Spontaneous Chromosome Doubling Research Articles

Development of In Vitro Anther Culture for Doubled Haploid Plant Production in Indica Rice (Oryza sativa L.) Genotypes.

Anther culture is an efficient biotechnological tool in modern plant breeding programs to produce new varieties and parental lines in hybrid seed productions. However, some bottlenecks-low induction rate, genotype dependency, albinism-restrict the widespread utilization of in vitro anther culture in rice breeding, especially in Oryza sativa ssp. indica (indica) genotypes, while an improved efficient protocol can shorten the process of breeding. Three different induction media (N6NDK, N6NDZ, Ali-1) and four plant regeneration media (mMSNBK1, MSNBK3, MSNBKZ1, MSNBKZ2) were tested with five indica rice genotypes to increase the efficiency of in vitro androgenesis (number of calli and regenerated green plantlets). The production of calli was more efficient on the N6NDK medium with an average 88.26 calli/100 anthers and N6NDZ medium with an average of 103.88 calli/100 anthers as compared to Ali-1 with an average of 6.96 calli/100 anthers. The production of green plantlets was greater when calli was produced on N6NDK medium (2.15 green plantlets/100 anthers) compared when produced on to N6NDZ medium (1.18 green plantlets/100 anthers). Highest green plantlets production (4.7 green plantlets/100 anthers) was achieved when mMSNBK1 plant regeneration medium was used on calli produced utilizing N6NDK induction medium. In the best overall treatment (N6NDK induction medium and mMSNBK1 plant regeneration medium), four tested genotypes produced green plantlets. However, the genotype influenced the efficiency, and the green plantlets production ranged from 0.4 green plantlets/100 anthers to 8.4 green plantlets/100 anthers. The ploidy level of 106 acclimatized indica rice plantlets were characterized with flow cytometric analyses to calculate the percentage of spontaneous chromosome doubling. Altogether, 48 haploid-, 55 diploid-, 2 tetraploid- and 1 mixoploid plantlets were identified among the regenerant plantlets, and the spontaneous chromosome doubling percentage was 51.89%. Utilization of DH plants have been integrated as a routine method in the Hungarian rice breeding program. The tetraploid lines can be explored for their potential to offer new scopes for rice research and breeding directions in the future.

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding.

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the '1009' genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.

Production of Doubled Haploid through Anther Culture of the Male-Fertile Lines in the CMS System of Eggplant with the Cytoplasm of <i>Solanum grandifolium</i>

To produce doubled haploid homozygous male-sterile line of eggplant with the cytoplasm of Solanum grandifolium, anther culture of the male-fertile (MF) BC5 in the S. grandifolium induced CMS system was done. Anthers were cultured in an embryo induction medium and were heated to 35℃ for 3 d in the dark and then transferred to 25℃ under a 16 h photoperiod. After 20 d the anthers were transferred to a germination medium. The presence of pollen in the anther, number of chromosomes in the root tip cells, size of the stomatal guard cells and pollen fertility of the regenerated plants were investigated. The frequency of embryo formation in the MF line was 1.02%. From the embryos, one regenerated plant showed pollen non-formation type male sterility. Root tip chromosome number and stomatal guard cell size confirmed that the regenerated male-sterile plant is a microspore originated doubled haploid through spontaneous chromosome doubling. The present study demonstrates the first successful production of a doubled haploid homozygous male-sterile line in a CMS system of eggplant with the cytoplasm of S. grandifolium. Therefore, this material could be a useful breeding material for eggplant and improve the speed of conventional breeding.

Breeding an underutilized fruit crop: a long-term program for Hylocereus.

