Anther culture response of F1 durum × bread wheat hybrids after colchicine application

The effect of colchicine on the androgenic response of durum × bread wheat F₁ hybrids was studied. For this, three Greek durum wheat cultivars, exhibiting good yield and pasta quality, were crossed to two bread wheat cultivars with good response to anther culture. Spikes of the resulting hybrids, containing microspores in the mid to late uninucleate stage, were selected and cultured on two different media (W₁₄ and solid potato‐2) with and without colchicine. A negative effect on anther culture response was observed when colchicine was added to the W₁₄ induction medium. A similar effect was observed in two of the hybrids when they were cultured on to potato‐2 induction medium. In both induction media, green plant production was influenced negatively by colchicine treatment. The same was observed in albino plant production. The results of the present study support the view that anther culture response is strongly genotype dependent. Finally, potato‐2 induction medium was the most suitable one for the material studied.     

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.     

In vitro Selection for Tolerance to Fusarium in F1 Microspore Populations of Wheat

Microspore populations of eight Fhybrids of winter wheat (Triticum aestivum L.) whose parents had different levels of resistance to Fusarium were screened in vitro, using phytotoxins of Fusarium as biochemical probe. Two selection methods were compared for the in vitro selection: either embryoids and calli were first initiated from anthers in toxin-free medium and then grown on medium with 0.3—0.9 %Fusarium toxin; or anthers were immediately cultured in modified liquid potato-2 medium in the presence of 1.5, 2.0 and 2.5ml Fusarium toxin per liter culture medium, a concentration which reduced the number of calli and embryoids to about 10 % compared to the toxin-free controls. Microspores from donor hybrids which were produced from very susceptible cultivars were killed by lower toxin concentrations than micro-spores from hybrids of less susceptible parents. From surviving calli and embryoids, originally initiated from 242,000 anthers in both procedures a total of 375 green lines could be regenerated. The results indicate that it is possible to enrich the fraction of regenerating microspores by those which contain the gene complex responsible for reduced susceptibility to Fusarium by the use of a pathotoxin.     

Androgenesis in Hexaploid Spring Wheat F2 Populations and Their Parents Using a Multiple-Step Regeneration System

The main goals of the wheat androgenesis experiment were to study the main phenomena of in vitro androgenesis in anther culture of F₂ populations (10) and their parents (6), to compare the usage of P-4 and C-17 media for the formation of embryo/callus and to demonstrate a new plant regeneration system. The P-4 induction medium was found to be significantly better than the C-17 in the number of responsive anthers (RA) and calli induced (CI) at the 1 % and 0.1 % level, respectively. Genotypic effect was evident in both RA and CI. The yields of F₂ populations in RA and CI were significantly higher than those of their parents regarding both media. The data confirmed the existence of heterosis for RA and CI in F₂ populations. The ratio of green/albino plant regeneration was more favourable in the C-17 derived embryo/calli than in the P-4 derived ones. The frequency of plantlet regeneration was enhanced in the group of unresponsive calli by the application of the multiple-step regeneration system. In this system the calli lacking well developed morphogenic structure were transferred to a new regeneration medium, containing a higher concentration of the same cytokinin, other cytokinin or basic medium, before the occurrence of irreversible changes in their physiology.     

Single and combined effects of colchicine, L-proline and post-inoculation low temperature on anther culture of wheat, Triticum aestivum L.

This investigation was undertaken to study the effects of colchicine, L-proline and a post-inoculation cold temperature of 14°C for 7 days, either as a single or as combined treatments, on the parameters of anther culture of the DH83Z118.32 wheat genotype. Results indicate that the addition of 100 mg/1 colchicine to the induction medium for a period of 3 days and at an incubation temperature of 28°C yielded the highest success index. This treatment, although it significantly reduced embryogenesis, improved embryoid quality so that not only the total regeneration, but also the percentage of green plantlets increased significantly, In addition, it raised the percentage of doubled haploids and, consequently, led to a significantly better final success index. Combining the colchicine treatment with a reduced post-inoculation temperature did not intensify its beneficial effect, although a treatment of reduced temperature alone was superior to normal temperature (28°C) for most parameters studied. The beneficial effects of adding L-proline (200 mg/1) to the induction medium, combined with a low temperature (10°C for 4 days) treatment, were diminished when this treatment was combined with a colchicine treatment.     

