Haploid Production Research Articles

Genetic analysis of anther culture response and identification of QTLs associated with response traits in wheat (Triticum aestivum L.).

Anther culture is the most effective tool for doubled haploid production of wheat. This investigation was conducted to estimate genetic parameters of anther culture response in wheat and identification of putative Quantitative trait loci (QTLs) associated with response traits. Two varieties of wheat, namely ICR-DH (P1) and Sle 1 × 15 (P2) and their F1 and F2 progenies were used in the present investigation to estimate genetic parameters of anther culture response. Two molecular marker systems, SRAP and SSR markers were used to detect the polymorphism between two anther donor parents. Single marker analysis (SMA) and Composite interval mapping (CIM) were used to localize the putative QTL associated with four anther culture response in wheat using 100 plants of F2 population derived from F1 cross 'ICR-DH' × 'Sel 1 × 15'. Analyses of variance indicated significant differences between four populations (P1, P2, F1 and F2) for callus induction (CAL), number of green plants per 100 anther (GR), number of albino plant per 100 anther (AR) and total regenerated plants per 100 anther (TR). The additive effects were more important than dominance effects in controlling these traits. The two molecular marker systems were sufficient in detecting polymorphism between two parents. Thirty two putative QTLs were detected on eight linkage groups. Our study indicated that the additive effects of genes and detection of new QTLs permit marker-assisted selection of genotypes with high green plantlet regeneration efficiency in anther culture, and therefore favor efficient use of anther culture in wheat breeding programs.

ФЕДЕРАЛЬНЫЙ НАУЧНЫЙ ЦЕНТР ОВОЩЕВОДСТВА: ВЕКОВАЯ ИСТОРИЯ КАК ФУНДАМЕНТ РАЗВИТИЯ (обзор)

The review presents the history of the Gribovskaya Vegetable Breeding Experimental Station, the first in Russia and the USSR for vegetable breeding and seed production, on the basis of which the All-Russian Research Institute of Vegetable Breeding and Seed Production was established, farther reorganized into the Federal Research Center for Vegetable Growing. The center’s activity dates back to 1920, when, under the leadership of Sergei I. Zhegalov, a theoretical and practical basis for the development of domestic breeding was laid. The century-old anniversary of the selection of vegetable crops allows us to trace the way of its formation in Russia, successes and future development. Since 1920, scientists paid much attention to the development and improvement of breeding methods that increase the efficiency of selection, as well as to accelerate the selection process to create targeted varieties and hybrids. With regard to the main vegetable crops, methods have been developed for interspecific hybridization (N.I. Timin et al., 2013; A.F. Agafonov et al., 2018), molecular labeling (T.P. Suprunova et al., 2011; E.A. Domblides et al., 2015), clonal micropropagation and production of doubled haploids successfully used in breeding (M.S. Bunin et al., 2004). Basic protocols have been proposed for in vitro culture of microspore for most cabbage crops (E.A. Domblides et al., 2016) and non-pollinated ovules for Cucurbitaceae (N.A. Shmykova et al., 2015). A technology has been developed for the production of doubled haploids in carrots in in vitro cultures of anthers, non-pollinated ovules and microspores (T.S. Wurtz et al., 2016). The economic benefit of modern biotechnological in vitro methods when creating hybrids has been proven: the time for creating hybrids is reduced from 12 to 6 years, financial costs are reduced 2 times (A. Mineikina et al., 2019; T. Vurtz et al., 2019). The aggravated situation with plant diseases and the expansion of the areas of new harmful pathogens on vegetable crops are discussed. Based on immunological, molecular and morphophysiological tests at artificial, provocative and natural infections, the sources of resistance to economically significant diseases are identified, in cabbage to Plasmodiophora brassicae, in table beet to Cercospora beticola, in vegetable beans to viral diseases, in onions to Peronospora destructor (I.A. Engalycheva et al., 2019). Physiological and biochemical methods are widely used when creating varieties with a high content of biologically active substances and antioxidants. Technologies have been developed for obtaining functional food products, including new types of teas with a therapeutic and prophylactic effect, soft drinks, food dyes, and confectionery (M.S. Gins et al., 2017). Recipes for gluten-free bakery products have been created using introduced yacon, amaranth and daikon cultures. Technologies for selenium enrichment of vegetable crops for fresh consumption and as raw materials for functional products have been developed (N.A. Golubkina et al., 2018). The intellectual potential accumulated over a hundred-year history is inextricably linked with the traditions laid down at the experimental station. Nowadays the Federal Research Center for Vegetable Growing coordinates scientific research on the selection, production and processing of vegetable and melon crops in Russia within the framework of state programs for the development of the industry and ensuring food security.

