Anther-culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.)

An in vitro anther-culture method has been improved by using young mother plants and by using frequent subcultures, thus increasing the androgenic yield in different Capsicum annuum L. genotypes. An assortment of peppers was used, composed of 15 genotypes (four breeding lines, seven cultivars and four F₁ hybrids). A new system for qualifying the androgenic response was established. For use in practical breeding, a minimum of 5 % of plant regeneration was proposed as the criterion for a fair response. Accordingly, one excellent, one good and eight fair responses were identified among the genotypes investigated. As compared to the standard cultivar. 2 genotypes gave a significantly better response, i.e. ‘Fehérözön’ (75.8%) and ‘Szechuan 90716’ (21.0%). In comparative investigations, F₁ hybrids, produced from crosses between poor/non-responsive and responsive genotypes, showed a fair level of response, even the case of a poor response in donor parent. The ploidy level of the resulting plants was determined by flow-cytometric analysis.     

Analysis of simple sequence repeat markers in homozygous lines of apple

Haploids in apple were induced using anther culture and in situ parthenogenesis followed by in vitro culture of immature embryos or cotyledons. In addition to an earlier characterization of the zygosity state using isozymes, the purpose of this work was an analysis of simple sequence repeats (SSRs) to describe the homozygosity more comprehensively. Five previously described SSR primer combinations were applied. Initially, polymerase chain reaction amplification with the primer pairs was performed on the donor genotypes ‘Alkmene’ and ‘Remo’ and on a further four apple cultivars. Only those primer pairs that amplified two different contrasting alleles could be used for investigation of the zygosity state. According to these analyses both the androgenic and parthenogenic regenerated shoots are homozygous.     

Microspore Embryogenesis Induced by Low Gamma Dose Irradiation in Apple

Flower buds and extracted anthers of two apple genotypes, ‘Golden Delicious’— a previously known non-androgenic genotype — and ‘Topred’— a known androgenic genotype —, were irradiated by gamma-rays at doses ranging from 5 to 20 Gy, after 0 to 5 weeks of cold pre-treatment (3 °C). When the extracted anthers were irradiated, both microspore embryogenesis and callogenesis were limited for the two genotypes. For ‘Golden Delicious’, androgenic embryogenesis was induced from the flower buds irradiated at 10 to 20 Gy and after 3 to 5 weeks of cold pre-treatment. For ‘Topred’, androgenic embryogenesis was slightly improved after flower bud irradiation at 5 to 10 Gy following cold pre-treatment from 0 to 3 weeks; it was limited after 5 weeks of cold pre-treatment.     

QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice

Anther culturability of rice is a quantitative trait controlled by nuclear‐encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F₂ populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F₂ populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.     

The Effect of Sugars and Growth Regulators on Embryoid Formation and Plant Regeneration from Barley Anther Culture

The influence of carbon source and growth regulator composition in induction medium on anther culture response was investigated using spring barley genotypes. Anthers were cultured on BAC3, Ficoll-containing medium, supplemented with one of the following carbohydrates: sucrose, maltose, cellobiosc and melibiose (6 % w/v). The use of either maltose or cellobiose resulted in a significantly higher anther response, calli and/or embryoid production and green plant regeneration compared to the incubation of anthers on a medium containing sucrose. Contrary to these results, the replacement of sucrose by melibiose in BAO medium, drastically reduced the efficiency of anther culture. As an average for the three genotypes tested, the frequency of green plants per 100 anthers plated was 9- to 22-fold higher on medium supplemented with sucrose or cellobiose than on medium containing melibiose as a sole carbohydrate. Among the growth regulators tested, the combination of auxin NAA (2 mg/l) and cytokinin BAP (1 mg/1) performed much better than the employment of auxin 2,4-D combined either with zeatin riboside or BAP as cytokinins. The beneficial effect of medium supplemented with NAA and BAP was associated with better embryoid formation compared to the other growth regulator combinations tested. The hormone-free combination gave a similar anther response and production of calli as any medium supplemented with growth regulators, but the regeneration capacity of calli produced on hormone-free medium was much lower, resulting in the drastic reduction of the number of both total and green regenerants.     