This review describes three decades of introduction, agro-technology development, breeding and selection of Hylocereus species, known as pitaya or dragon fruit, as an example of a holistic program aimed to develop the horticultural potential of a perennial underutilized fruit crop. Interspecific homoploid and interploid crosses and embryo rescue procedures produced improved hybrids, some of which have been released to farmers. Molecular tools and morphological and phenological comparisons between the parental species and the resulting hybrids provided valuable information on dominant/recessive traits and on genetic relationships that could be exploited for further hybridizations. In addition, Hylocereus were crossed with species of the closely related genus Selenicereus, producing valuable intergeneric hybrids. In situ chromosome doubling resulted in the production of autopolyploid lines, from which an understanding of the effect of increased ploidy on fruit traits and metabolomic profiles was obtained. Gamete-derived lines were produced, adding to our biobank homozygote lines that were subsequently used for further hybridization. Spontaneous chromosome doubling occurred in haploid gamete-derived Hylocereus monacanthus lines and in interspecific interploid Hylocereus megalanthus × H. undatus hybrids obtained from an embryo rescue procedure, resulting in plants with double the expected ploidy. Challenging technical problems were addressed by the development of protocols for DNA isolation, flow cytometry, in situ chromosome doubling, androgenesis, gynogenesis and embryo rescue following interspecific and interploidy crosses. Current research leading to the development of genomics and molecular tools, including a draft genome of H. undatus, is also presented. Perspectives for further development of Hylocereus species and hybrids are discussed.

Advances in Gene Editing of Haploid Tissues in Crops.

Emerging threats of climate change require the rapid development of improved varieties with a higher tolerance to abiotic and biotic factors. Despite the success of traditional agricultural practices, novel techniques for precise manipulation of the crop’s genome are needed. Doubled haploid (DH) methods have been used for decades in major crops to fix desired alleles in elite backgrounds in a short time. DH plants are also widely used for mapping of the quantitative trait loci (QTLs), marker-assisted selection (MAS), genomic selection (GS), and hybrid production. Recent discoveries of genes responsible for haploid induction (HI) allowed engineering this trait through gene editing (GE) in non-inducer varieties of different crops. Direct editing of gametes or haploid embryos increases GE efficiency by generating null homozygous plants following chromosome doubling. Increased understanding of the underlying genetic mechanisms responsible for spontaneous chromosome doubling in haploid plants may allow transferring this trait to different elite varieties. Overall, further improvement in the efficiency of the DH technology combined with the optimized GE could accelerate breeding efforts of the major crops.

In vitro kultura antera kao efikasno primenjena tehnika za proizvodnju dvostrukih haploida pšenice (Triticum aestivum L.)

In vitro anther culture is the most commonly-used technology for doubled haploid production in wheat (Triticum aestivum L.) and various cereals. The technology application allows the genetic attainment of homozygous pure lines from heterozygous breeding material in a single generation, and has become a rapid alternative to the conventional breeding methods. This technology also contributes to more accurately assess QTL × environmental interactions and has been applied in: genetic researches for marker-trait association study, genomics and as a target for transformation, genetic engineering, gene mapping, and mapping of quantitative trait loci (QTLs). This paper reviews the most important factors that limit the efficiency of winter wheat (Triticum aestivum L.) anther culture, such as genotype dependency and albinism, in addition to other affecting factors including the collection time of tillers and physiological growth environments of plants. Factors that improve the efficiency of wheat anther culture in mitigating both genotypic dependency and albinism have been included in this review, too, for example, genetic improvements by following a strategy to realize this purpose through using responsive breeding material for crossing, application of convenient stress pre-treatments (cold pre-treatments, heat shock treatment, colchicine, hormones, and chemicals) to induce androgenesis, and improvement of the composition of anther culture media and culture conditions. We have also highlighted the methods of chromosome doubling for the haploid wheat produced by anther culture androgenesis, such as spontaneous chromosome doubling or use of colchicine and anti-mitotic chemicals at an early stage of anther culture or as root immersion treatment.

Production of Doubled Haploid Sugar Beet (Beta vulgaris L.) Plants Through Gynogenesis.

Haploid and doubled haploid plant production through unpollinated ovule culture, with diverse benefits and applications, is considered among the most effective and advantageous breeding method for sugar beet (Beta vulgaris L.). It is known that sugar beet is not responsive to androgenesis, which is widely used for most plant species. Sugar beet is a recalcitrant plant in vitro due to the very low spontaneous chromosome doubling and low gynogenesis rate. Thus, a steadily increasing gynogenesis efficiency has always been an important target for an efficient sugar beet breeding program. Given the scarcity of published papers focusing on gynogenesis in sugar beet, this chapter describes haploid and doubled haploid production through ovule culture of unfertilized flowers as a practical method.