FEDERAL RESEARCH CENTER FOR VEGETABLE GROWING — 100-YEAR HISTORY AS A BASIS FOR FUTURE DEVELOPMENTS (review)

The review presents the history of the Gribovskaya Vegetable Breeding Experimental Station, the first in Russia and the USSR for vegetable breeding and seed production, on the basis of which the All-Russian Research Institute of Vegetable Breeding and Seed Production was established, farther reorganized into the Federal Research Center for Vegetable Growing. The center’s activity dates back to 1920, when, under the leadership of Sergei I. Zhegalov, a theoretical and practical basis for the development of domestic breeding was laid. The century-old anniversary of the selection of vegetable crops allows us to trace the way of its formation in Russia, successes and future development. Since 1920, scientists paid much attention to the development and improvement of breeding methods that increase the efficiency of selection, as well as to accelerate the selection process to create targeted varieties and hybrids. With regard to the main vegetable crops, methods have been developed for interspecific hybridization (N.I. Timin et al., 2013; A.F. Agafonov et al., 2018), molecular labeling (T.P. Suprunova et al., 2011; E.A. Domblides et al., 2015), clonal micropropagation and production of doubled haploids successfully used in breeding (M.S. Bunin et al., 2004). Basic protocols have been proposed for in vitro culture of microspore for most cabbage crops (E.A. Domblides et al., 2016) and non-pollinated ovules for Cucurbitaceae (N.A. Shmykova et al., 2015). A technology has been developed for the production of doubled haploids in carrots in in vitro cultures of anthers, non-pollinated ovules and microspores (T.S. Wurtz et al., 2016). The economic benefit of modern biotechnological in vitro methods when creating hybrids has been proven: the time for creating hybrids is reduced from 12 to 6 years, financial costs are reduced 2 times (A. Mineikina et al., 2019; T. Vurtz et al., 2019). The aggravated situation with plant diseases and the expansion of the areas of new harmful pathogens on vegetable crops are discussed. Based on immunological, molecular and morphophysiological tests at artificial, provocative and natural infections, the sources of resistance to economically significant diseases are identified, in cabbage to Plasmodiophora brassicae, in table beet to Cercospora beticola, in vegetable beans to viral diseases, in onions to Peronospora destructor (I.A. Engalycheva et al., 2019). Physiological and biochemical methods are widely used when creating varieties with a high content of biologically active substances and antioxidants. Technologies have been developed for obtaining functional food products, including new types of teas with a therapeutic and prophylactic effect, soft drinks, food dyes, and confectionery (M.S. Gins et al., 2017). Recipes for gluten-free bakery products have been created using introduced yacon, amaranth and daikon cultures. Technologies for selenium enrichment of vegetable crops for fresh consumption and as raw materials for functional products have been developed (N.A. Golubkina et al., 2018). The intellectual potential accumulated over a hundred-year history is inextricably linked with the traditions laid down at the experimental station. Nowadays the Federal Research Center for Vegetable Growing coordinates scientific research on the selection, production and processing of vegetable and melon crops in Russia within the framework of state programs for the development of the industry and ensuring food security.

Trichostatin A Affects Developmental Reprogramming of Bread Wheat Microspores towards an Embryogenic Route.

Microspores can be developmentally reprogrammed by the application of different stress treatments to initiate an embryogenic pathway leading to the production of doubled haploid (DH) plants. Epigenetic modifications are involved in cell reprogramming and totipotency in response to stress. To increase microspore embryogenesis (ME) efficiency in bread wheat, the effect of the histone deacetylase inhibitor trichostatin A (TSA) has been examined in two cultivars of wheat with different microspore embryogenesis response. Diverse strategies were assayed using 0–0.4 µM TSA as a single induction treatment and after or simultaneously with cold or mannitol stresses. The highest efficiency was achieved when 0.4 µM TSA was applied to anthers for 5 days simultaneously with a 0.7 M mannitol treatment, producing a four times greater number of green DH plants than mannitol. Ultrastructural studies by transmission electron microscopy indicated that mannitol with TSA and mannitol treatments induced similar morphological changes in early stages of microspore reprogramming, although TSA increased the number of microspores with ’star-like’ morphology and symmetric divisions. The effect of TSA on the transcript level of four ME marker genes indicated that the early signaling pathways in ME, involving the TaTDP1 and TAA1b genes, may be mediated by changes in acetylation patterns of histones and/or other proteins.

