Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

An evaluation of seed and seedling drought tolerance screening tests in wheat

Summary A series of experiments was performed in order to evaluate the significance of seed germination and seedling growth in osmotic media as screening methods for drought tolerance.Ten spring wheat (Triticum aestivum L. em Thell.) and one durum wheat (Triticum durum Desf.) were tested under controlled environments, using polyethylene glycol-6000 (PEG) solutions as the moisture stress inducing media.Tolerance in the rate of endosperm utilization, under stress, prior to the onset of germination varied among cultivars.Germination rate or injury to germination at various concentrations of PEG differed significantly among cultivars. Cultivar rating with respect to injury to germination changed with stress levels. Injury to germination did not correlate with endosperm utilization rate in PEG or in water.Germinating seedlings were tolerant to extreme desiccation up to the stage of emergence of the first leaf from the coleoptile.Growth of photosynthesizing seedlings was monitored as they were carried through an increasing concentration gradient of PEG solutions, ranging from –5.9 to –11.3 bars of water potential. Cultivars significantly differed in seedling growth tolerance to increasing levels of water stress. Seedling growth tolerance across cultivars was not correlated with their germination responses under srress.It is concluded that tolerance to water stress in growing seedlings can be screened for by using PEG-containing nutrient solutions. It can not be predicted from germination tests in osmotica.Work was done under a US-Israel Binational Science Foundation (BSF) Grant no. 1654/78.Contribution from Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 192-E, 1979 series.     

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.     

Assessment of genetic diversity in bread wheat genotypes for tolerance to drought using AFLPs and agronomic traits

Moisture stress greatly limits the productivity of wheat in many wheat-growing regions of the world. Knowledge of the degree of genetic diversity among parental materials for key selection traits will facilitate the development of high yielding, stress tolerant wheat cultivars. The objectives of this study were to: (i) use amplified fragment length polymorphisms (AFLPs) to assess genetic diversity among bread wheat lines and cultivars with different responses to drought stress in two distinct environments and, (ii) compare genetic diversity estimated by AFLPs with diversity evaluated on agronomic performance under drought stress. Twenty-eight genotypes, 14 from Iran and 14 developed or obtained by CIMMYT, were evaluated in the study. Phenotypic data on the 14 Iranian lines were obtained in Iran, and data on the 14 CIMMYT lines were collected in Mexico. Ten AFLP primer pairs detected 335 polymorphic bands among the 28 cultivars. At the 5th fusion level of the resulting dendrogram, 6 genotype clusters were identified. Thirteen of the 14 CIMMYT genotypes grouped into one cluster while 4 of the remaining groups were comprised only of Iranian genotypes. When the agronomic performance of the Iranian materials was compared with the AFLP diversity analysis, 5 of the 6 drought susceptible genotypes clustered together in the agronomic dendrogram, and were located in the same cluster in the AFLP dendrogram. However, the drought tolerant Iranian materials did not show the same degree of relationship. The CIMMYT materials did not demonstrate a significant association between agronomic performance and genetic diversity determined using AFLPs. Clearly these data show that there are genotypes with similar agronomic performance and different genetic constitutions in this study that can be combined in a breeding program to potentially improve tolerance to drought stress.     

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     

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.     

Doubled haploid production in winter wheat and triticale genotypes,using the Hordeum bulbosum system

Summary An attempt was made to produce doubled haploids on 16 winter wheat and six spring and winter triticale genotypes thought to carry genes for interspecific incompatibility. The potential for haploid production was maximized by the use of Hordeum bulbosum genotypes selected for high crossability on crossable wheat genotypes, the use of two post-pollination applications of gibberellic acid and by the pollination of immature florets.A low frequency of seed was set on both the wheat and the triticale genotypes, having mean seed sets of 0.20 per cent and 0.27 per cent respectively. Although the frequency of embryos (seed quality) was high, doubled haploid production was further limited by poor embryo differentiation and regeneration. Haploid plantlets were obtained from the wheat cultivars Moulin and Renard, although successful chromosome doubling and doubled haploid production was achieved in Moulin only.     