Somaclonal Variation in Tissue Culture of Triticum aestivum×Agropyron desertorum F1 Hybrid

Callus induced from immature inflorescences of the partially self-fertile hybrids (2 n = 35; ABDPP) between Triticum aestivum (2n = 42; AABBDD) and Agropyron desertorum (2n = 28; PPPP) led to the regeneration of 88 plants on MS medium supplemented with 2 mg/l of 2,4-D. These regenerants were used to investigate somaclonal variation and to obtain more selfed derivatives. Immature inflorescences at the stage of developing floral primordia gave the best response in terms of callus induction and plant regeneration. The regenerants exhibited great variability for most morphological traits. Although the regenerants did not exhibit variation in chromosome number, they did show a higher degree of meiotic instability than the initial hybrid. In particular, the regenerants gave much higher selfed seed-set (5.49 %) than the donor hybrid (0.46 %), so that a total of 484 selfed seeds were obtained.     

Heterosis performance of yield and fibre quality in F1 and F2 hybrids in upland cotton

Because of the difficulty of producing F₁ hybrid seeds by hand emasculation and pollination, wide use of heterosis in cotton production has been limited in China. The objective of this study was to evaluate the potential of F₂ hybrids for yield and fibre quality. A half diallel involving eight parents and their F₁ and F₂ hybrids was grown in replicated studies at Linqing and Nanjing in 1999 and Nanjing in 2000. Yield and fibre quality was determined for all 64 entries. Fibre quality was also determined for parents and F₁s, but only for Zhongmiansuo 28 (ZMS28), Xiangzamian 2 (XZM2) and Wanmian 13 (WM13) F₂s. These three F₂ hybrids are extensively planted in China and provide experimental controls with which to compare the performance of new hybrids. Average yield heterosis for F₁s and F₂s was 15.9 and 9.2%, respectively. Inbreeding depression for yield varied but some F₂s greatly out‐yielded the best variety. Average F₁ heterosis was 6.7, 6.2 and 2.9%, respectively for number of bolls per unit area, boll weight, and lint percentage. The average F₂ heterosis for the same traits was 4.4, 3.3 and 1.6%, respectively. F₁ heterosis for fibre traits was low. In general, parental average was a good indicator of the yield and fibre quality of F₁ hybrids. These encouraging results suggest there is sufficient heterosis for yield to use F₂s in China.     

Heterosis for yield and other agronomic traits of winter triticale F1 and F2 hybrids

Triticale is generally treated as a self‐pollinating crop and line breeding is practised. Hybrid breeding has been discussed for some time, but there is little information for winter triticale. This study investigated heterosis for eight agronomic traits in F₁ and F₂ hybrids grown together with their parents as drilled plots in three environments. On average, grain yield heterosis was 12.5 dt/ha (a relative 10.5%) compared with the mid‐parent value for F₁ hybrids, and 6.2 dt/ha (5.0%) for F₂ hybrids and withawide range of 4.4–17.1 dt/ha for F₁ hybrids. A positive contribution to the heterosis of yield was made by kernels/spike and 1000‐kernel weight, whereas spikes/m² showed negative heterosis. Hybrid plants in F₁ and F₂ were taller than mid‐parents (8.3 cm and 5.3 cm, respectively), with a tendency to earlier heading. The negative heterosis for falling number in F₁ and F₂ hybrids could be a problem for commercial production of triticale hybrids. By selecting parents for combining ability and the identification of heterotic patterns, grain yield heterosis of 20% appears feasible.     

Spike pretreatments, anther culture conditions, and anther culture response of 17 German varieties of spring wheat (Triticum aestivum L.)

The aim of this work was to establish an in vitro regeneration system from anther cultures of different German varieties of spring wheat (Triticum aestivum L.). Using ‘Nandu’ the most widely grown spring wheat cultivar in Germany, different culture conditions were investigated with regard to their influence on anther culture response. The best results were obtained when applying a cold pretreatment to the donor spikes and using the synthetic L3 induction medium, liquid or solidified with gelrite. The highest rates obtained in these experiments with ‘Nandu’ were 8.6% responding anthers, 22.3% embryoid induction, 15.3% albino regeneration and 5.5% green plant regeneration (all rates related to the number of cultured anthers). Of the ‘Nandu’ plants analysed, 51.1% were haploid and 44.3% were diploid, probably as a consequence of spontaneous chromosome doubling. When screening a further 16 commercial German varieties of spring wheat, 10 exhibited good anther culture response and four of these (‘Eta’‘Jondolar’, ‘Mieka’, and ‘Star’) proved to be highly responsive, reaching embryoid induction rates between 4.3 and 10.3% and rates of green plant regeneration between 5.4 and 10.7%.     