Maternal haploid induction in African marigold (Tagetes erecta L.) through in vitro culture of un-fertilized ovules

A protocol for production of haploids from two heterozygous male sterile lines of Tagetes erecta using un-pollinated ovules as explant was standardized. Various factors affecting the in vitro gynogenic response viz., growth regulators, ovule developmental stage, basal media, sucrose concentration, photoperiod and cold shock duration were assessed in Tagetes erecta. Direct induction of parthenogenic embryos occurred in cultivar ‘Local Orange’ when the un-pollinated ovules were cultured in EMS (MS medium enriched with coconut water, AgNO3, PVP etc.) medium supplemented with 2 mg l−1 BAP and 0.5 mg l−1 NAA or 2 mg l−1 BAP along with 2 mg l−1 2,4-D. The developmental stage of the flower buds was vital in embryo induction from the excised ovules. The ovules collected from half-open flower buds were more responsive to gynogenesis as compared to ovules collected from un-open and fully open flower buds. EMS basal medium was found best for gynogenesis over the other commercially available basal media. Cold pre-treatment of flower buds at 4 oC had no stimulatory effect and negatively affected the gynogenesis. The dark culture condition was imperative for direct parthenogenic embryo induction than 16 h light duration. The ploidy levels of 17 regenerants were determined by cytological studies, revealed 47.06% as diploids, 41.18% as haploids and 11.76% as mixoploids. These results were reconfirmed with flow cytometry analysis. The determination of ploidy level by counting the number of chloroplasts in stomatal guard cells of marigold was also established for rapid screening of haploids. The resultant haploids were successfully diploidised and are being utilized in the hybrid breeding programme at our institute. The standardized protocol for doubled haploids (DHs) production by un-pollinated ovule culture paved the way for Tagetes F1 hybrid breeding. This is the first successful report of in vitro gynogenesis in Tagetes spp for production of doubled haploids (DHs). The protocol for obtaining high frequency parthenogenic embryo induction from unpollinated ovules was standardized and validated for rapid production of homozygous lines.

Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.

The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.

Opportunities and Challenges in Doubled Haploids and Haploid Inducer-Mediated Genome-Editing Systems in Cucurbits

Doubled haploids have played a major role in cucurbit breeding for the past four decades. In situ parthenogenesis via irradiated pollen is the preferred technique to obtain haploid plantlets whose chromosomes are then doubled in Cucurbitaceae, such as melon, cucumber, pumpkin, squash and winter squash. In contrast to doubled haploid procedures in other species, in situ parthenogenesis in cucurbits presents many limiting factors which impede efficient production of haploids. In addition, it is very time-consuming and labor-intensive. However, the haploid inducer-mediated genome-editing system is a breakthrough technology for producing doubled haploids. Several reports have described using the CRISPR/Cas9 system in cucurbit species, and although its application has many bottlenecks, the targeted knock-out of the CENH3 gene will allow breeders to obtain haploid inducer lines that can be used to obtain parthenogenetic embryos. In this review, we discuss the progress made towards the development of doubled haploids and haploid inducer genotypes using CRISPR/Cas9 technologies in cucurbit species. The present review provides insights for the application of haploid inducer-mediated genome-editing system in cucurbit species

Doubled haploid production in maize under Sub-montane Himalayan conditions using R1-nj-based haploid inducer TAILP1

The use of in vivo haploid induction system makes the doubled haploid (DH) technology easier to adopt for the conventional maize breeders. However, despite having played an important role in the initial developmental phases of DH technology, Indian maize research has yet to harvest its benefits. Haploid Inducer Lines (HILs) developed by CIMMYT are being widely used in maize breeding programmes in many countries including India. There, however, is no published information on the efficiency of DH line production using CIMMYT HILs in Indian maize breeding programmes. In the present study, the efficiency of DH production using CIMMYT’s tropically adapted inducer line TAILP1 was investigated with eight source populations including two of sweet corn. The average haploid induction rate (HIR) of TAILP1 was 5.48% with a range of 2.01 to 10.03%. Efficiency of DH production ranged from 0.14 to 1.87% for different source populations with an average of 1.07%. The information generated will be useful for maize breeders intending to use DH technology for accelerated development of completely homozygous lines.

