Haploid Plants Research Articles

Construction of Tissue Culture System of Onobrychis viciaefolia Scop ‘Mengnong’ Anthers

Background: To accelerate the establishment of a genetic improvement system for Onobrychis viciaefolia Scop. by cultivating haploid plants and producing pure parental material. Methods: We endeavored to set up the Onobrychis viciaefolia Scop. anther culture system, examine the suitable anther pre-treatment method and the factors influencing medium type and hormone ratio during anther regeneration. Result: The regenerative capacity of intact anthers was preserved by 48h low temperature (4°C) medium treatment. Or the broken anthers were obtained when the regenerated grass was budding and preserved in the medium after 48 hours of low temperature treatment; callus induction was based on MS and the callus rate was 18.33% when 1.0 mg/L 2,4-D + 0.75 mg/L 6-BA + 0.8 mg/L NAA + 2.0 mg/L KT was added. With N6 as the basic medium, the addition of 2.0 mg/L 2,4-D + 0.75 mg/L 6-BA + 0.4 mg/L NAA + 0.5 mg/L KT resulted in a healing rate of 16.19%; the addition of 0.5 mg/L 6-BA + 1.0 mg/L KT to the MS medium resulted in a differentiation rate of 68%; during the construction of the anther histoponic system, browning and waterlogging of the healing tissues and vitrification of the seedlings hindered the formation of regenerated plants.

Loss-of-function of gynoecium-expressed phospholipase pPLAIIγ triggers maternal haploid induction in Arabidopsis.

Production of in planta haploid embryos that inherit chromosomes from only one parent can greatly increase breeding efficiency via quickly generating homozygous plants, called doubled haploid. One of the main players of in planta haploid induction is a pollen-specific phospholipase A, which is able, when mutated, to induce invivo haploid induction in numerous monocots. However, no functional orthologous gene has been identified in dicots plants. Here, we show that loss-of-function of gynoecium-expressed phospholipase AII (pPLAIIγ) triggers maternal haploid plants in Arabidopsis, at an average rate of 1.07%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome. Promoter activity of pPLAIIγ shows enriched expression in the funiculus of flower development stages 13 and 18, and pPLAIIγ fused to yellow fluorescent protein reveals a plasma-membrane localization Interestingly, the polar localized PIN1 at the basal plasma membrane of the funiculus was all internalized in pplaIIγ mutants, suggesting that altered PIN1 localization in female organ could play a role in maternal haploid induction.

Targeting Disease Susceptibility Genes in Wheat Through wide Hybridization with Maize Expressing Cas9 and Guide RNA.

Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight and tan spot, Septoria nodorum blotch, and spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15 to 33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool, not only for targeting susceptibility genes to improve disease resistance without regulatory issues, but also for understanding gene function in wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Production of double haploid watermelon via maternal haploid induction.

Production of double haploid watermelon via maternal haploid induction.

Near infrared spectroscopy (NIR) and chemometrics methods to identification of haploids in maize

Haploids in maize are selected through visual analysis, whether by use of expression of the R1-navajo marker in seeds, or by the phenotype of haploid plants in the field. These methods of identification of haploid plants demand space, resources, and time. Near infrared spectroscopy (NIR), together with multivariate calibration methods, is an alternative for identification of haploid seeds and plants in a quick and accurate way. The aim of the present study was to use NIR to classify haploid and diploid seeds and differentiate haploid, doubled haploid, and diploid plants originating from different genotypes used as donor. In the first experiment, haploid and diploid maize seeds were analyzed from four genotypes used as parents. In another experiment, diploid, haploid, and doubled haploid plants from 34 source populations were evaluated and differentiated. Spectral data were collected from all the seed and leaf samples using the portable MicroNIR device. Then the data were pre-processed by principal component analysis and by partial least squares discriminant analysis (PLS-DA). The use of the portable MicroNIR, together with the PLS-DA method, enabled classification of haploid and diploid seeds and haploid, doubled haploid, and diploid plants with 100% accuracy, constituting a fast, simple, non-destructive, and reliable methodology for identification of haploids in maize, and it is more effective than the visual analysis.