Retrospect on in vitro androgenesis of sorghum (Sorghum bicolor)

Development of sorghum hybrids can immensely benefit from an enhanced anther culture protocol since the resultant doubled haploids would accelerate, as in other major cereal crops, the attainment of homozygosity and fixation of alleles. Production of the hybrids that have got high yielding, stress tolerance and other traits of industrial significance in a shorter time as compared to the current trend can hence be realized. This review presents the advances that have been made in the development of such a protocol, with closer attention to androgenic induction components of pre-treatment, culture media composition and conditions as well as ploidy determination and eventual chromosome doubling. From the findings of this review, it is clear that further work is desired to ensure a protocol, that to a large degree, breaks the genotype dependency trend that has been widely cited to impede the usability of the tested protocols.     

Regeneration of anther-derived plants from pentaploid hybrids of Festuca arundinacea × Lolium multiflorum

Anther culture was used to generate progeny from pentaploid F1 hybrids of Festuca arundinacea (2n = 6x = 42) with Lolium multiflorum (2n = 4x = 28). Of 6360 anthers cultured, 676 green and 3460 albino plantlets were regenerated. Large differences in the anther culture response were observed among the F1 hybrids with the regeneration efficiency ranging from 0 to 28.8 green plants per 100 anthers cultured. All regenerants were less vigorous than the F1 hybrids and considerable morphological variation was present in such characters as plant height, leaf length and width, and the type and length of inflorescences. Among 295 plants which flowered during the first growing season only 38 were male fertile with pollen stainability ranging from 7.7% to 84.7%. Chromosome numbers in 170 analyzed regenerants ranged from 14 to 42 and fell into three groups: 14 to 21, 28, and 30 to 42. The experiment demonstrated that generation of large populations of androgenic progeny was feasible. This revised version was published online in July 2006 with corrections to the Cover Date.     

Haploidy breeding and mutagenesis for drought tolerance in wheat

Several intraspecific crosses between known drought tolerant wheat varieties and stable high yielding recombinants were made with the objective to develop improved cultivars for the moisture stressed rainfed areas of Pakistan. Five of these crosses were selected for further creation of useful mutations through the application of low doses of gamma rays and development of doubled haploids through anther culture. Anther culture response of the selected irradiated F₁ generations was studied on liquid and solid induction media. The highest number of calli among almost all crosses was produced on Potato-2induction medium. All the crosses varied greatly in response to callus induction and maximum calli (75%) were obtained from Lyl-73/vee’s’ cross. Similarly, genotypic differences were found for green vs. albino regenerants. The highest number of green plantlets (12.1%) was recorded for Lu-26/3062. From the developed doubled haploid population 25 DH-mutants were initially selected and nine lines were finally included in multi-locational field tests. Two DH-mutants (i.e. DHML-50 and DHML-9) have potential for better grain yield, earliness, disease resistance and moisture stress tolerance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of salt tolerant rice breeding line via doubled haploid

Summary A haploid breeding program was initiated to develop doubled haploid salt tolerant rice breeding line via anther culture. Two sensitive breeding lines BR4608-R₁-R₂ and BR4909-R₁-R₂ were crossed with a salt tolerant line IR13146-13-3-3 to transfer its salt tolerant character to the doubled haploids.Anther from confirmed F₁s of the two crosses were cultured in defined medium for callus induction and eventual plant regeneration. Fifteen doubled haploid (DH) lines were obtained from two crosses. Test for salt tolerance were done in vitro. Five out of 15 lines were found tolerant at the level of 8–10 decisiemens/m (ds/m) while the rests were sensitive to that level of salinity.Field experiment was conducted to evaluate the doubled haploids under saline and non saline soil. Five salt tolerant lines produced comparable yield with the resistant control (BR 23) under saline condition, whereas these lines yielded even higher in non saline soil under irrigated condition when evaluated with other 10 sensitive DH linesAbbreviations LSD Least Significant Difference - NAA Napthalene Acetic Acid     

Plant regeneration from anther culture in Canadian cultivars of flax (Linum usitatissimum L.)