Diallelic Analysis of Tropical Maize Germplasm Response to Spontaneous Chromosomal Doubling.

Chromosome doubling is an important step in the production of maize doubled haploid (DH) lines to induce fertility in the male and female reproductive organs of haploid plants. Chromosomal doubling is routinely accomplished by treating haploid seedlings with mitosis-inhibiting chemicals. However, chromosomal doubling involves several labor-intensive steps and toxic chemicals. Spontaneous chromosomal doubling without any chemical treatments occurs at high frequency in haploids from a few maize genotypes. This study focused on elucidating the genetic components of two traits important for using spontaneous doubling in maize-breeding programs, namely, haploid male fertility (HMF) and haploid fertility (HF). In two different sets of diallel crosses, haploids were derived and assessed for HMF and HF in two environments in replicated trials. The results revealed significant genotypic variations for both traits. The general combining ability (GCA) and specific combining (SCA) were significant for both traits. Significant and positive GCA effects of up to 14% and 9% were found for HMF and HF, respectively. No significant reciprocal effects and genotype-by-environment (G×E) interactions were found for HF in both experiments, but HMF showed significant effects for both in one of the experiments. The GCA effects were more important than the SCA effects for HMF and HF across environments, implying that selection could facilitate their improvement. The high correlations between F1-hybrid performance and mid-parent values, as well as that between F1-hybrid performance and GCA effects, also supports the assumption that these traits are controlled by a few genes. SCA effects also played a role, especially when lines with low spontaneous doubling were used as parents. Overall, spontaneous doubling can be introgressed and improved in elite germplasm with selection, and it has the potential to be employed in DH pipelines.

Haploidy in Tobacco Induced by PsASGR-BBML Transgenes via Parthenogenesis.

Background: Engineering apomixis in sexually reproducing plants has been long desired because of the potential to fix hybrid vigor. Validating the functionality of genes originated from apomictic species that contribute to apomixis upon transfer to sexually reproducing species is an important step. The PsASGR-BABYBOOM-like (PsASGR-BBML) gene from Pennisetum squamulatum confers parthenogenesis in this apomict, and its functionality was demonstrated in several sexually reproducing monocots but not in any dicots. Methods: We introduced the PsASGR-BBML gene regulated by egg cell-specific promoters, either AtDD45 or AtRKD2, into tobacco, and analyzed progeny of the transgenic lines resulting from self-pollination and crossing by flow cytometry. Results: We identified haploid progeny at a frequency lower than 1% in the AtDD45pro lines, while at a frequency of 9.3% for an octoploid (2n = 8x) AtRKD2pro line. Haploid production in the T2 generation, derived from the tetraploid T1 offspring of this original octoploid AtRKD2pro line, was also observed. Pollinated by homozygous transgenic tobacco carrying a DsRed marker gene, 4x progeny of the AtRKD2pro line yielded parthenogenetic embryos identified as DsRed negative. We verified that the DsRed negative seedlings recovered were haploid (2x). Conclusion: The PsASGR-BBML gene regulated by egg cell-specific promoters could enable parthenogenesis in tobacco, a dicotyledon species.

Grain barley starch as a gelling agent of nutrient media for cultivation of anthers and isolated embryos of H. vulgare L.

Aim. Determination the possibility of agar replacement with normal and waxy type starches produced from barley seeds in the nutrient media for spring barley haploid production. Methods. Anthers of spring barley line DH00-126 were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose (9.0 %) and differed in solidifying agents (agar, starch from barley seeds or chemically modified starch D5a-1). Trophic capacities of barley starch were determined both in anther and embryo culture of barley. Results. It was determined that barley starch of waxy type was not suitable for solidifying of nutrient medium. Use of normal type barley starch (6.5 %) resulted in sufficient decrease in the frequency of green plant regeneration. At the same time, this modification had no effect on the number of morphogenic anthers. Barley starch was inferior to pea starch in the ability to support seedling growth in embryo culture in vitro. Conclusions. Barley starch of normal type has worse trophic capacities in comparison to pea starch in embryo culture. It was appeared barley starch to be a less suitable gelling agent of medium for spring barley haploid production in anther culture in vitro than agar and chemically modified starch D5a-1.
 Keywords: Hordeum vulgare L., anther culture in vitro, embryo culture in vitro, agar, barley starch.