CIMMYT's approach to breed for drought tolerance

Summary About 32% of the 99 million ha wheat grown in developing countries experiences varying levels of drought stress. Three major drought types have been identified: Late drought (LD) is common in the Mediterranean region, early drought (ED) is found in Latin America and wheat is produced on residual soil moisture (RM) in the Indian subcontinent and part of Australia. Until 1983, CIMMYT selected all germplasm under near optimum conditions for its yield potential and tested only advanced lines under drought. In spite of many critics, this approach proved to be successful, since in the mid 80's CIMMYT germplasm was grown on 45% of the wheat area in LC with annual rainfall from 300–500 mm and on 21% in areas with less than 300 mm. Since 1983, CIMMYT's drought breeding methodology is to alternate segregating populations between drought stressed and fully irrigated conditions (FI) and to test advanced lines under a line source irrigation system. To compare the efficiency of these approach, yield of four, mostly leading varieties, from each of the regions with LD, ED, RM, and FI and twelve recent CIMMYT cultivars selected for high yield under FI and RM conditions (ALT) were compared under four different moisture regimes (FI, LD, ED, and RM) in 89–90 and 90–91 in Yaqui Valley, Mexico. Genotypic correlation between yield and days to flowering, days to maturity, height, grains m^-2, TKW, test weight and grain fill period were calculated.Mean grain yield of the four best lines in the ALT group was highest under all moisture stress regimes, followed by the FI-group. However, the highest yielding cultivar within each moisture regime was from the FI-group under FI, from the LD-group under LD, and from the ALT-group under ED and RM conditions. Estimates for genetic advance suggest that FI is the best environment for increasing grain yield even in all three drought environments. This indicates that yield potential per se is beneficial also in drought environments. The highest yield in drought environments was realized by the CIM cultivars selected under FI and RM. Simultaneous evaluation of the germplasm under near optimum conditions, to utilize high heritabilities and identify lines with high yield potential, and under stress conditions to preserve alleles for drought tolerance seem at present the best strategy.     

Selection for yield in wheat breeding

Summary If selection based on F₃ yield tests is to be effective, the yield tests must be successful in discriminating among yield genotypes. The available literature indicates that simple tests with limited or no replication are not very effective, although more extensive, replicated tests may be.Data from an experiment comparing F₃ yield tests with a single seed descent procedure showed that F₃ selection based on a two-replicate test with single seed descent procedure did not justify the extra work involved.     

Chromosomal regions associated with green plant regeneration in wheat (Triticum aestivum L.) anther culture

Genetic capacity for green plant regeneration in anther culture were mapped in a population comprising 50 doubled haploid lines from a cross between two wheat varieties ‘Ciano’ and ‘Walter’ with widely different capacity for green plant regeneration. Bulked segregant analysis with AFLP markers and composite interval mapping detected four QTLs for green plant percentage on chromosomes 2AL (QGpp.kvl-2A), 2BL (QGpp.kvl-2B.1 and QGpp.kvl-2B.2) and 5BL (QGpp.kvl-5B).The three QTLs detected on chromosome 2AL and 2BL all derived their alleles favouring green plant formation from the responsive parent ‘Ciano’.The remaining QTL on chromosome 5BL had the allele favouring green plants from the low responding parent ‘Walter’. In a multiple regression analysis the four QTLs could explain a total of 80% of the genotypic variation for green plant percentage. None of the chromosomal regions with QTLs for green plant percentage showed significant influence on either embryo formation or regeneration frequencies from the anther culture. The three major QTLs located on group two chromosomes were fixed in a second DH population derived from two parents ‘Ciano’ and ‘Benoist’,both with high capacity to produce green plants. A QTL explaining31.5% of the genetic variation for green plant formation were detected on chromosome 5BL in this cross as well. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative and molecular characterization of heat tolerance in hexaploid wheat

Understanding the genetic basis of tolerance to high temperature is important for improving the productivity of wheat (Triticum aestivum L.) in regions where the stress occurs. The objective of this study was to estimate inheritance of heat tolerance and the minimum number of genes for the trait in bread wheat by combining quantitative genetic estimates and molecular marker analyses. Two cultivars, Ventnor (heat-tolerant) and Karl92 (heat-susceptible), were crossed to produce F₁, F₂, and F₃populations, and their grain-filling duration (GFD) at 30/25 ^°C 16/8 h day/night was determined as a measure of heat tolerance. Distribution of GFD in the F₁ and F₂ populations followed the normal model (χ², p > 0.10). A minimum of 1.4 genes with both additive and dominance effects, broad-sense heritability of 80%, and realized heritability of 96%for GFD were determined from F₂ and F₃ populations. Products from 59primer pairs among 232 simple sequence repeat (SSR) pairs were polymorphic between the parents. Two markers, Xgwm11 andXgwm293, were linked to GFD by quantitative trait loci (QTL) analysis of the F₂ population. The Xgwm11-linked QTL had only additive gene action and contributed 11% to the total phenotypic variation in GFD in the F₂population, whereas the Xgwm293-linked QTL had both additive and dominance action and contributed 12% to the total variation in GFD. The results demonstrated that heat tolerance of common wheat is controlled by multiple genes and suggested that marker-assisted selection with microsatellite primers might be useful for developing improved cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genotypic differences in wheat Al tolerance