The effect of culture of anthers at 35 °C for one to four days prior to culture at 25 °C in darkness, genotype, anther orientation on callus induction and shoot regeneration in anther culture of flax was investigated. The influence of type and concentrations of cytokinins in the regeneration medium on shoot regeneration was also investigated. The results suggested that culture of anthers at 35 °C prior to continuous culture at 25 °C in darkness did not significantly improve the percentage of anthers producing calli. However, culture of anthers at 35 °C for one day significantly increased the overall efficiency of regeneration compared to no culture temperature treatment. Genotypic effects were significant for the percentage of anthers producing calli and the overall efficiency of regeneration. Anther orientation showed no significant differences. The regeneration medium containing 4.5 μM zeatin had significantly higher percentage of calli forming shoots than the same basal medium containing 0.01 μM TDZ. The importance of these findings for flax breeding was discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Efficient application of in vitro anther culture for different European winter wheat (Triticum aestivum L.) breeding programmes

The efficiency of our anther culture protocol was tested with high‐ and low‐responding genotypes, ‘Svilena’ and ‘Berengar’, and 93 F₁ winter wheat crosses in 2010 and 2011. Based on data for these genotypes, the effect of genotype influenced the number of embryo‐like structures, regenerated plantlets and green plantlets, while the number of albino plantlets was affected by genotype, year and environmental factors. Although genotype also influenced the production of green plantlets from breeding crosses, with green plantlets per 100 anthers ranging from 0.04 to 28.67, the average regeneration rate over all crosses was 5.3 green plantlets/100 anthers, which resulted in a total of 11 416 well‐rooted green plantlets. The survival rate of green plantlets following acclimatization was 97.21% in 2010 and 96.34% in 2011. In this study, the phenomenon of albinism and genotype dependency did not hinder the production of more than five thousand green plantlets each year. In our experiments, anther culture proved to be an efficient method in winter wheat breeding programmes with lower costs than alternative technologies.     

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

To 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.     

Androgenic capability among genotypes of winter and spring barley

Variable efficiency of androgenesis remains a serious problem in many species of cereals. It is still unclear what makes certain genotypes more amenable to androgenesis than others. This study was undertaken to quantify the previously suspected advantage of winter barley genotypes over spring ones with regard to regeneration efficiency in anther culture. The material consisted of 40 barley hybrids originating from Polish breeding companies. The number of androgenic structures per 100 anthers did not differ significantly between analysed groups (119 vs. 152 non‐significant), but the average regeneration of green plants per 100 anthers was five times higher in winter genotypes (6.4 vs. 1.3). The incidence of albinism was lower for the winter than for the spring materials (70% vs. 90%), while the rate of spontaneous chromosome doubling was similar in both groups (58% vs. 56%). The results strongly support the notion that winter genotypes are more amenable to androgenesis and this may be a consequence of their better adaptation to stress conditions.     

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

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

Development of the components of a cytoplasmic male sterility hybrid system in rye through anther culture

Anther culture was applied as a method to develop the essential components of a cytoplasmic male sterility hybrid system in rye (Secale cereale L.). These components are the male sterile seed parent (A line), its isogenic maintainer counterpart (B line) and the restorer pollen parent (R line). Australian rye cultivars were crossed reciprocally to the cultivar ‘Luchs’ which carries the Pampa male sterile cytoplasm (cms-P). Anthers of the F1s in the cms-P cytoplasm (primary cross) and their reciprocals in the normal cytoplasm (reciprocal cross) were cultured in a modified C17 medium. Male sterile and male fertile doubled haploids were obtained from the anther culture of the F1s in the cms-P cytoplasm. Testcrosses indicated that the male sterile doubled haploids were A lines and the male fertile doubled haploids were R lines (restorers). The anther culture of genotypes in the normal cytoplasm (reciprocal cross) gave all male fertile doubled haploids. Testcrosses indicated that the male fertile doubled haploids were R lines (restorers) in the normal cytoplasm. The expected maintainer B lines were not identified because of the limited number of doubled haploids obtained from the anther culture of reciprocal crosses. Experimental single cross hybrids between male sterile and restorer male fertile doubled haploids showed high levels of heterosis. The results of this study have significant economic implications especially in the production of hybrids in several species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient low-cost DNA extraction and multiplex fluorescent PCR method for marker-assisted selection in breeding