Influence of Polyamines on Red Beet (Beta vulgaris L. ssp. vulgaris) Gynogenesis

The influence of polyamines (PAs), putrescine (Put) and spermidine (Spd) on the efficiency of gynogenesis in ovule cultures of red beet (syn. beetroot) (Beta vulgaris L. vulgaris) cultivar “Czerwona Kula” and breeding accessions no. 3/2010 and no. 7/2008 was investigated. The effect of Put on the process of plant regeneration from gynogenetic embryos was studied. The response to the applied PAs was strongly dependent on the genotype. In “Czerwona Kula”, an increase in the number of obtained embryos was achieved by using each of the two PAs in the B5 medium. The effect of Spd was stronger. Put added to the regeneration medium at the concentration of 0.5 mg L−1 increased the number of obtained plants. All shoots placed on the rooting medium supplemented with 160 mg L−1 Put formed roots. The distribution of ploidy and homozygosity of gynogenetic plants depended on the genotype. Of the tested genotypes, the highest number of haploid plants, 68%, was obtained in red beet “Czerwona Kula”. The highest percentage of homozygotes, 69% for the glucose phosphate isomerase (GPI, E.C.5.3.1.9) isoenzyme and 100% for the aspartate aminotransferase (AAT, E.C.2.6.1.1) isoenzyme, was obtained in the population of gynogenetic plants of cultivar “Czerwona Kula”.

Identification of Arabidopsis thaliana haploid plants by counting the chloroplast numbers in stomatal guard cells

Identification of Arabidopsis thaliana haploid plants by counting the chloroplast numbers in stomatal guard cells

Haploid male fertility is restored by parallel spindle genes in Arabidopsis thaliana.

Doubled haploid technology can accelerate plant breeding and its two main steps are haploid induction and subsequent doubled haploid production from fertile haploid plants. Although haploid female fertility is present to some extent in plants, the lack of haploid male fertility is a bottleneck. Herein, we demonstrate that mutations in the parallel spindle genes are sufficient to restore haploid male fertility in Arabidopsis with no impact on haploid female fertility.

Synthetic apomixis with normal hybrid rice seed production

Synthetic apomixis with normal hybrid rice seed production

A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases

Crop breeding schemes can be significantly accelerated by using (doubled) haploid plants. In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes, and haploidization rates are generally very low. Therefore, methodological improvements to and new concepts for haploidization are required. Here, we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases (ECSs). We show that after successful sperm–egg cell fusion, ECSs play a critical role to ensure male and female nucleus fusion after fertilization. The ecs1 ecs2 double mutant can induce haploids by both selfing and hybridization in Arabidopsis and ECS mutation is also capable of producing haploids in rice. In summary, our study develops a novel approach for maternal haploidization and provides new insights into the molecular basis of fertilization.

Agronomic performance, yield stability and selection of doubled haploid rice lines in advanced yield trials

<abstract> <p>Anther culture is a widely utilized technique in rice breeding because of its simplicity and effectiveness in rapidly obtaining pure lines in the form of doubled haploid plants. The selection of doubled haploid (DH) rice lines derived from anther culture in advanced yield trials is an important step for obtaining superior DH lines. We aimed to determine agronomic performance, including yield and yield stability in order to select lowland DH rice lines that are high yield and have good agronomic performance based on the selection index method. The research was conducted in Indonesia at three locations, i.e., Bogor (West Java), Indramayu (West Java) and Malang (East Java) from July to December 2022. The genotypes tested were 29 DH lines and three check varieties (Inpari-42 Agritan GSR, Inpari-18 Agritan and Bioni63 Ciherang Agritan) using a randomized complete block design (RCBD) with genotypes as a single factor and three replications. High heritability values are found in all agronomic characters, except the percentage of filled grain/panicle, the percentage of empty grain/panicle and productivity. The yield stability based on the Kang method showed that 15 lines were stable and had high productivity. Phenotypic correlation analysis showed that the number of productive tillers, days to flowering, days to harvesting, number of filled grains/panicle and percentage of filled grains all had positive values and significantly correlated with productivity. Phenotypic path analysis showed that the character of days to harvesting, number of filled grains/panicle, number of productive tillers and percentage of filled grains/panicle directly affected the productivity. Based on the weighted selection index, 12 DH lines were selected due to having a positive and higher index (8.54 to 0.28) than the Bioni63 Agritan and Inpari 18 check varieties. Among those lines, 9 DH lines were also stable based on the Kang Method.</p> </abstract>

Doubled Haploids: Contributions of Poland’s Academies in Recognizing the Mechanism of Gametophyte Cell Reprogramming and Their Utilization in Breeding of Agricultural and Vegetable Species

Abstract Diverse processes leading to doubled haploid (DH) plant production, such as microspore embryogenesis, gynogenesis, and distant hybridization followed by genome elimination, are based on the unique ability of plant cells to form haploid embryos without fertilization. All of these are possible because of various in vitro culture systems that enable the growth and development of tissues or single cells outside of the parental organism. The possibility of re-directing cell development from its original pathway to embryogenesis brings several benefits to many research areas, but the most important is the possibility of its implementation in breeding programs. This review summarizes the achievements of Polish research groups in studies of the mechanisms of haploid/DH embryo development and demonstrates the practical applications of these systems in basic studies and plant breeding. It shows the results of studies on economically important crops including barley ( Hordeum vulgare L.), oilseed rape ( Brassica napus L.), triticale (× Triticosecale Wittm.), oat ( Avena sativa L.), rye ( Secale cereale L.), sugar beet ( Beta vulgaris ssp. vulgaris L.), and some vegetable species, including carrot ( Daucus carota L.), onion ( Allium cepa L.), red beet ( Beta vulgaris L.), and members of the Brassicaceae.

