Haploid Production Research Articles

Synergistic induction of fertilization-independent embryogenesis in rice egg cells by paternal-genome-expressed transcription factors.

In flowering plants, rapid activation of the zygotic genome occurs after fertilization1-3, but there is limited knowledge of the molecular pathways underlying embryo initiation4. In rice, a key role is played by the transcription factor BABY BOOM 1 (OsBBM1), initially expressed from the paternal genome1. Ectopic OsBBM1 expression in the egg cell can override the fertilization requirement, giving rise to parthenogenetic progeny5. Here we show that the WOX-family transcription factor DWARF TILLER1 (OsDWT1)/WUSCHEL-LIKE HOMEODOMAIN 9 (OsWOX9A)6, another gene paternally expressed in zygotes, is a strong enhancer of embryo initiation by OsBBM1. Co-expression of OsWOX9A and OsBBM1 in egg cells results in 86-91% parthenogenesis, representing 4- to 15-fold increases over OsBBM1 alone. These results suggest that embryo initiation is promoted by the synergistic action of paternal-genome-expressed transcription factors in the fertilized egg cell. These findings can be utilized for the efficient production of haploids, as well as clonal hybrid seeds in crop plants7,8.

Production and identification of melon double haploid induced by wide hybridization between melon and cucumber

Production and identification of melon double haploid induced by wide hybridization between melon and cucumber

Evaluating the effect of cupric sulphate on morphogenic structure induction and plant regeneration in spring barley anther culture in vitro

Aim. Investigation was aimed to ascertain possibility to increase the efficiency of spring barley haploid production in anther culture in vitro by addition of cupric sulphate to the solution for cut tiller cold pretreatment and by increase of cupric sulphate concentrations in the media for morphogenic structure induction and plant regeneration. Methods. Cut tillers were cold pretreated for 5 days at 4ºC in water and in the solutions contained 5 µM and 10 µM of cupric sulphate. Isolated anthers were cultivated on agar solidified nutrient medium containing N6 macro-and MS micronutrients, physiologically active substances, maltose (9.0 %) and on the media with increased (10 µM) content of cupric sulphate. Results. No effect of cupric sulphate addition on cut tiller viability and morphogenesis in anther culture in vitro was found. The lowest plant regeneration frequency was obtained when cupric sulphate was added both to inductive and regenerative media. Сonclusions. Addition of cupric sulphate to the pretreatment solution as well as increase of this microelement concentration (up to 10 µM) in the nutrient media can’t be considered as the methodological approach suitable to enhance the efficiency of spring barley haploid production in anther culture in vitro.

Large-scale production of rice haploids by combining superior haploid inducer with PTGMS lines

Large-scale production of rice haploids by combining superior haploid inducer with PTGMS lines

Improving detached tiller culture for doubled haploid production in wheat (T. aestivum L.) by post‐pollination fungicides application and UV radiation treatment

AbstractThis work aims at improving the proven protocol for doubled haploid production in wheat. This procedure has some deficits, for example, microbial infection of ears as well as of the nutrient culture medium. In the present study, the potential effect of sterilization by detached tiller culture fungicides of ears as well as of nutrient medium to control microbial infection, and its effect on the formation of caryopses and embryos (CFF, EFF) was studied. In a further experiment, the UV‐light was used as physical sterilant for the nutrient medium. All of the fungicides and their concentrations studied were found to be effective to control infection on ears except the concentration 0.05% tilt. 0.1% tilt gave the highest CFF (79.5%) followed by 0.1% Bayleton (78.0%). The lowest CFF was recorded in control treatment (48.8%). Among Bavistin treatments, the 0.2% Bavistin gave maximum CFF (68.5%) as well as EFF (29.1%) followed by 0.1% Bavistin (64.9%, 23.7%). The higher concentration of tilt (0.2%) showed detrimental effects on EFF (17.5%). Bavistin in tiller culture nutrient medium reduced infection in medium and was shown to improve CFF and EFF (67.4%, 29.8%).

New Observations of the Effects of the Cytoplasm of Aegilops kotschyi Boiss. in Bread Wheat Triticum aestivum L.