Effect of different in vitro and in vivo variables on the efficiency of doubled haploid production in Triticum aestivum L. using Imperata cylindrica-mediated chromosome elimination technique

Development of doubled haploid (DH) plants in wheat is an important technique for establishment of completely homozygous lines in the shortest time frame. In this study, we standardized the in vitro and in vivo variables for the production of wheat DH plants via crossing of three spring wheat genotypes with a wild grass (Imperata cylindrica). The experiment involves testing new treatments and protocol modifications for 2 years. Wheat florets were pollinated with I. cylindrica pollen after 2 days of emasculation, and next day pollinated spikes were injected with different auxin analogues: 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 100 mg/l 3,6-dichloro-2-methoxybenzoic acid (dicamba) or 50 mg/l 2,4-D + 50 mg/l dicamba. A combination of 50 mg/l 2,4-D and 50 mg/l dicamba was superior to other treatments having maximum haploid embryos and haploid plants developed per emasculated florets. The responsiveness of embryo formation to the addition of silver nitrate (AgNO3) (the ethylene inhibitor) with the auxin treatments showed that the presence of silver nitrate in the medium may not be essential for wheat embryo induction. Time of embryo rescue had a significant effect on embryo and haploid plant production. The most successful time for embryo rescue was 24 days after pollination (DAP) resulting in maximum haploid plants developed (40% in wheat variety AKYK92 and 25% each in variety HD2888 and HS542). The immature embryos were cultured in different media, i.e. Murashige and Skoog (MS), half-strength MS, modified MS media, Gamborg’s B5 and modified B5 media. Maximum number of haploid plants were found in half-strength MS media in all accessions (60.0%) followed by modified MS media (40.00%). For colchicine concentration, during 2016–2017, maximum numbers of DHs with less plant mortality were obtained at 0.08 and 0.06% dose. Taking into account the first year results, refinement of the dose in the second year showed that colchicine doses of 0.08 and 0.075% were the best dose for maximizing DH induction in wheat; however, in this case the number of examined plants was very small. Therefore, we can treat the two doses as a guide for further research on larger samples.

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.

Homozygous Transgenic Barley (Hordeum vulgare L.) Plants by Anther Culture.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

Haploid Bio-Induction in Plant through Mock Sexual Reproduction.

SummaryHaploidization is invaluable for basic genetic research and crop breeding. The haploid bio-induction principle is an important topic that remains largely unexplored. In this study, both CenH3 RNAi and in vitro inhibition were used to simulate and induce haploids in allopolyploid crop. Notably, in vitro CenH3 inhibition showed that the results were much the same to that of RNAi in phenotype, chromosome behavior, microspore production, and haploid induction. Cytological analyses of RNAi and inhibitor-treated progenies revealed elimination of chromosomes, defective microspores with empty nuclei, thereby giving rise to pseudo male gametes, and haploid parthenogenesis induction. We found distinct defective empty microspores that were positively correlated with the decrease of CenH3 during RNAi manipulation. Investigation through both in vivo and in vitro studies revealed that haploidization was induced through the pseudo male gamete-mediated mock sexual reproduction. The present results provide insights for the haploid parthenogenesis induction process.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

Puzzling out plant reproduction by haploid induction for innovations in plant breeding.

Mixing maternal and paternal genomes in embryos is not only responsible for the evolutionary success of sexual reproduction, but is also a cornerstone of plant breeding. However, once an interesting gene combination is obtained, further genetic mixing is problematic. To rapidly fix genetic information, doubled haploid plants can be produced: haploid embryos having solely the genetic information from one parent are allowed to develop, and chromosome doubling generates fully homozygous plants. A powerful path to the production of doubled haploids is based on haploid inducer lines. A simple cross between a haploid inducer line and the line with gene combinations to be fixed will trigger haploid embryo development. However, the exact mechanism behind in planta haploid induction remains an enduring mystery. The recent discoveries of molecular actors triggering haploid induction in the maize crop and the model Arabidopsis thaliana pinpoint an essential role of processes related to gamete development, gamete interactions and genome stability. These findings enabled translation of haploid induction capacity to other crops as well as the use of haploid inducer lines to deliver genome editing machinery into various crop varieties. These recent advances not only hold promise for the next generations of plant breeding strategies, but they also provide a deeper insight into the fundamental bases of sexual reproduction in plants.

