Search Results (27)

Abiotic stress can reprogram the gametophytic pathway; the mechanisms by which floral bud pre-treatment influences microspore embryogenesis initiation remain unclear. In this study, we use bisulfite sequencing, sRNA-seq, and RNA-seq to analyze the dynamic changes in rice microspores under different cold treatment durations. Our results showed that a 10-day cold treatment is essential for CXJ microspore embryogenesis initiation. DNA methylation levels showed a slight change at CG, CHG, and CHH sites under cold treatment. The number of both hyper- and hypomethylated DMRs increased over cold treatment, with more hypermethylated DMRs at 5 and 10 dpt. Hypermethylated DMRs were more frequently in the TSS region compared to hypomethylated DMRs. The proportion of 24 nt sRNAs increased upon cold stress, with more downregulated than upregulated sRNAs at 10 dpt. The number of DMR target DEGs increased from 5 to 10 dpt. Promoter hypomethylation at the CHH site was more frequently associated with DEGs. These outcomes suggested that the RdDM pathway participates in the initiation of rice ME. GO analysis indicated that DMR target DEGs at 10 dpt were enriched in responses to chemical stimuli, biological processes, and stress responses. An auxin-related gene, OsHOX28, was further identified. Its upregulation, potentially mediated by the RdDM pathway, may play a crucial role in the initiation of rice ME. This study provides more information on epigenetic mechanisms during rice ME. Full article

Brassicas are considered the third most important source of vegetable oil globally. With the escalating production of Brassica varieties, there is growing demand for high-yielding genotypes. Doubled haploid (DH) techniques have become very popular in various Brassica breeding programs. Such DH techniques can play a significant role in plant breeding by accelerating the production of homozygous lines and increasing selection efficiency. Among these methods, isolated microspore culture stands out as the most effective, facilitating the generation of a higher number of embryos compared to conventional methods of plant breeding. Different chemical compounds such as herbicides, brassinosteroids, and polyethylene glycol have an antimitotic effect and have been found to generate DH plants and improve microspore embryogenesis in Brassica species. Colchicine and trifluralin have proven to be efficient chromosome-doubling agents as well as important supplements that can increase the rate of embryogenesis. This review serves as a comprehensive summary and effectiveness evaluation of the latest research findings in the Brassica oil crops to help increase efficiency of the future research focusing on DH methods and application of antimitotic agents in the various oilseed species of the genus Brassica. Full article

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid–liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines. Full article

The use of doubled haploid (DH) technology enables the development of new varieties of plants in less time than traditional breeding methods. In microspore embryogenesis (ME), stress treatment triggers microspores towards an embryogenic pathway, resulting in the production of DH plants. Epigenetic modifiers have been successfully used to increase ME efficiency in a number of crops. In wheat, only the histone deacetylase inhibitor trichostatin A (TSA) has been shown to be effective. In this study, inhibitors of epigenetic modifiers acting on histone methylation (chaetocin and CARM1 inhibitor) and histone phosphorylation (aurora kinase inhibitor II (AUKI-II) and hesperadin) were screened to determine their potential in ME induction in high- and mid-low-responding cultivars. The use of chaetocin and AUKI-II resulted in a higher percentage of embryogenic structures than controls in both cultivars, but only AUKI-II was superior to TSA. In order to evaluate the potential of AUKI-II in terms of increasing the number of green DH plants, short and long application strategies were tested during the mannitol stress treatment. The application of 0.8 µM AUKI-II during a long stress treatment resulted in a higher percentage of chromosome doubling compared to control DMSO in both cultivars. This concentration produced 33% more green DH plants than the control in the mid-low-responding cultivar, but did not affect the final ME efficiency in a high-responding cultivar. This study has identified new epigenetic modifiers whose use could be promising for increasing the efficiency of other systems that require cellular reprogramming. Full article

Among various methods stimulating biological progress, double haploid (DH) technology, which utilizes the process of microspore embryogenesis (ME), is potentially the most effective. However, the process depends on complex interactions between many genetic, physiological and environmental variables, and in many cases, e.g., winter wheat, does not operate with the efficiency required for commercial use. Stress associated with low-temperature treatment, isolation and transfer to in vitro culture has been shown to disturb redox homeostasis and generate relatively high levels of reactive oxygen species (ROS), affecting microspore vitality. The aim of this study was to investigate whether controlled plant growth, specific tiller pre-treatment and culture conditions could improve the potential of microspores to cope with stress and effectively induce ME. To understand the mechanism of the stress response, hydrogen peroxide levels, total activity and the content of the most important low-molecular-weight antioxidants (glutathione and ascorbate), as well as the content of selected macro- (Mg, Ca, NA, K) and micronutrients (Mn, Zn, Fe, Cu, Mo) were determined. These analyses, combined with the cytological characteristics of the microspore suspensions, allowed us to demonstrate that an increased microspore vitality and stronger response to ME induction were associated with higher stress resistance based on more efficient ROS scavenging and nutrient management. It was shown that a modified procedure, combining a low temperature with mannitol and sodium selenate tiller pre-treatment, reduced oxidative stress and improved the effectiveness of ME in winter wheat lines. Full article