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding.

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the '1009' genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.

Haploid induction and plant production in bottle gourd by pollination with gamma irradiated pollen

Bottle gourd [Lagenaria siceraria (Molina) Standley] has been used exclusively as rootstocks for Cucurbit crops. Haploid technique increases the selection efficiency for developing resistant bottle gourd varieties. This work focused on haploid induction in bottle gourd through in situ parthenogenesis by pollination with gamma-irradiated pollen. Pollination were carried out for six genotypes of bottle gourd with 50, 75, 100, 125 and 150 Gy (Gy) doses of gamma rays. Production of in vitro haploid plants was influenced by irradiation dose, genotype and embyo stage. Seeds at 17 to 21 days after pollination was found to be optimum for embryo rescue. Pollination with irradiated pollen at 100 to 125 Gy was effective for haploid induction. The ploidy level of the 11 parthenogenetic plantlets was confirmed by flow cytometry and 36.3% haploid and 9% triploid plantlets were obtained. This work provides valuable germplasm resources for bottle gourd genetic analysis and breeding programs. Haploid plantlets through in situ parthenogenesis were obtained in bottle gourd. Irradiation doses between 100-150 Gy were best for parthenogenesis induction in bottle gourd.

Rapid creation of CENH3-mediated haploid induction lines using a cytosine base editor (CBE).

Haploid induction (HI) can create true-breeding lines in a single generation, which can significantly accelerates the breeding process. In recent years, scientists have developed a variety of new techniques to induce haploids through manipulation of CENH3, a variant of the centromere-specific histone H3. One alternative approach is based on CENH3 point mutations derived from EMS/TILLING, which is not lethal and yet is responsible for inducing haploids. However, most residues have been obtained by EMS mutagenesis over a long period of time. Recently, a new approach called 'base editing' was developed for plants. Here, we report a new method that uses a cytosine base editor (CBE) to create a point mutation of CENH3 as a haploid induction line, which substitutes adenine (A) for guanine (G). As proof of the extreme simplicity of this approach to create haploid-induced lines, we identified an L130F substitution within the histone fold domain in Arabidopsis thaliana. Subsequently, we tested the haploid-inducing potential of homozygous L130F plants by pollinating them with Col-0, and obtained 2.9% paternal haploid plants. In brief, our innovative technology provides a new perspective for the promotion of CENH3-mediated haploid induction in crops, and also provides a variety of options for breeders. Such conserved point mutations as L130F could be developed into a general instrument for haploid induction in a wide range of plant species. Extending these systems would represent a major advance over haploid production.

The Production of Helianthus Haploids: A Review of Its Current Status and Future Prospects.

The genus Helianthus comprises 52 species and 19 subspecies, with the cultivated sunflower (Helianthus annuus L.) representing one of the most important oilseed crops in the world, which is also of value for fodder and technical purposes. Currently, the leading direction in sunflower breeding is to produce highly effective heterosis F1 hybrids with increased resistance to biotic and abiotic stresses. The production of inbred parental lines via repeated self-pollination takes 4-8 years, and the creation of a commercial hybrid can take as long as 10 years. However, the use of doubled haploid technology allows for the obtainment of inbred lines in one generation, shortening the time needed for hybrid production. Moreover, it allows for the introgression of the valuable genes present in the wild Helianthus species into cultivated sunflowers. Additionally, this technology makes it possible to manipulate the ploidy level, thereby restoring fertility in interspecific hybridization. This review systematizes and analyzes the knowledge available thus far about the production of haploid and dihaploid Helianthus plants using male (isolated anther and microspore cultures) and female (unpollinated ovaries and ovules culture) gametophytes, as well as by induced parthenogenesis using γ-irradiated pollen and interspecific hybridization. The genetic, physiological, and physical factors influencing the efficiency of haploid plant production are considered. A special section focuses on the approaches used to double a haploid chromosome set and the direct and indirect methods for determining the ploidy level. The current analyzed data on the successful application of haploid sunflower plants in breeding are summarized.

Doubled-haploid induction generates extensive differential DNA methylation in Arabidopsis.