In vivo colchicine manipulation for enhancing DH production efficiency in Triticum durum using Imperata cylindrica-mediated chromosome elimination approach

Durum wheat × Imperata cylindrica hybridization was carried out utilizing two durum wheat genotypes as maternal parents (A-9-30-1 and PDW 314) and I. cylindrica as a pollen source followed by in vivo application of colchicine to establish a standard protocol for colchicine application aimed at enhancement of doubled haploid production in durum wheat. Various concentrations of colchicine ranging from 500 to 7000 ppm were injected to the pollinated spikes at different time interval viz., 24 h, 48 h and 72 h after pollination. Application of 5000–7000 ppm colchicine at 48 h after pollination was established as the optimum colchicine dose for chromosome doubling as confirmed through cytological studies in durum wheat using I. cylindrica-mediated chromosome elimination approach. PDW 314 was identified as a durum wheat genotype having more potential for doubled haploid induction via in vivo colchicine application than A-9-30-1. Identification of optimum dose of colchicine for application at in vivo level for induction of doubled haploids has opened new vistas in durum wheat improvement programme.

Broccoli various genotypes regenerated plants (R0) seed set after geitonogamy

Relevance. Broccoli cabbage ( Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) is widely spread around the world due to its dietetic attribute, preventive and healing effect, cooking simplicity. F 1 hybrids production is the main stream in modern crop breeding as they surpass varieties in evenness and yield quality. However, it takes up to 6 years of inbreeding to obtain pure parent lines capable for hybrid creation. Thus biotechnological methods, especially doubled haploids production technology shell be implemented in selection process. 100% homozygous line can be worked out in one generation by this technology. Nevertheless earlier researches revealed such plants low seed set that hinder their application in mass production. Therefore after pollination seed set level identification is an important step in applying lines gained via in vitro process. Materials and methods. Broccoli cabbage regenerated plants produced by isolated microspore in vitro culture method were used as raw material in our study. Flower and bud pollination was held in climate cells. Results. Study of 11 broccoli cabbage DH-lines disclosed genotype influence the ability to set seeds after bud pollination. 93.3% genotype A and 95.5% A1.3 pods were seedless in our experiments. Most genotypes demonstrated 50.3-85.7% seed set level. Low and middle ability to form seeds prevailed mainly. This peculiarity should be considered while dealing with regenerated plants by raising the number of crossings for successful breeding.

A novel approach for haploid generation in Lolium

AbstractDevelopment of haploid and/or dihaploid methodologies for Lolium sp. is important to future improvement efforts as the genus includes the majority of the world's cool‐season annual and perennial turf and forage grass species. Microspore or anther culture haploid induction systems can result in the generation of either maternal or paternal haploids that can be effectively used to advance genetic gains through a breeding or selection program. The successful and traditional approach for the production of haploids and eventually dihaploids in Lolium sp. is most successful when microspore culture approaches are applied. Unfortunately, this approach is labor‐intensive, expensive and often restrictive to amendable genotypes. Within the Lolium‐Festuca genome complex, there is a need for modern breeding approaches that can facilitate the rapid development of haploids or dihaploids for the development of improved germplasm or cultivars. It has been suggested that an in vivo approach could facilitate further advances in Lolium sp. genetic studies, molecular breeding and superior cultivar development. The recent development of a two novel annual ryegrass (Lolium perenne L. subsp. multiflorum (Lam.) Husnot [syn. L. multiflorum Lam.]) line that when hybridized to tall fescue (Schedonorus arundinaceus (Schreb.) Dumort., nom. cons.) has been previously demonstrated. This research investigates and reports on the potential generation of haploids and/or dihaploids through this novel ryegrass when hybridized with L. multiflorum or L. perenne and production of the first invivo haploid/dihaploid Lolium sp. approach.