This study aimed to evaluate the effects of dimethyl sulfoxide (DMSO) on microspore embryogenesis and green plant regeneration in wheat anther culture. Five culture media, as well as the inclusion of 1% DMSO in the surface disinfection solution, were investigated in three winter wheat genotypes. Our results showed that the Altındane genotype produced the highest number of embryoids, 215 per 100 anthers cultured in CHB-3 medium, whereas the Dariel and Pehlivan genotypes produced 6.6 and 0 embryoids, respectively, from 100 anthers cultured. On the other hand, the addition of 1% DMSO to the same medium adversely affected embryoid production compared to the medium without DMSO. A 70% ethanol solution with 1% DMSO for the surface disinfection of spikes was effective in increasing the embryoids from approximately 0 to 17.8% and from 1 to 48.4% in CHB-3 + 1%DMSO and CHB-3 medium, respectively. Furthermore, the Altındane genotype produced 22.2 plantlets/100 anthers (17.7 albino and 4.4 green plants) and 17.7 albino plantlets per 100 anthers in CHB-3 and CHB-3 + DMSO, respectively. Our results suggested that the inclusion of 1% DMSO in the disinfection solution increased the number of embryoids without supporting the production of green plants. Full article

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore. Full article

Reproduction studies, particularly embryology, represent basic information of any plant. However, the current embryological information is fragmentary for Kentucky bluegrass (Poa pratensis L.). Here, paraffin sections were used to examine the cyto-embryological characteristics, including microsporogenesis, microgametogenesis, megasporogenesis, megagametogenesis, and apomixis, of wild Kentucky bluegrass germplasm from Gannan (GN) and Longnan (LN) in Gansu Province. The study found no significant differences in pollen diameter, characteristics, viability, and stigma receptivity between the two germplasm materials. The Kentucky bluegrass consisted of three anthers, and each contained four pollen sacs that were divided into left and right halves. After meiosis, the microspore mother cells formed dyads and tetrads, which were primarily symmetrical and underwent mitosis to form three-celled pollen. Kentucky bluegrass has a one-locular ovary, two-feathery stigmas, thick nucleolar and anatropous ovules, and a typical polygonum embryo sac as its reproductive organs. The main type of apomixis observed was apospory, resulting in the coexistence of multiple embryo sacs. Polyembryonic seeds were frequently observed in Kentucky bluegrass due to apospory. Most importantly, our research found that apospory caused early embryogenesis during fertilization, which is a vital embryological feature for identifying sexual reproduction and apomixis in Kentucky bluegrass. Sexual reproduction followed strict double fertilization, while in apomixis a complete seed was only formed through pseudogamy. These embryological characteristics are documented here, and their study can aid in understanding the evolution of Kentucky bluegrass. Full article

Rapeseed is one of the most important oil crops in the world. Increasing demand for oil and limited agronomic capabilities of present-day rapeseed result in the need for rapid development of new, superior cultivars. Double haploid (DH) technology is a fast and convenient approach in plant breeding as well as genetic research. Brassica napus is considered a model species for DH production based on microspore embryogenesis; however, the molecular mechanisms underlying microspore reprogramming are still vague. It is known that morphological changes are accompanied by gene and protein expression patterns, alongside carbohydrate and lipid metabolism. Novel, more efficient methods for DH rapeseed production have been reported. This review covers new findings and advances in Brassica napus DH production as well as the latest reports related to agronomically important traits in molecular studies employing the double haploid rapeseed lines. Full article

Brassica napus is a species of high agronomic interest, used as a model to study different processes, including microspore embryogenesis. The DH4079 and DH12075 lines show high and low embryogenic response, respectively, which makes them ideal to study the basic mechanisms controlling embryogenesis induction. Therefore, the availability of protocols for genetic transformation of these two backgrounds would help to generate tools to better understand this process. There are some reports in the literature showing the stable transformation of DH12075. However, no equivalent studies in DH4079 have been reported to date. We explored the ability of DH4079 plants to be genetically transformed. As a reference to compare with, we used the same protocols to transform DH12075. We used three different protocols previously reported as successful for B. napus stable transformation with Agrobacterium tumefaciens and analyzed the response of plants. Whereas DH12075 plants responded to genetic transformation, DH4079 plants were completely recalcitrant, not producing any single regenerant out of the 1784 explants transformed and cultured. Additionally, an Agrobacterium rhizogenes transient transformation assay was performed on both lines, and only DH12075, but no DH4079 seedlings, responded to A. rhizogenes infection. Therefore, we propose that the DH4079 line is recalcitrant to Agrobacterium-mediated transformation. Full article

