Anatomical and hormonal factors determining the development of haploid and zygotic embryos of oat (Avena sativa L.)

A critical step in the production of doubled haploids is a conversion of the haploid embryos into plants. Our study aimed to recognize the reasons for the low germination rate of Avena sativa haploid embryos obtained by distant crossing with maize. Oat cultivars of ‘Krezus’ and ‘Akt’ were investigated regarding embryo anatomy, the endogenous phytohormone profiles, and antioxidant capacity. The zygotic embryos of oat were used as a reference. It was found that twenty-one days old haploid embryos were smaller and had a less advanced structure than zygotic ones. Morphology and anatomy modifications of haploid embryos were accompanied by extremely low levels of endogenous auxins. Higher levels of cytokinins, as well as tenfold higher cytokinin to auxin ratio in haploid than in zygotic embryos, may suggest an earlier stage of development of these former. Individual gibberellins reached higher values in ‘Akt’ haploid embryos than in the respective zygotic ones, while the differences in both types of ‘Krezus’ embryos were not noticed. Additionally to the hormonal regulation of haploid embryogenesis, the poor germination of oat haploid embryos can be a result of the overproduction of reactive oxygen species, and therefore higher levels of low molecular weight antioxidants and stress hormones.

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.

Lipid anchoring and electrostatic interactions target NOT-LIKE-DAD to pollen endo-plasma membrane

Phospholipases cleave phospholipids, major membrane constituents. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD, also known as MTL or ZmPLA1) leads to peculiar defects in sexual reproduction, notably the induction of maternal haploid embryos. Contrary to previous reports, NLD does not localize to cytosol and plasma membrane of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. After pollen tube burst, NLD localizes at the apical region of the egg apparatus. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and lipid biosensors indicated that phosphatidylinositol-4,5-bisphosphate is enriched in the endo-PM, uncovering a unique example of how membrane electrostatic properties can define a specific polar domain (i.e., endo-PM), which is critical for plant reproduction and gamete formation.

The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio–dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio–mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio–dependent genes respond directly to the N/C ratio through multiple modes of regulation.

Production of haploid embryos and plants in Iranian melon (Cucumis melo L.) through irradiated pollen-induced parthenogenesis.

The irradiated pollen technique (IPT) is the most successful haploidization technique within Cucurbitaceae. The influence of gamma-ray doses (250, 350, 450 and 550 Gy), genotypes and stage of development of embryos obtained by IPT on the induction of haploid embryos were studied in several Iranian melon cultivars as well as their hybrids with alien cultivars. Female flowers were pollinated using pollen that had been irradiated with gamma rays. Different shapes and stages of embryos were excised 21-25 days after pollination and cultured on E20A medium. Direct culture, liquid culture and integrated culture methods were used; integrated culture and liquid culture methods showed advantages in increasing the efficiency of haploid plant production in melon breeding programmes. Results revealed that 550 Gy of gamma irradiation was successful in inducing parthenogenesis and fruit development, whereas lower irradiation doses were not effective in inducing haploid embryos. The percentages of embryos per seed were the highest in 'Samsoori' (1.2%) and 'Saveh' (1.1%) cultivars. Some of the heart-shaped and cotyledon-shaped embryos developed into haploid plants. In total, 52 parthenogenic melon plantlets were recovered from 274 embryos via IPT. Production of haploid embryos and haploid plants was strongly influenced by gamma-ray dose, embryo stage and genotype. Indirect methods and chromosome counting performed on the root cells of regenerated plants showed that these plants were haploid (n = x = 12).

A chimeric gene paternally instructs female sex determination in the haplodiploid wasp Nasonia

Various primary signals direct insect sex determination. In hymenopteran insects, the presence of a paternal genome is needed to initiate female development. When absent, uniparental haploid males develop. We molecularly and functionally identified the instructor sex-determination gene, wasp overruler of masculinization (wom), of the haplodiploid wasp Nasonia vitripennis. This gene contains a P53-like domain coding region and arose by gene duplication and genomic rearrangements. Maternal silencing of wom results in male development of haploid embryos. Upon fertilization, early zygotic transcription from the paternal wom allele is initiated, followed by a timely zygotic expression of transformer (tra), leading to female development. Wom is an instructor gene with a parent-of-origin effect in sex determination.

Lipid anchoring and electrostatic interactions target the phospholipase NOT-LIKE-DAD to pollen endo-plasma membrane

Phospholipases are ubiquitous enzymes that cleave phospholipids, one major constituent of membranes. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD) leads to peculiar defects in sexual reproduction. Indeed, pollination of a wild-type female with mutant pollen generates haploid embryos containing solely maternal genetic information. Contrary to previous reports NLD does not localize to cytosol and plasma membrane (PM) of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions between membrane and NLD are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and anionic lipid bio-sensors indicated that phosphatidylinositol-4,5-bisphosphate (PIP(4,5)P2) is enriched in the endo-PM as compared to the PM. Our results uncover a unique example of how membrane electrostatic properties can specify a unique polar domain (i.e. endo-PM), which is critical for plant reproduction and gamete formation.