The use of AFLP markers for the identification of carrot breeding lines and F1 hybrids

Four inbred lines of carrot (cytoplasmic male‐steriles and corresponding maintainers) and eight of their F₁ hybrids were studied with the amplified fragment length polymorphism (AFLP) technique to examine their genetic relationship and produce markers useful for testing hybrid seed purity. Eighty‐six polymorphic amplicons were identified in bulked DNA samples using eight primer pair combinations. Genetic distance was estimated on the basis of the presence or absence of polymorphic bands. The dendrogram plotted on the basis of the AFLP data closely represented the pedigree relationships of the lines and their hybrids. From one to six amplicons specific for a breeding line were identified. Most of them were also present in the DNA bulks of respective F₁ hybrids. However, screening performed on individual plants of two parental lines and the corresponding hybrid indicated insufficient uniformity of parental lines, limiting the applicability of AFLP markers for testing hybrid seed purity.     

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.     

Cytogenetic analysis of F1, F2 and BC1 plants from intergeneric sexual hybridization between Sinapis alba and Brassica oleracea by genomic in situ hybridization

By intergeneric sexual hybridization between Sinapis alba and Brassica oleracea , F₁, F₂ and BC₁ progeny plants were produced. S. alba plants (genome SS, 2n = 24) were pollinated with B. oleracea (genome CC, 2n = 18), and the fertile F₁ plants were pollinated with B. oleracea to obtain BC₁ plants. GISH analysis showed that 10 out of 12 F₁ plants had 12 S. alba chromosomes (one full S chromosome set) and nine B. oleracea chromosomes (one C chromosome sets), representing the expected hybrids. However, two F₁ plants had 12 S chromosomes and 18 C chromosomes (two C chromosome sets), indicating unexpected hybrids. A maximum of three trivalents between C and S chromosomes were identified at metaphase I of semi-fertile F₁ pollen mother cells (PMCs), which indicates homology and chromosome pairing between these two genomes. The C genome had obviously been doubled in two F₂ plants from selfed semi-fertile F₁ plants. BC₁ plants consisted of 18 C chromosomes and different numbers of one, five and six additional S chromosomes, respectively. Monosomic alien addition lines developed in the present study can be used for B. oleracea breeding and Sinapis alba gene mapping.     

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.     

Anther and isolated microspore culture response of wheat lines from northwestern and eastern Europe

Hexaploid wheat genotypes from north-western Europe show low responses to current anther culture techniques. This phenomenon was investigated on 145 north-western European wheat lines. Twenty-seven lines from eastern Europe were included to observe the response pattern of wheat from an area, where the technique has been used successfully. On average, eastern European wheat lines produced 3.6 green plants per 111 anthers, while only 1.4 green plants per 111 anthers were obtained in north-western European lines. This difference was due to the high capacity for embryo formation among the eastern European lines, while the ability to regenerate green plants was widespread in both germplasm groups. Isolated wheat microspore culture performed on 85 of these wheat lines gave an average 3.7-fold increase in green plants per anther compared with the anther culture response. The increased recovery of green plants was due to improved plant regeneration and increased green plant percentage from embryos derived from isolated microspore culture.     

Addition of Colchicine to Wheat Anther Culture Media to Increase Doubled Haploid Plant Production

Chromosome doubling is critical for obtaining doubled-haploid plants from wheat (Triticum aestivum L.) anther culture. The most common doubling method applies colchicine to the plant. However, colchicine is phytotoxic and can induce a high frequency of plant death. In this experiment, anthers from two wheat genotypes (“Pavon 76” and ‘Centurk’) were placed on nine embryoid initiation media having three sugar sources (maltose, sucrose, and maltose + glucose) with three colchicine concentrations (0.0, 0.1, and 0.2 g · l^-1). Wheat starch was used as a gelling agent. After three days, the anthers were washed and moved to fresh media without colchicine. Increasing the colchicine concentration decreased the number of embryoids produced from 77.4 embryoids/100 anthers to 29.9 embryoids/100 anthers, but did not significantly affect the frequency of plant regeneration (0.49 green plants/embryoid to 0.40 green plants/embryoid), and increased the frequency of doubled-haploid plants (19.0 doubled-haploid plants/100 green plants to 72.3 doubled-haploid plants/100 green plants). Considering the total number of doubled-haploid plants produced, low levels of colchicine added to the initiation media were very effective.     