Doubled haploids (DHs) fix traits from hybrids in one generation. DH induction includes two changes in ploidy levels typically associated with variation in DNA methylation. However, DNA methylation patterns in DH plants and their biological significance are largely unknown. We generated three DH lines in Arabidopsis thaliana by crossing a haploid inducer with the accession Col-0, thus removing tissue culture and hybridization as a variable. DH induction produced thousands of differentially DNA methylated regions (DMRs), most of which were stochastic. Both haploidization and colchicine-induced genome duplication produced DMRs; the former mainly yielded DMRs at non-CG contexts, whereas the latter affected differential gene body methylation. Spontaneous genome doubling of haploid plants also induced DMRs in greater numbers than self-propagation. Our results provide the first evidence that haploid induction and genome doubling result in differential DNA methylation, offering a novel approach to induce epialleles.

In Vitro Doubled Haploid Production of Bacterial Blight Resistant Plants from BC2F1 Plants (Ranbir Basmati X Pau148) Through Anther Culture

Doubled haploid plants are very important for the development of complete homozygous plants from heterozygous parents in one generation as they possess duplicate copy of haploid chromosome. Haploid production is easily obtained from in vitro anther culture. The present study was undertaken with the objective to develop doubled haploids using anthers for in vitro induction of callus on N6 medium supplemented with various combinations and concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5-2.5 mg/L), Kinetin (0.5-1.0 mg/L) and Naphthalene acetic acid (NAA) (2.0 mg/L) as callus induction medium (CIM). The highest callus induction frequency was obtained when N6 medium fortified with 2,4-D (2.5 mg/L), Kinetin (0.5 mg/L) and NAA (2 mg/L) of 10.07 per cent. The induced callus was sub cultured for shoot regeneration on Murashige and Skoog medium (MS) supplemented with growth regulators: Kinetin and NAA (0.5 mg/L each) in combination with BAP (0.0 - 2.5 mg/L). MS medium supplemented with NAA (0.5 mg/L), Kinetin (0.5 mg/L) and BAP (1.5 mg/L) was most responsive exhibiting regeneration frequency of 28.1 per cent which resulted in maximum regeneration of green plantlets and only 5.21 per cent of albinos. Individual plantlets were separated and immersed in liquid MS medium augmented with NAA (0.5-1.0 mg/L) and BAP (0.5-1.0 mg/L). Maximum rooting was observed in MS medium with NAA (0.5 mg/L) and BAP (1.0 mg/L). The survival rate of in-vitro raised plants was 51.51 per cent. Of these surviving plants, 21 plants were observed to have the sterility percentage above 50 percent and hence can be considered as the doubled haploid plants. Plant DH8 is susceptible and DH20 is heterozygous for gene Xa21. Two plants are susceptible for gene xa13

The Recent Development in Technologies for Attaining Doubled Haploid Plants In Vivo

Haploid plants with a doubled set of chromosomes (doubled haploid (DH)) significantly speed up the selection process by the fixation of genetic traits in each locus in the homozygous state within one generation. Doubled haploids are mainly attained by the formation of plants from the cultured gametophytic (haploid) tissues and cells in vitro, or by targeted reduction in the parent chromosome during intra- or interspecific hybridization. Since then, DH has become one of the most powerful tools to support various basic research studies, as well as applied research. This review is focused on the recent development of the production of doubled haploids in vivo and their fundamental bases. The various mechanisms and approaches responsible for the formation of haploids in vivo are discussed, particularly the induction of parthenogenesis by BBM-like proteins, the long constructed Salmon system of wheat, the usage of patatin-like phospholipases MTL/PLA1/NLD, the IG1 system, uniparental genome elimination during interspecific hybridization, and the perspective technology of centromeric histone 3 (CENH3) modification.

Haploid induction by nanobody-targeted ubiquitin-proteasome-based degradation of EYFP-tagged CENH3 in Arabidopsis thaliana.

The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox, we tested whether targeted in vivo degradation of CENH3 protein can be harnessed to generate haploids in Arabidopsis thaliana. We show that a recombinant anti-GFP nanobody fused to either heterologous F-box (NSlmb) or SPOP/BTB ligase proteins can recognize maternally derived enhanced yellow fluorescent protein (EYFP)-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the genomic CENH3-EYFP-complemented cenh3.1 mother with plants expressing the GFP-nanobody-targeted E3 ubiquitin ligase resulted in a haploid frequency of up to 7.6% in pooled F1 seeds. EYFP-CENH3 degradation occurred independently in embryo and endosperm cells. In reciprocal crosses, no haploid induction occurred. We propose that the uniparental degradation of EYFP-fused genomic CENH3 during early embryogenesis leads to a decrease in its level at centromeres and subsequently weakens the centromeres. The male-derived wild type CENH3 containing centromere outcompetes the CENH3-EYFP depleted centromere. Consequently, maternal chromosomes undergo elimination, resulting in haploids.