Doubled Haploid Production in Cereals Using Microspore Culture

Doubled haploids are extremely useful in plant breeding since they provide rapid homozygosity. However, the success rate of doubled haploid production in cereals is still not high enough, and there is a special problem involving the formation of high percentage of albino plants. Nevertheless, the success rate in microspore culture in cereals is higher than classical anther cultures, and the method has the advantage of spontaneous chromosome doubling. On the other hand, this method has some critical stages such as pretreatments and microspore isolation, and these stages need to be optimized for the successful use of the technique in plant breeding. For this aim, there have been studies in recent years about combining the pretreatment practices, supplementing growth media with a variety of ingredients, improving the various co-culturing practices and decreasing the albino plant percentage. This technique has been commonly used in the world especially in barley and wheat breeding. Improving the success rate of the technique will be useful for its integration into modern breeding techniques such as apomictic crops and transgenics.

QTL Mapping and Prediction of Haploid Male Fertility Traits in Maize (Zea mays L.).

Chromosome doubling of maize haploids is a bottleneck in the large-scale application of doubled haploid (DH) technology. Spontaneous chromosome doubling (SCD) of haploid has been taken as an important method in the production of DH lines and low haploid male fertility (HMF) is a main limiting factor for the use of SCD. To study its genetic basis, haploids of 119 DH lines derived from a cross between inbred lines Qi319 and Chang7-2 were used to map the quantitative trait locus (QTL) contributing to HMF. Three traits including anther emergence rate (AER), anther emergence score (AES) and pollen production score (PPS) of the haploid population were evaluated at two locations. The heritability of the three traits ranged from 0.70 to 0.81. The QTL contributing to AER, AES and PPS were identified on the chromosomes 1, 2, 3, 4, 5, 7, 9 and 10. Five major QTL, qAER5-1, qAER5-2, qAES3, qPPS1 and qPPS5, were found and each could explain more than 15% of the phenotypic variance at least in one environment. Two major QTL, qPPS1 and qPPS5, and two minor QTL, qAES2 and qAER3, were repeatedly detected at both locations. To increase the application efficiency of HMF in breeding programs, genomic prediction for the three traits were carried out with ridge regression best linear unbiased prediction (rrBLUP) and rrBLUP adding QTL effects (rrBLUP-QTL). The prediction accuracies of rrBLUP-QTL were significantly higher than that by rrBLUP for three traits (p < 0.001), which indirectly indicates these QTL were effective. The prediction accuracies for PPS were 0.604 (rrBLUP) and 0.703 (rrBLUP-QTL) across both locations, which were higher than that of AER and AES. Overall, this study provides important information to understand the genetic architecture of SCD of maize haploids.

High spontaneous male-fertility-restorer frequency in a maize recurrent selection experiment

Using the crosses of the DH lines derived from a single cross hybrid Xianyu 335 as source materials for haploid induction in the recurrent selection scheme, we found that the rate of spontaneous chromosome doubling in the tassels of the haploid plants increased significantly in the second cycle and showed a cumulative genetic effect, and through using DH recurrent selection technique, the doubling rate of the haploid tassels can be doubled. Proved performing continuous recurrent selection in a narrow single cross background, the genetic makeup of the haploids had been enriched for spontaneous doubling restoration. Using hybrids between DH lines from two cycles haploid selection as source materials for haploid induction, the haploid tassel doubling rate had reached 85.15% and haploid ears showed seed set had reached 66.18%, representing a rate increase of 6.99 folds and 9.86 folds of its original source population Xianyu335.The genetic gain of set seeds plant frequency is 29.74% per cycle. Also, because the DH lines had been selected from two cycles of haploids or whole genome gamete selection based on the haploid phenotypes, it was effectively eliminated many deleterious and bad genes, and also cumulatively combined many superior genes for agronomy and plant growth vigor. These new DHs can be directly used as parents for germplasm enhancement and new hybrid selection and breeding. The discovery of the enrichment for spontaneous chromosome doubling and fertility restoration in of the tassels of the haploid plants tassels that were derived from DH recurrent selection technique and create high spontaneous doubling rate source materials may have showed a significant application value in maize breeding. It shows significant advantages for construct maize two cycle. Recurrent selection with high haploid spontaneous doubling rate can to speed up breeding output and cumulative genetic improvements.

The Effect of Caffeine and Trifluralin on Chromosome Doubling in Wheat Anther Culture.