Cytogenetic study of Helianthus laevigatus and its F1 and BC1F1 hybrids with cultivated sunflower, Helianthus annuus

Although the wild sunflower species Helianthus laevigatus has not been extensively studied it may be considered for sunflower breeding as a potential source of desirable genes for Sclerotinia stalk rot resistance and high contents of proteins and linoleic acid in the seed. A set of six H. laevigatus populations was crossed to cultivated sun~ower lines and produced nine F₁ (2-14 plants) and 66 BC₁F₁ hybrid combinations (1-13 plants). Male sterility occurred in F₁ and BC₁F₁ hybrid combinations and pollen viability was lower in the progenies than in the parents (51.6-77.2%in F₁ and in F₁ and 4.8-34.0% in BC₁F₁). Meiosis was normal in the H. laevigatus populations It was found that this tetraploid species also occurred in a hexaploid form Numerous irregularities were observed in the meiosis of the F₁ interspecific hybrids During diakinesis, quadrivalents and hexavalents were recorded in addition to bivalents Dislocated chromosomes and chromosome bridges were present in the other phases The chromosome number in F₁ was 68 (tetraploid). Irregularities in chromosome pairing were observed in the interspecific hybrids at BC₁F₁. There were many univalents, and trivalents quadrivalents and hexavalents were also present The chromosome number in the BC₁F₁ generation ranged from 34 to 60. The occurrence of meiotic irregularities in the F₁ and BC₁F₁ interspecific hybrids indicates that H. laevigatus and the cultivated sunflower differ in genome constitution.     

Segregation patterns of AFLP markers in F1 hybrids of a cross between tetraploid and diploid species in the genus Malus

Malus xiaojinensis, one of the most important wild genotypes in the genus Malus, is resistant to a variety of stresses such as Fe deficiency chlorosis, drought and cold. However, lack of knowledge of its genetic background prevents using genetic analysis to study those agronomic traits and corresponding gene functions. Here, as the first step towards construction of the linkage map of M. xiaojinensis, genetic analysis of the F1 triploid hybrids (M. xiaojinensis × M. baccata) was performed with amplified fragment length polymorphism (AFLP) markers. Using 15 EcoRI‐ MseI primer combinations, 1110 AFLPs were identified, with 31.3% of M. xiaojinensis‐, 12.7% of M. baccata‐specific markers, 54.9% of common markers, and 1.2% of non‐parental markers; 93.3% of the AFLP markers exhibit the expected segregation ratio. Thirty‐two M. xiaojinensis‐specific markers and 47 common markers display a 5 : 1 and 11:1 segregation ratios, respectively, suggesting that M. xiaojinensis is an autotetraploid, or at least an isosyndetic allotetraploid.     

Relationship of Root and Shoot Characters in Parent, F1 and F2 Populations of Rice

The direct and indirect contributions of root characters — root length, roots/plant, fresh and dry root weight on grain yield/plant were worked out from a 7 × 7 diallel set of rice hybrids. The materials were grown in pots with four replications. Path analysis was done at genotypic level of correlation.
The grain yield/plant showed positive correlation with all the root characters in parent, F₁ and F₂ except with roots/plant in F₁ population. Fresh root weight demonstrated positive direct effect on grain yield/plant in all the three generations. Roots/plant had highly positive direct effect in F₂. Direct effects were negative in respect of root length and dry root weight in F₂ generation.     

Performance Studies in F6 Lines of Autotetraploid Fenugreek

Hybridization followed by pedigree selection was carried out between two autotetraploid strains of fenugreek (Trigonella foenum-graecum L.). Morphological and cytological investigations were carried out in two autotetraploid parents, their F₁ hybrids and F₆ lines derived from F₁ after selfing. Cytomorphological data of F₆ lines were compared with those of F₁, hybrids and of their parents. The hybrid plants were intermediate with respect to different vegetative traits, however, they showed increased seed set as compared to their autotetraploid parents. The mean pod number per plant in F₆ was one and half times as high as one of the high yielding tetraploid parents and approximately twice as high as that of F₁ hybrid. Meiosis in F₆ was more regular than in F, and the parents. Thus, hybridization followed by pedigree selection is helpful not only in regularizing the meiotic behavior, but vigour and seed fertility have also improved in autotetraploid fenugreek.     

Durum wheat × maize crosses for haploid wheat production: Influence of parental genotypes and various experimental factors

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars of Triticum turgidum ssp. durum (female parent) were crossed with eight genotypes of Zea mays. After pollination, plant stems were either maintained in situ or cut near the base and kept in a 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining them in situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.