism of action of different factors in order to throw some light on the process of haploid induction.

Haploid Plant Development from Anthers and In Vitro Embryo Culture of Wheat1

Experiments were designed to identify cultivars of wheat (Triticum aestivum L., aestivum group) and culture media most suitable for successful in vitro anther culture and development of haploids. Anthers containing uninucleate pollen from 17 cultivars grown in the greenhouse and five lines grown in the field were excised and cultured on a potato (Solanum tuberosum L.) medium. ‘Kitt’, ‘Downy’, ‘Centurk’, and ‘GWO‐1809’ produced the highest number of embryoids from microspores.Calli removed from anthers cultured in vitro were differentiated into plantlets. Over 50 plants have been recovered. Preliminary cytological determinations proved that the plants were haploid. The results demonstrate the feasibility of the anther culture techniques for the development of haploids in agronomically important wheats.Embryos from presoaked and surface‐sterilized seed of 26 wheat lines were excised and planted on defined agar media to measure in vitro growth rates. Embryonic cells from mature wheat seeds divided and produced partially differentiated tissues on standard tissue culture media containing supplements of 2,4‐dichlorophenoxyacetic acid (2,4‐D), 2‐isopentenyladenosine, and naphthaleneacetic acid. Transfer of the calli to a medium with less 2,4‐D or to a medium with 1 mg/liter of indole‐3‐acetic acid and kinetin enhanced differentiation and the differentiated tissues ultimately produced whole plants. There were statistically significant differences in the growth rates, measured as fresh weight, among different lines. Among the promising lines for in vitro cultures were Kitt, GWO‐1809, ‘Baart’, ‘Coker 75‐6’, and others. Isolated embryos from some cultivars did not form calli in vitro. Kitt and GWO‐1809 grew well as partially differentiated calli.

Plant regeneration from leaf protoplasts of haploid Hyoscyamus muticus L. produced via anther culture

Haploid plants of Hyoscyamus muticus L. (egyptian henbane) obtained by conventional anther culture technique were propagated in vitro via axillary buds. Protoplasts isolated from leaves of such haploids were freed of debris by density gradient centrifugation on Percoll and cultured by a multidish system. Up to 35% divisions could be obtained in the protoplast population. Small calli from protoplast-derived cells rapidly regenerated plantlets. The high regeneration capacity of H. muticus in culture suggests it to be a good system for studies in morphogenesis and possibly for studies in genetic modification and secondary product biosynthesis.

Performance of Anther‐Derived Dihaploids and Their Conventionally Inbred Parents as Lines, in F1 Hybrids, and in F2 Generations1

Dihaploid lines of tobacco (Nicotiana tabacum L.) were developed from anther cultures of two long‐term, conventionally inbred cultivars. Ten dihaploids from ‘Coker 139’ and 10 dihaploids from ‘NC 95’ were selfed and randomly crossed to produce 10 dihaploid F1's. Each F1 was selfed to produce a dihaploid F2. The conventional cultivars were crossed to produce a conventional F1, which was selfed to produce an F2. All genetic materials were evaluated for agronomic performance and chemical composition.Dihaploid lines grew more slowly, yielded approximately 15% less, and were agronomically inferior to their conventional pure‐line parents. Dihaploid F1's and F2's yielded less than the conventional F1 and F2. Enforced complete homozygosity in tobacco may not be entirely responsible for the reduction in yield of dihaploid lines developed by anther culture methods. The dihaploid technique as presently employed may be mutagenic.Reduced yields of dihaploid lines produced by the anther culture technique and the inferiority of crosses of dihaploid lines compared to conventional entries, suggest that this technique may not be suitable for a tobacco breeding program, where increased yield is of primary interest.