Curly kale (Brassica oleracea L. convar. acephala var. sabellica), the most common type of edible kale, characterized by providing rich nutrition and health care functions, is sought after and has been listed as top of the healthiest vegetables in recent trends, and has aroused the interest of breeders in cultivating new varieties. However, it usually takes more than six years to obtain a homozygous kale inbred line for commercial seed production through conventional breeding procedures due to its long growth and development period. The isolated microspore culture (IMC) technique could be a time-saving alternative method for producing doubled haploid (DH) lines that are genetically homozygous. In this study, we successfully utilize the efficient cytokinin thidiazuron (TDZ) to promote microspore embryogenesis and plant regeneration in two curly kale cultivars (‘Winterbor F₂’ and ‘Starbor F₂’). Compared with the control (0 mg/L TDZ), all tested TDZ concentrations (0.1, 0.2, 0.3, 0.4 mg/L) had no adverse effects on embryogenesis, and 0.2 mg/L TDZ had an optimal effect on embryo survival and plant regeneration of the two genotypes. For ‘Starbor F₂’, 0.2 mg/L TDZ treatment achieved the highest embryogenesis rate (1.83-fold higher than the control group) and direct seeding rate (1.61-fold increase), and the lowest mortality rate. Likewise, 0.2 mg/L TDZ increased the embryogenesis rate of ‘Winterbor F₂’ by 1.62 times, the direct seeding rate by 1.61 times, and the mortality rate fell to the lowest. A 1/2 Murashige and Skoog (MS) medium with 0.2 mg/L 1-Naphthaleneacetic acid (NAA) can significantly promote the rooting of the regenerated seedlings. These results provide new insights into the practical application of the IMC technique in shortening the breeding cycle of kale. Full article

Although zygotic embryogenesis is usually studied in the field of seed biology, great attention has been paid to the methods used to generate haploid embryos due to their applications in crop breeding. These mainly include two methods for haploid embryogenesis: in vitro microspore embryogenesis and in vivo haploid embryogenesis. Although microspore culture systems and maize haploid induction systems were discovered in the 1960s, little is known about the molecular mechanisms underlying haploid formation. In recent years, major breakthroughs have been made in in vivo haploid induction systems, and several key factors, such as the matrilineal (MTL), baby boom (BBM), domain of unknown function 679 membrane protein (DMP), and egg cell-specific (ECS) that trigger in vivo haploid embryo production in both the crops and Arabidopsis models have been identified. The discovery of these haploid inducers indicates that haploid embryogenesis is highly related to gamete development, fertilization, and genome stability in ealry embryos. Here, based on recent efforts to identify key players in haploid embryogenesis and to understand its molecular mechanisms, we summarize the different paths to haploid embryogenesis, and we discuss the mechanisms of haploid generation and its potential applications in crop breeding. Although these haploid-inducing factors could assist egg cells in bypassing fertilization to initiate embryogenesis or trigger genome elimination in zygotes after fertilization to form haploid embryos, the fertilization of central cells to form endosperms is a prerequisite step for haploid formation. Deciphering the molecular and cellular mechanisms for haploid embryogenesis, increasing the haploid induction efficiency, and establishing haploid induction systems in other crops are critical for promoting the application of haploid technology in crop breeding, and these should be addressed in further studies. Full article

Cut flower ornamental kale (Brassica oleracea var. acephala) is a biennial cultivar, which completes a sexual reproductive generation in two years. Isolated microspore culture (IMC) can accelerate plant homozygosity instead of self-pollinations. However, the application of IMC in cut flower ornamental kale was rare since its low rate of embryogenesis. It is proved that histone acetylation might affect the gene expression in microspores and led to the transformation of microspores from pollen development pathway to embryogenesis. In this paper, microspores, derived from three varieties of cut flower ornamental kale, Crane Bicolor (CB), Crane Pink (CP) and Crane Feather Queen (CFQ), were treated with histone deacetylation inhibitor (HDACI) trichostatin A (TSA). Results revealed that the appropriate concentration of TSA was 10 nM for CB with obtaining 5.39 embryos per bud, while for CP and CFQ was 5 nM with acquiring 10.89 and 16.99 embryos per bud, respectively. TSA treatment also reduced the embryonic mortality, of which 10 nM TSA treatments CB was the optimal and the embryonic mortality decreased to 25.01%. The double haploid (DH) proportion of regenerated plants reached 37.3%. These results contribute to improving the technology for IMC in cut flower ornamental kale. Full article

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage ‘01-88’ under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment. Full article

The European radish is one of the most unresponsive crops in the Brassicaceae family to embryogenesis in in vitro microspore culture. The aim of this work was to study the process of embryogenesis of European radish and its biological features. In this study, the embryogenesis of European radish is described in detail with illustrative data for the first time. For the first time for the entire family Brassicaceae, the following were found: microspores with intact exines with ordered-like divisions; microspores completely free of exines; and a new scheme of suspensors attachment to the apical parts of embryoids. The morphology of double and triple twin embryoids was described, and new patterns of their attachment to each other were discovered. Uneven maturation of European radish embryoids at all stages of embryogenesis was noted. The period of embryoid maturation to the globular stage of development corresponded, in terms of time, to the culture of B. napus, and into the cotyledonary stage of development, maturation was faster and amounted to 17–23 days. The rate of embryoid development with and without suspensors was the same. Full article