The cytoplasm of Aegilops kotschyi is known for the induction of male sterility and haploidy in wheat. Both systems originally appeared rather simple, but manipulation of the standard chromosome constitution of the nuclear genome revealed additional interactions. This study shows that while there is little or no allelic variation at the main fertility restorer locus Rfmulti on chromosome arm 1BS, additional genes may also be involved in the nuclear-mitochondrial genome interactions, affecting not only male fertility but also the growth rate, from pollen competition for fertilization and early endosperm divisions all the way to seed size and plant maturity. Some of these effects appear to be of a sporophytic nature; others are gametophytic. Induction of parthenogenesis by a rye inducer in conjunction with the Ae. kotschyi cytoplasm is well known. However, here we show that the cytoplasmic-nuclear interactions affect all aspects of double fertilization: producing maternal haploids from unfertilized eggs, diploids from fertilized eggs or synergids, embryo-less kernels, and fertilized eggs without fertilization of the double nucleus in the embryo sack. It is unclear how frequent the inducers of parthenogenesis are, as variation, if any, is obscured by suppressors present in the wheat genome. Genetic dissection of a single wheat accession revealed five distinct loci affecting the rate of maternal haploid production: four acting as suppressors and one as an enhancer. Only when the suppressing haplotypes are confirmed may it be possible to the identify genetic variation of haploidy inducers, map their position(s), and determine their nature and the mode of action.

First Gynogenesis of Vanilla planifolia for Haploid Production and Ploidy Verification Protocol.

Vanilla orchids are members of the Vanilloideae orchid subfamily, and they hold significant economic value as a spice crop in tropical regions. Despite the presence of 180 known species within this subfamily, commercial production focuses on only three species (Vanilla planifolia, V. odorata, and V. pompona) and one hybrid (V. × tahitensis), prized for their aromatic qualities and bioactive compounds. Limited modern breeding initiatives have been undertaken with vanilla orchids, although recent advancements in genomic research are shedding light on this crop's potential. The protracted breeding cycle of vanilla, coupled with increasing demand for germplasm, underscores the importance of research and breeding efforts in vanilla. This paper outlines a protocol for haploid production in V. planifolia using unfertilized ovaries in tissue culture conditions. Additionally, we present a methodology to confirm the haploid nature of putative haploid lines through stomatal size comparison, chromosome counting, and flow cytometry analysis, proving the successful development of haploid vanilla plants. These findings contribute to the advancement of breeding programs and genetic improvement strategies for the vanilla industry.

Androgenesis and gynogenesis in tomato (<I>Solanum lycopersicum</I> L.) <I>in vitro</I>

Tomato (Solanum lycopersicum L.) is one of the most consumed vegetable crops worldwide. Tomato fruits are rich in vitamins, minerals, and pigments, including lycopene. The high demand and the need to enhance tomato production call for new improved cultivars and F1 hybrids.Biotechnological methods reduce the time for source material development and the labor intensity of breeding efforts. Obtaining doubled haploid plants makes it possible to fix and analyze new gene combinations faster than with conventional breeding techniques, and produce homozygous genotypes. Tomato is highly unsusceptible to haploid induction, which has been continuously studied for more than 40 years and is still of special interest. The main methods for producing haploids are based on androgenesis and gynogenesis. Androgenesis is the production of haploids from the cells of the male gametophyte, and gynogenesis from the cells of the female gametophyte.The objective was to review the research on the induction of tomato haploids based on androgenesis and gynogenesis. No standardized, efficient or reproducible protocols are currently available to produce doubled haploids of tomato. It is necessary to determine the incubation conditions, physicochemical environments, dependence of the genotype in vitro, physiological state of the donor plant, and development of the anther, which affect the reproducibility of protocols to achieve haploid induction. Anther culture for obtaining haploid tomato plants has not yielded successful results, and the studies on microspore culture were too few, so it is difficult to understand the effectiveness of this technique. The method of gynogenesis is poorly investigated, but the culture of unfertilized ovules can become a successful way to obtain tomato haploids, with more research on this subject.

Genome-Wide Identification and Expression Analysis of the DMP and MTL Genes in Sweetpotato (Ipomoea batatas L.).

Sweetpotato (Ipomoea batatas L.) is a strategic crop with both economic and energy value. However, improving sweetpotato varieties through traditional breeding approaches can be a time-consuming and labor-intensive process due to the complex genetic nature of sweetpotato as a hexaploid species (2n = 6x = 90). Double haploid (DH) breeding, based on in vivo haploid induction, provides a new approach for rapid breeding of crops. The success of haploid induction can be achieved by manipulating specific genes. Two of the most critical genes, DMP (DUF679 membrane proteins) and MTL (MATRILINEAL), have been shown to induce haploid production in several species. Here, we identified and characterized DMP and MTL genes in sweetpotato using gene family analysis. In this study, we identified 5 IbDMPs and 25 IbpPLAs. IbDMP5 and IbPLAIIs (IbPLAIIκ, IbPLAIIλ, and IbPLAIIμ) were identified as potential haploid induction (HI) genes in sweetpotato. These results provide valuable information for the identification and potential function of HI genes in sweetpotato and provide ideas for the breeding of DH lines.