Summary Aluminium tolerance of 83 genotypes from Croatian and Yugoslav Triticum aestivum germplasm was evaluated in nutrient solutions having Al³⁺ activities of 0, 12.5 and 25 M. Relative root length (25 M Al³⁺/0 Al) of various genotypes ranged from 2 to 97% (from very sensitive to tolerant to Al). No genotype with Al tolerance close to that of very tolerant cultivar Atlas-66 was found. Soil, climatic, fertilization, and liming effects that wheat plants giving seeds for the nutrient solution Al-tolerance screening had been subjected to during their growth cycle did not influence the Al-tolerance ranking. Significant correlation was found between screening wheat for Al tolerance in nutrient solutions and in acid Pseudogley soil amended with five rates of limestone in a greenhouse experiment. Seed protein concentration was significantly related to the Al-tolerance ranking (r² = 0.962). Such a significant correlation was not obtained in a case of rheological and other quality characteristics of seeds. Al-tolerant wheat genotypes identified in this study will be used in breeding for improved Al tolerance.Abbreviations HSD Tukey's Honestly Significant Difference - RRL-2 relative root length, in % (12.5 M Al³⁺/0 Al) - RRL-4 relative root length, in % (25 M Al³/0 Al)     

Heat tolerance for yield and its components in different wheat cultivars

Summary Twenty one diverse, standard and experimental cultivars of common spring wheat (Triticum aestivum L.) were tested for the effect of heat stress on phenology, yield and its components by growing the materials for 2 years under full irrigation during the hot summer (offseason), and the cool winter (normal) conditions. Heat tolerance was estimated for each variable by the heat susceptibility index (S) which scales the reduction in cultivar performance from cool to hot conditions relative to the respective mean reduction over all cultivars.Genotypes differed significantly in S for yield and its components. The ranking of cultivars in S over the 2 years was consistent for yield, kernels per spike and kernel weight, but not for spike number. Of the three yield components, the greatest genotypic variation in S was expressed for kernels per spike. However, S for yield could not be simply attributed to S in a unique component across all cultivars. On the other hand, a general linear model regression of summer yield on its components revealed that the most important yield component affecting yield variation among cultivars under heat stress was kernel number per spike. Kernel number per spike was positively associated across cultivars with longer duration and greater stabilty of thermal time requirement from emergence to double ridge. It is therefore concluded that kernel number per spike under heat stress is a reasonable estimate of heat tolerance in yield of wheat and that this tolerance is operative already during the first 2 to 3 weeks of growth.     

Differential tolerance to Fe and Zn deficiencies in wheat germplasm

Tolerance to Fe deficiency of wheat genotypes exhibiting differential tolerance to Zn deficiency is not known, even though the relationship between Fe nutrition and differential tolerance of wheat genotypes to Zn deficiency has been hypothesised frequently. In the present experiment, eight Triticum aestivum and two T. turigidum L. conv. durum cultivars were grown in nutrient solution deficient in either Znor Fe. Three indices of tolerance to nutrient deficiency were compared: relative [(-nutrient/+nutrient) × 100] shoot growth, shoot dry weight under nutrient deficiency and relative shoot/root dry weight ratio. Genotypes Aroona, Excalibur, Stilleto and Trident were classified as tolerant to both Zn and Fe deficiency, while durum wheats Durati and Yallaroi were sensitive to Zn deficiency and moderate to sensitive to Fe deficiency. Genotypes Excalibur, Stilleto and Trident come from the same breeding programme and have the common parent (line MEC3 =Sonora64//TZPP/YAQUI54) that could have been the donor of the genes for tolerance to Zn deficiency. When Fe-deficient, all wheat genotypes were severely chlorotic but kept producing shoot and root dry matter at a relatively high rate, making the relationship between the relative shoot growth and the relative leaf chlorophyll content poor. This is the first report of wheat genotypes exhibiting multiple tolerance to Zn and Fe deficiencies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Applications of pedigree-based genome mapping in wheat and barley breeding programs