Challenges for wheat doubled haploid (DH) production using anther culture include genotype variability in green plant regeneration and spontaneous chromosome doubling. The frequency of chromosome doubling in our program can vary from 14% to 80%. Caffeine or trifluralin was applied at the start of the induction phase to improve early genome doubling. Caffeine treatment at 0.5 mM for 24 h significantly improved green plant production in two of the six spring wheat crosses but had no effect on the other crosses. The improvements were observed in Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Caffeine had no significant effect on chromosome doubling, despite a higher frequency of doubling in several caffeine treatments in the first experiment (67–68%) compared to the control (56%). In contrast, trifluralin significantly improved doubling following a 48 h treatment, from 38% in the control to 51% and 53% in the 1 µM and 3 µM trifluralin treatments, respectively. However, trifluralin had a significant negative effect on green plant regeneration, declining from 31.8 green plants per 20 anthers (control) to 9–25 green plants per 20 anthers in the trifluralin treatments. Further work is required to identify a treatment regime with caffeine and/or anti-mitotic herbicides that consistently increases chromosome doubling in wheat without reducing green plant regeneration.

Production of doubled haplois in Brassica purpuraria

Relevance.In recent years vegetable crop Brassica rapa ssp. chinensis var. purpuraria (synonyms: Brassica campestris L. var.purpurea Bailey; Brassica rapa L. ssp. chinensis var. purpurea) is gaining popularity as an object of genetic and molecular researches, and as an economically valuable vegetable plant due to the high content of biologically active compounds and distinctive economically valuable traits. Effective technology for development DH-plants to accelerate the breeding process for this culture has not been developed yet, so research in this area is relevant.Materials and methods. The study included two varieties from the collection of Vavilov AllRussian Research Institute of Plant Industry (VIR): No. 1301 (China) and No. 1357(Netherlands). Both protocols standard unmodified and with addition of silver nitrate (AgNO3) in the medium for embryogenesis induction were used in experiments for production of DH plants from isolated microspore in vitro. Direct chromosome counting in meristem cells and flow cytometry were used to determine the ploidy of regenerating plants.Results. As a result of the study embryogenesis in B. purpuraria culture can develop with the use of a standard protocol as well as with the addition of silver nitrate that showed a positive effect on the induction of embryogenesis. The yield of embryoids varied depending on the genotype of the individual plant within the variety accession. The highest yield of embryoids was 40 embryoids/petri dish. The main problem at the stage of regeneration is that about half of the regenerating plants occurred to be albinos and were not viable. We show a high degree of spontaneous chromosome doubling in regenerated plants (all analyzed plants were doubled haploids). In total 38 regenerated plants were obtained from accession No. 1301. It was shown that four DH-plants had self-incompatibility after self-pollination, but seed progeny from other plants was obtained. The created material was taken for genetics study and breeding work.

Haploid spontaneous diploidization during apogamy of male gametophytes in Pyropia haitanensis (Bangiales, Rhodophyta)

Cytological observations were systematically employed for detecting cellular morphogenesis and chromosomal behavior of apogamous development of new sporophytes from individually cultured male gametophytes of Pyropia haitanensis. The results showed that the apical parts of premature male gametophytes initially underwent obvious apogamy, which primarily appeared in the functionally specialized spermatangial mother cells (SSMCs) that would not develop into highly differentiated spermatangia but change their developmental fate and become brownish red in color. These brownish red spermatangial mother cells (SMCs) were released accompanying the spermatia dissociated from mature spermatangia and then rarely formed circular carpospore-like cells (CLCs) and carpospore-like germlings (CLGs) that eventually developed into normal apogamous sporophytic conchocelis. Chromosome counts indicated that spermatangia and spermatia were haploid (n = 5), whereas SSMCs, CLCs, and CLGs were diploid (2n = 10). Furthermore, filamentous conchocelis and conchosporangial branch cells of apogamous sporophytes were diploid and no haploid, aneuploid, and polyploid were observed, suggesting that spontaneous chromosome doubling occurred in the process of apogamy and initiated rarely SSMCs rather than released spermatia. Moreover, pit connections were observed in the whole development of apogamous sporophytes like in heterozygous sporophytes. Mitotic chromosomal behaviors appeared during the first two divisions of conchospores that normally grew into haploid tetrads and developed into unisexual male gametophytes with the characteristics of apogamy in the aging cultures.