Formation of Different Polyploids Through Disrupting Meiotic Crossover Frequencies Based on cntd1 Knockout in Zebrafish.

Polyploidy, a significant catalyst for speciation and evolutionary processes in both plant and animal kingdoms, has been recognized for a long time. However, the exact molecular mechanism that leads to polyploid formation, especially in vertebrates, is not fully understood. Our study aimed to elucidate this phenomenon using the zebrafish model. We successfully achieved an effective knockout of the cyclin N-terminal domain containing 1 (cntd1) using CRISPR/Cas9 technology. This resulted in impaired formation of meiotic crossovers, leading to cell-cycle arrest during meiotic metaphase and triggering apoptosis of spermatocytes in the testes. Despite these defects, the mutant (cntd1-/-) males were still able to produce a limited amount of sperm with normal ploidy and function. Interestingly, in the mutant females, it was the ploidy not the capacity of egg production that was altered. This resulted in the production of haploid, aneuploid, and unreduced gametes. This alteration enabled us to successfully obtain triploid and tetraploid zebrafish from cntd1-/- and cntd1-/-/- females, respectively. Furthermore, the tetraploid-heterozygous zebrafish produced reduced-diploid gametes and yielded all-triploid or all-tetraploid offspring when crossed with wild-type (WT) or tetraploid zebrafish, respectively. Collectively, our findings provide direct evidence supporting the crucial role of meiotic crossover defects in the process of polyploidization. This is particularly evident in the generation of unreduced eggs in fish and, potentially, other vertebrate species.

On the issue of producing doubled haploids of table beet (<i>Beta vulgaris</i> L. var. <i>conditiva</i> Alef.) (a review)

Currently, hybrid table beet seeds make up a significant part of the seeds sold in the world due to their high synchrony, root uniformity, and the effect of heterosis. Heterosis breeding of table beet in Russia is developed insufficiently. One of the reasons is the lack of a well-studied homogeneous linear material. Another reason is a long and labor-consuming process of obtaining parent components for classical crossing due to a 2-year cycle of crop development, a pronounced self-incompatibility system, and inbreeding depression. In vitro production of doubled table beet haploids makes it possible to obtain homozygous material in a short time. It can be used in breeding programs as an alternative to traditional inbred lines. Therefore, introduction of the haploidization technology into the table beet breeding programs is of great importance. This article discusses various approaches to the production of doubled beet haploids and describes crucial achievements, major problems, and the ways to solve them. Methods for producing doubled haploids of table beet has not been studied profoundly enough, so they require additional in-depth research aimed at improving their efficiency and reproducibility.

Creation of rice doubled haploids with low amylose content using in vitro anther culture.

In vitro androgenesis is a unique model for producing homozygous doubled haploid plants. The use of haploid biotechnology accelerates to obtain of doubled haploid plants, which is very important in rice breeding. The purpose of this work is to improve the production of doubled haploids in rice anther culture in vitro and selection of doubled haploid plants with valuable traits. The study the influence of nutrient media on the production of calli and plant regeneration processes in anther culture of 35 rice genotypes was revealed a significant influence of nutrient media on callus production. It was shown that the addition to culture medium phytohormones ratio with high level of cytokinin (5.0 mg/L BAP) and a low level of auxin (0.5 mg/L NAA), supplemented with amino acid composition promotes high production of green regenerated plants (68.75%) compared to albino plants (31.25%). As a result, doubled haploid lines of the glutinous variety Violetta were selected, which characterized by a low amylose content variation (from 1.86 to 2.80%). These doubled haploids are superior to the original variety in some yield traits and represent valuable breeding material.