Marker‐assisted selection (MAS) is an increasingly important tool in current breeding efforts for improved crop plants and animal breeds. It enables detection of favourable alleles in early developmental stages and thus may result in substantial cost savings. Until now, however, the high costs of the required chemicals and materials, together with the still very labour‐intensive methods, have been an obstacle to widespread application of MAS. A new multiplex‐polymerase chain reaction (PCR)‐based method has been developed for reliable low‐cost, high‐throughput screening. By its use 3366 apple seedlings were screened with an average hands‐on time from DNA extraction to data ready for analysis of < 4 h per 96 plants, and at a cost below US$ 0.5 per marker per plant. Factors that have a strong effect on segregation ratios such as elevated levels of outcrossing are easily detected, as a significant correlation was observed between deviation from expected segregation ratios in some affected markers and the level of outcrossing in a cross. The new method is suitable for many crop species and, provided that suitable buffers are used for DNA extraction, for animals too.     

Barley mutant heterosis and fixation of'F1-performance'in doubled haploid lines

The phenomenon of mutant heterosis was studied in barley using F₁ hybrids developed from crosses involving two mutants derived from the same parent variety (067AR× 032AR), or from crosses of mutants with their parent varieties (Diva × 228DV; 437MG × Mg 4170). The agronomic performance (plant height, tillers per plant, kernel number and weight per plant) of F₁ hybrids was evaluated in relation to the corresponding parent variety used for mutant development. Wide-space sowing (0.O6 m²/plant) was applied to allow the full expression of hybrid vigour. The hybrids significantly exceeded their parent varieties in various yield parameters in each year of the experiment, Depending on the cross, the level of heterosis ranged from 39.45% to 82.1% for kernel number and from 44.45% to 91.2% for kernel weight, as an average of observations over three years. The increased yield of hybrids was accompanied by an increase in spikes/plant, without significant changes, in most cases, in yield components such as kernels/spike and 1000 kernel weight. The height of F₁ plants remained similar to the corresponding parent variety. Doubled haploicls (DH) were developed through anther culture from three F₁ hybrids to determine whether the heterotic effect could be fixed, even partly, in homozygous lines. Twenty-eight, 42 and 68 DH lines per cross were produced, and evaluated in the field, space-planted at 0.06 m²/plant, together with hybrids and parent varieties. The best-performing DH lines were evaluated again the following season in a conventional field trial. We were able to select 1.5 -14.3% of‘F₁,-performing’DH lines per hetorotic cross combination, which, in two years of studies, reached the yield of heterotic hybrids and significantly out-yielded the parent variety in plot. The yield potential of DH lines was better expressed under a wide sowing density, but even under normal growing conditions, the yield of the best selected DH lines surpassed their corresponding parent varieties by 17.77ndash;30.1%.     

Field Performance of Androgenetic Doubled Haploid Spring Wheat Lines in Comparison with Lines Selected by the Pedigree System

A total of 316 doubled haploid lines (DH) of spring wheat were compared with 621 lines selected in a pedigree system (PS) under field conditions in a breeding nursery. The lines originated from 21 crosses and the samples tested represent mean values for variables comprising the main breeding goals such as disease resistance, baking quality, and agronomic traits. In general, the DH lines were later in days to heading and shorter than PS lines of different homozygosity levels. Except for leaf rust (Puccinia recondite), the DHs were more resistant to the artificially infected diseases such as powdery mildew (Erysiphe graminis), stripe rust (Puccinia striiformis), and septoria nodorum blotch (Septoria nodorum). These differences, which were relatively small for practical selection purposes, were probably due to easier and more precise disease assessment of the homozygous DH lines. For quality characters, the DHs had a higher protein level, while the rest of the parameters were similar for both origins. The data analyzed suggest so far that androgentic doubled haploids in spring wheat are very similar to lines selected in a pedigree system in respect to all the agronomic characters tested. However, the DH lines were produced in a much shorter period of time. It is suggested that androgenetic doubled haploids be produced from F₁ hybrids and that the well-established bulk method should continue to allow selection bulk method should continue to allow selection for rare recombinants as soon as homozygosity is reached.