Genome-wide association study to identify genomic regions influencing spontaneous fertility in maize haploids

Efficient production and use of doubled haploid lines can greatly accelerate genetic gains in maize breeding programs. One of the critical steps in standard doubled haploid line production is doubling the haploid genome using toxic and costly mitosis-inhibiting chemicals to achieve fertility in haploids. Alternatively, fertility may be spontaneously restored by natural chromosomal doubling, although generally at a rate too low for practical applications in most germplasm. This is the first large-scale genome-wise association study to analyze spontaneous chromosome doubling in haploids derived from tropical maize inbred lines. Induction crosses between tropicalized haploid inducers and 400 inbred lines were made, and the resulting haploid plants were assessed for haploid male fertility which refers to pollen production and haploid fertility which refers to seed production upon self-fertilization. A small number of genotypes were highly fertile and these fertility traits were highly heritable. Agronomic traits like plant height, ear height and tassel branch number were positively correlated with fertility traits. In contrast, haploid induction rate of the source germplasm and plant aspect were not correlated to fertility traits. Several genomic regions and candidate genes were identified that may control spontaneous fertility restoration. Overall, the study revealed the presence of large variation for both haploid male fertility and haploid fertility which can be potentially exploited for improving the efficiency of doubled haploid derivation in tropical maize germplasm.

Haploid male fertility and spontaneous chromosome doubling evaluated in a diallel and recurrent selection experiment in maize.

Mainly additive gene action governed inheritance of haploid male fertility, although epistatic effects were also significant. Recurrent selection for haploid male fertility resulted in substantial improvement in this trait. The doubled haploid (DH) technology offers several advantages in maize breeding compared to the traditional method of recurrent selfing. However, there is still great potential for improving the success rate of DH production. Currently, the majority of haploid plants are infertile after chromosome doubling treatment by antimitotic agents such as colchicine and cannot be selfed for production of DH lines. Improvement in haploid male fertility (HMF) by selection for a higher spontaneous chromosome doubling rate (SDR) has the potential to increase DH production efficiency. To investigate the gene action governing SDR in two breeding populations, we adapted the quantitative-genetic model of Eberhart and Gardner (in Biometrics 22:864-881. https://doi.org/10.2307/2528079 , 1966) for the case of haploid progeny from ten DH lines and corresponding diallel crosses. Furthermore, we carried out three cycles of recurrent selection for SDR in two additional populations to evaluate the selection gain for this trait. Additive genetic effects predominated in both diallel crosses, but epistatic effects were also significant. Entry-mean heritability of SDR observed for haploid progeny of these populations exceeded 0.91, but the single-plant heritability relevant to selection was low, ranging from 0.11 to 0.19. Recurrent selection increased SDR from approximately 5-50%, which suggests the presence of few QTL with large effects. This improvement in HMF is greater than the effect of standard colchicine treatment, which yields at maximum 30% fertile haploids. Altogether, the results show the great potential of spontaneous chromosome doubling to streamline development DH lines and to enable new breeding schemes with more efficient allocation of resources.

Morphogenesis and spontaneous chromosome doubling during the parthenogenetic development of haploid female gametophytes in Pyropia haitanensis (Bangiales, Rhodophyta)

Pyropia haitanensis is one of the most economically important seaweeds growing in the coastal regions of south China. Previous studies revealed that parthenogenesis could emerge in P. haitanensis under certain culture conditions. In the present study, detailed cytological observations were made of morphogenesis and chromosomal behaviors of female gametophytes of P. haitanensis during parthenogenesis. Unisexual cultures of female gametophytes, derived from isolated somatic cells of wild gametophytes or obtained from cloned sporophytes, ultimately led to their development into parthenosporophytes. Based on the changes in shape and color of the cellular chromatophore, as well as the metamorphosis and condition of the released cells, the parthenogenesis of P. haitanensis was divided into six developmental stages: vegetative cells (VCs) with multiple stellate chromatophores, carpogonium-like cells (CLCs) with dispersed chromatophores, carpogonium-like protoplasts (CLPs) released post-cell wall dissolution, carpospore-like cells (CCs) with regenerated cell wall, carpospore-like germlings (CGs), and parthenosporophytes. Chromosomal observations showed that VCs were haploid (n = 5), small proportions of CLCs, and CLPs and CCs were diploid (2n = 10), while all the CGs, filamentous conchocelis cells, and conchosporangial branch cells of parthenosporophytes were diploid (2n = 10). Haploid, aneuploid, and polyploidy were not observed in the latter group of cells. Spontaneous chromosome doubling occurred during the parthenogenesis of P. haitanensis, first in highly differentiated CLCs. Furthermore, pit connections could be seen throughout the parthenosporophyte development process similar to that in heterozygous sporophytes. The chromosomes were seen undergoing meiosis during the germination of conchospores released from mature parthenosporophytes, leading to the generation of tetrad cells, which developed into haploid female gametophytes. These results provide evidence for better understanding the mechanism underlying parthenogenesis in Pyropia.