The aim of the pedigree-based genome mapping project is to investigate and develop systems for implementing marker assisted selection to improve the efficiency of selection and increase the rate of genetic gain in breeding programs. Pedigree-based whole genome marker application provides a vehicle for incorporating marker technologies into applied breeding programs by bridging the gap between marker–trait association and marker implementation. We report on the development of protocols for implementation of pedigree-based whole genome marker analysis in breeding programs within the Australian northern winter cereals region. Examples of applications from the Queensland DPI&F wheat and barley breeding programs are provided, commenting on the use of microsatellites and other types of molecular markers for routine genomic analysis, the integration of genotypic, phenotypic and pedigree information for targeted wheat and barley lines, the genomic impacts of strong selection pressure in case study pedigrees, and directions for future pedigree-based marker development and analysis.     

History of wheat breeding for a half century in Japan

Summary Wheat breeding efforts for a half century in Japan were investigated by using of 129 varieties registered in MAFF from 1929 to 1984 in the view point of case history for three years. Varieties released in each breeding station are classified apparently by growth habit associated closely with duration of the cold requirement. Heading date is earlier in varieties released in southwest breeding stations (southern varieties) than in varieties released in northern breeding stations (northern varieties). Culm length is higher and the pre-harvest sprouting is more sensitive in northern varieties. In quality characters, northern varieties has the higher milling rate, lower apparent amylose content and larger particle size of flour. In varieties released in the Tohoku district, trend of breeding direction with time is smaller ear numbers and larger 1,000-grain weight and greater resistance to powdery mildew. In varieties released in the Kyushu district, a significant correlation with released year was clearly observed with regard to early maturity, short culm length, less grain crude protein content, less apparent amylose content and higher milling rate on breeding advancement.     

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.     

Limited application of homozygous genotypes in apple breeding

Apple is a temperate fruit species with several breeding disadvantages such as the highly heterozygous nature of the genome and a long reproductive cycle. It is expected that homozygous apple genotypes can increase breeding efficiency, but inbreeding is prevented by the gametophytic self‐incompatibility mechanism. In this paper, the production and characterization of homozygous genotypes, generated from anther culture in Malus × domestica cv. ‘Braeburn’, is described as well as their potential for use in breeding programmes. After determination of large scale anther culture efficiency, anther culture‐derived genotypes were simple sequence repeat (SSR)‐fingerprinted to determine their homozygous nature and confirm their androgenic origin. Their value for breeding was estimated based on growth and fertility measurements. The use of homozygous androgenic genotypes from ‘Braeburn’ in apple breeding programmes is currently not a realistic approach, partly because of the low efficiency of anther culture, but mainly because of the reduced vigour and severe sterility of the androgenic genotypes produced.     

Methods for characterization of preharvest sprouting resistance in a wheat breeding program

Preharvest sprouting (PHS) can be a serious problem in western Canada resulting in economically important yield and grade losses for wheat producers. Improved PHS resistance is an important breeding objective and wheat breeders require effective and reliable methods to select for PHS resistance. The objective of this study was to evaluate whether differences exist between field and artificial weathering in the evaluation of PHS resistance in breeding lines and to determine whether sprouting scores are a good estimator of PHS resistance when compared to Hagberg falling numbers. Results suggest that both field and artificial weathering can be effectively used to screen for PHS resistance in wheat breeding lines. Although in this study, field weathering resulted in greater sprouting damage than artificial weathering, the ability to control conditions may make artificial weathering the more reliable test for PHS resistance in a breeding program. Mean sprouting scores greater than 7.5 consistently identified lines with low PHS resistance; hence, sprouting scores can be used to discard the most sprouting susceptible lines. However, falling numbers appear to be more reliable to evaluate the PHS resistance of advanced lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of colchicine treatment on microspore division and microspore-derived embryo differentiation in wheat (Triticum aestivum L.) anther culture

Summary The relationship between division symmetry andin vitro microspore embryo genesis was studied using two winter wheat varieties of high embryogenic capacity. Anther cultures were treated with colchicine added to the induction media at concentrations of 0.01%, 0.02% and 0.04%. As a result of the colchicine treatment, the rate of symmetrical divisions increased significantly which was followed by a significant increase in the microspore-derived embryo frequency. The effect of colchicine was not dependent on the concentrations used. On the basis of this it can be supposed that there is a clear relationship between symmetric divisions and microspore-derived embryo differentiation.