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.

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.

Cytological Investigation in Two Mutants of the Hybrid Grapevine ‘Niagara’

‘Niagara’ is a hybrid table grape (V. vinifera × V. labrusca). Several spontaneous somatic mutants from this grapevine have already been described. However, cytological information about these mutants is scarce. Therefore, studies in the microsporogenesis have been carried out in two of these mutants, a seedless and a large berry mutant (giant mutant). A low percentage of abnormalities such as precocious chromosome segregation, bivalent not aligned, laggard and stickiness chromosomes were seen in both mutants. A merging process of microspores was observed in several tetrads in the seedless mutant: the microspore came together by large cytoplasmic bridges giving rise to a large and amorphous single-celled tetrad, which eventually collapsed. The giant mutant showed 10.29% of tetrads with microcytes. However, these tetrad abnormalities did not hinder pollen viability. Significant differences were seen concerning stomata and pollen grain sizes, stomata frequency and anther number per flower between the mutants. The chromosome count at diakinesis (n=19) and in diploid tapetal cells (2n=38) clarified the fact that the seedless somatic mutation was not associated with any ploidy event. Conversely, the giant mutant showed n=38 at diakinesis and 2n=76 in diploid tapetal cells, pointing to a case of spontaneous chromosome duplication. Since these abnormalities did not disturb the percentage of pollen viability very much, in both mutants (higher than 86%), the seedless one may be used as a male parent in crossing, regarding the desirable seedlessness character, while the mutant giant may be employed as female parent for the obtainment of seedless triploid plants. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Androgenic response of Brazilian wheat genotypes to different pretreatments of spikes and to a gelling agent

The objective of this work was to analyze the androgenic response of Brazilian wheat genotypes to different pretreatments of the spikes, prior to the culture of isolated microspores, and to the effect of a gelling agent in the induction culture medium. Five genotypes were evaluated for embryo formation, green plant regeneration, and spontaneous chromosome duplication. Wheat spikes were subjected to two pretreatments: cold, at 4ºC for 21 days; and 2-hydroxynicotinic acid, at 32ºC for two days. Culture media were evaluated with or without Ficoll as a gelling agent. Cold produced more embryos and green plants than the chemical pretreatment in four out of five genotypes. Only two genotypes treated with 2-hydroxynicotinic acid were able to produce plants, and one of them produced a single albino plant. Medium containing Ficoll produced more embryos than liquid medium and promoted a higher number of plants. Spontaneous chromosome duplication varies between genotypes and pretreatments, and shows high variability.

Effect of Pepper Types on Obtaining Spontaneous Doubled Haploid Plants via Anther Culture

The most successful technique used to obtain haploid plant in pepper is anther culture. The chromosome content of haploid plants can be doubled spontaneously or using colchicine. In this study, we compared the rate of spontaneous doubled haploidy of different pepper types. Seven charleston, six bell, eight capia, and seven green pepper genotypes were used as plant material. Murashige and Skoog (MS) nutrient medium with 4 mg·L−1 naphthaleneacetic acid (NAA), 0.5 mg·L−1 6-benzylaminopurine (BAP), 0.25% activated charcoal, 30 g·L−1 sucrose, and 15 mg·L−1 silver nitrate (AgNO3) was used. Ploidy levels of plants obtained through anther culture were detected using both flow cytometry and simple sequence repeats (SSR) markers. The results showed that different spontaneous doubled haploidy rates were obtained from different pepper types. The highest rate was observed in bell pepper type with 53.4% (mean of six genotypes) of haploid plants undergoing spontaneous chromosome doubling. This was followed by charleston and capia types with 31.9% and 30.4% doubling, respectively. Green pepper type gave the lowest spontaneous doubled haploidy rate with 22.2% doubling. The results obtained from this study will be useful both for future work on haploidy in pepper and for breeding programs.

THE KNOWN UK CASES OF ATAXIA TELANGIECTASIA-LIKE DISORDER (ATLD)

Ataxia telangiectasia-like disorder (ATLD) is a very rare autosomal recessive disease with only 25 patients recognised worldwide. ATLD is likened to Ataxia telangiectasia (A-T) due to an overlap of clinical presentations and cellular characteristics. The clinical hallmark of A-T and ATLD is progressive young onset cerebellar ataxia. Variably present characteristics include dysarthria, oculomotor apraxia, ocular telangiectasia, immunodeficiency, spontaneous chromosome abnormalities and a predisposition to malignancy. In contrast to A-T, ocular telangiectasia is absent. Furthermore, ATLD patients tend to have a later onset and slower progression of neurological signs than A-T. The ATM gene, that has a key role in genome stability, is mutated in A-T resulting in an increase cancer predisposition. In ATLD, gene MRE11 is mutated leading to deficient activation of ATM. A functional consequence of the MRE11 mutation is raised chromosomal radiosensitivity because functional ATM kinase is required to rejoin chromosome breaks. This poster/presentation will describe the clinical features and genetic analysis of the ATLD cases with progressive ataxia known in the UK. Undergraduate Prize Winner 2015

Production of Tetraploid Gynogenetic Loach Using Diploid Eggs of Natural Tetraploid Loach, Misgurnus anguillicaudatus, Fertilized with UV-Irradiated Sperm of Megalobrama amblycephala without Treatments for Chromosome Doubling

The gynogenesis phenomenon in nature mainly appears in the reproduction of fish and invertebrates. So far, gynogenesis has been successfully induced in many fish species with the aid of some physical or chemical methods for chromosome doubling. However, few fish can produce gynogenetic progenies, genetically identical or similar to the somatic cells of the mothers, without a treatment for the doubling of chromosomes, which may be related to apomixis, premeiotic endoreduplication, or premeiotic endomitosis. At present, no studies are available about fish with normal ovarian structures producing gynogenetic progenies that could spontaneously double their chromosomes. According to the analyses of flow cytometry, chromosome number, and microsatellites, we found that, with the use of UV-irradiated sperm of blunt snout bream Megalobrama amblycephala, tetraploid loach Misgurnus anguillicaudatus produced tetraploid gynogenetic progenies without any treatments for the doubling of chromosomes. To determine the genetic relationships of gynogenetic progenies and their maternal parent, microsatellite genotyping was conducted. The results indicated that the reason for spontaneous chromosome duplication in gynogenetic progenies may be cytokinesis or inhibition of the extrusion of the second polar body. This is the first report on fish with normal ovarian structures that can produce gynogenetic progenies which spontaneously double their chromosomes and which are genetically identical or similar to the somatic cells of the mothers.

Introductory studies of bacterial effects on the course of mitosis in adventitious roots of Allium cepa L.

The level of spontaneous chromosome aberrations and other phenomena concomitant with mitoses in the meristematic cells of <i>Allium cepa</i> L. adventitious roots grown in water, depends to a large extent on the intensity of bacteria multiplication. From the water culture two strains of bacteria, which were most numerous, were isolated - <i>Agrobacterium</i> and <i>Flavobacterium</i>. A supernatant from bacteria grown on Davis medium induced chromosome sticking together, c-mitoses and the formation of polyploid nuclei in the roots of onion.