Факторы индукции гиногенеза огурца (Cucumis sativus L.) в культуре семязачатков

Improving the efficiency of doubled haploid technology is an urgent task to expand the possibilities of fundamental research and to increase the selection rate of commercial F1 hybrids. This study investigated the effect of the components of the induction nutrient medium: 74 casein hydrolyzate (250 mg/l and 500 mg/l), glutathione (10 mg/l), a combination of growth regulators TDZ (0.04 mg/l) and 2,4-D (0.15 mg/l) on the gynogenic response of cucumber in unfertilized ovules culture. The results showed that the addition of 250 mg/l of casein hydrolysate to the induction medium resulted in a more than twofold increase in the frequency of gynogenic ovules in two samples. The addition of 10 mg/l of glutathione to the induction medium helps to increase the frequency of gynogenic ovules by 1.5 to 2 times in 3 out of 6 samples. The results showed that cultivation of cucumber ovary fragments on the induction medium supplemented with 0.5 mg/l of putrescine leads to decrease in frequency of gynogenic ovules. The obtained data on the influence of the components of the induction medium in the culture of ovules can be used to optimize the technology for the production of doubled haploids in cucumber.

A novel visual marker to distinguish haploids from doubled haploids in rice (Oryza sativa, L) at early growth stages

Doubled haploid technology, which enables the generation of homozygous lines in a single step, is one of the modern tools being employed for accelerating breeding processes in different crops. In rice, a globally important staple food crop, doubled haploid production through androgenesis is increasingly being employed in breeding programs. Amongst the androgenic rice lines, doubled haploids are formed spontaneously at about 50–60%, while the remaining 40–50% of plants remain as haploids. As haploids cannot be easily identified, it is routine to grow all the rice androgenic lines till maturity and harvest the seeds from the fertile doubled haploids. Therefore, the methods that facilitate easy identification of haploids at an early developmental stage in rice would enable treatment of such haploid lines with colchicine, to increase the efficiency of doubled haploid production. Further, it would also help in eliminating the operational cost involved in maintaining them till maturity. In the above context, a systematic study to identify easily observable physiological and morphological differences between haploid and doubled haploid rice lines was undertaken. Rice haploids were found to be noticeably different from doubled haploids in photosynthetic rate, transpiration rate, stomatal conductance, and morphology of lodicules, stigma and style, features which have not been reported before. Most importantly, rice haploids invariably have acute leaf apex which is easily distinguishable from the doubled haploids that have attenuated leaf apex shape. Very high per cent accuracy in the prediction of ploidy level was observed when haploids were identified at an early developmental stage based on leaf apex shape, and the results verified with flow cytometry perfectly matches with leaf apex shape. The study establishes ‘acute leaf apex’ shape as an accurate visual marker to rapidly identify haploid rice lines at an early developmental stage in a cost-effective manner.

In vitro double haploid production through anther culture in niger (Guizotia abyssinica L.F. Cass)

In vitro double haploid production through anther culture in niger (Guizotia abyssinica L.F. Cass)

Double haploid production using microspore culture is a useful breeding method in the modulation of glucosinolates contents in Radish (Raphanus sativus L.)

Double haploid production using microspore culture is a useful breeding method in the modulation of glucosinolates contents in Radish (Raphanus sativus L.)

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

Androgenic response of spring barley (Hordeum vulgare L.) varieties and lines – sources of high grain protein content

Aim. Protein content is known to be one of the most important characters of grain quality. Investigation was aimed to determine the capability to haploid production in anther culture in vitro of spring barley accessions which are the sources of high protein content in grain. Methods. Infrared spectroscopy was used for evaluation of variability of barley genotypes in protein content. Anthers isolated from cold-pretreated spikes were cultivated on agar solidified nutrient medium containing N6 macro-and MS micronutrients, physiologically active substances and maltose (9.0 %). Results. Three years testing showed that mean grain protein contents in six spring barley accessions varied from 12.6 to 19.1 %. Commercial variety Yavir, accessions IR06605 and DD-21 from germplasm collection of the National Plant Gene Pool of Ukraine were selected as genotypes with a stable expression of this char-acter. Response to androgenesis in vitro varied from 6.9 to 45.9 % of morphogenic anthers and from 0.64 до 14.7 green plants per 100 cultivated anthers in dependence of genotype and donor plant growth conditions. Conclusions. Because of association between high protein content in grains and a relatively high androgenic response, varieties Velikan and Yavir were considered to be the most valuable ones for use in spring barley breeding for improvement of protein content via anther culture in vitro.

Production of haploid and doubled haploid plants of Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci via in vitro gynogenesis

Production of haploid and doubled haploid plants of Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci via in vitro gynogenesis

Production of Haploids in Ancient, Local and Modern Wheat by Anther Culture and Maize Pollination

Production of Haploids in Ancient, Local and Modern Wheat by Anther Culture and Maize Pollination