Development and characterization ofTriticum turgidum–Aegilops umbellulataamphidiploids

AbstractThe U genome ofAegilops umbellulatais an important basic genome of genusAegilops. Direct gene transfer fromAe. umbellulatainto wheat is feasible but not easy.Triticum turgidum–Ae. umbellulataamphidiploids can act as bridges to circumvent obstacles involving direct gene transfer. SevenT. turgidum–Ae. umbellulataamphidiploids were produced via unreduced gametes for spontaneous doubling of chromosomes of triploidT. turgidum–Ae. umbellulata F1hybrid plants. Seven pairs of U chromosomes ofAe. umbellulatawere distinguished by fluorescencein situhybridization (FISH) probes pSc119.2/(AAC)5and pTa71. Polymorphic FISH signals were detected in three (1U, 6U and 7U) of seven U chromosomes of fourAe. umbellulataaccessions. The chromosomes of the tetraploid wheat parents could be differentiated by probes pSc119.2 and pTa535, and identical FISH signals were observed among the three accessions. All the parental chromosomes of the amphidiploids could be precisely identified by probe combinations pSc119.2/pTa535 and pTa71/(AAC)5. TheT. turgidum–Ae. umbellulataamphidiploids possess valuable traits for wheat improvement, such as strong tillering ability, stripe rust resistance and seed size-related traits. These materials can be used as media in gene transfers fromAe. umbellulatainto wheat.

Influence of different concentrations of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale

In this work, the effect of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale was studied. The study of androgenesis was conducted on two model samples YaTKh-327-11 and YaTKh-18-11. The research revealed that the addition of the antioxidant ascorbic acid at a concentration of 2–8 mg/L into the culture medium to induce embryogenesis enhanced the formation of androgen structures and regeneration of green plants. The addition of gibberellic acid into the culture medium to induce embryogenesis gave negative effects. The average yield of green plants in the anther culture of spring triticale was 2 pcs/100 anthers. According to the research, 277 green plants were received and spontaneous doubling of chromosomes was recorded in 26.7%.

Identification of all homoeologous chromosomes of newly synthetic allotetraploid Cucumis × hytivus and its wild parent reveals stable subgenome structure.

Allopolyploidy and homoeologous recombination are two important processes in reshaping genomes and generating evolutionary novelties. Newly formed allopolyploids usually display chromosomal perturbations as a result of pairing errors at meiosis. To understand mechanisms of stabilization of allopolyploid species derived from distant chromosome bases, we investigated mitotic stability of a synthetic Cucumis allotetraploid species in relation to meiosis chromosome behavior. The Cucumis × hytivus is an allotetraploid synthesized from interspecific hybridization between cucumber (Cucumis sativus, 2n = 14) and its wild relative Cucumis hystrix (2n = 24) followed by spontaneous chromosome doubling. In the present study, we analyzed the wild parent C. hystrix and the latest generation of C. hytivus using GISH (genomic in situ hybridization) and cross-species FISH (fluorescence in situ hybridization). The karyotype of C. hystrix was constructed with two methods using cucumber fosmid clones and repetitive sequences. Using repeat-element probe mix in two successive hybridizations allowed for routine identification of all 19 homoeologous chromosomes of allotetraploid C. hytivus. No aneuploids were identified in any C. hytivus individuals that were characterized, and no large-scale chromosomal rearrangements were identified in this synthetic allotetraploid. Meiotic irregularities, such as homoeologous pairing, were frequently observed, resulting in univalent and intergenomic multivalent formation. The relatively stable chromosome structure of the synthetic Cucumis allotetraploid may be explained by more deleterious chromosomal viable gametes compared with other allopolyploids. The knowledge of genetic and genomic information of Cucumis allotetraploid species could provide novel insights into the establishment of allopolyploids with different chromosome bases.

Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.