Haploid Plants Research Articles

Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey

Legumes are an alternative to animal foods due to their high protein content. Like other legumes, Vicia faba (faba bean) has high protein content. However, faba bean breeding with classical methods is challenging due to inbreeding depression, self-incompatibility, and abortive embryo formation. The inadequacy of classical breeding methods due to the problems in the fertilization biology of the plant limits the production of new varieties. Therefore, the importance of using in vitro haploidization technique in legume breeding is increasing. Anther culture is a widely preferred tissue culture technique for obtaining haploid plants. The initial and most crucial stage in anther culture is the identification of anthers containing microspores in the appropriate developmental stage. In the study, anther samples with single and triple-nucleated microspores were cultured in MS nutrient medium containing 2,4-D (0.5 mg L-1) and Kinetin (2.5 mg L-1). In the first month of culture, embryo and embryoid-like structures were obtained at the heart stage from anthers containing microspores in the mononuclear stage, while anther samples containing microspores in the trinucleated stage were observed to darken and did not develop.

Doublehaploidization Efficiency of Selected Pepper Genotypes Via in Vitro Anther Culture

This study was carried out to determine the effects of genotype and media on haploid plant formation of 12 pepper genotypes (Capsicum annuum L.) via in vitro anther culture. The buds were cultured in 2 different media (O1 and O2) and, the results revealed that the media had effects on the development of anthers, embryo and plantlet initiation. When the anther developmental status is examined, the lowest value was obtained from the SU-31 genotype with 2.47%, while cv. Flinta F1 produced the highest rate with 12.79%. The highest growth rates were obtained from the O2 medium, and the cv. Dolphin variety produced a remarkable result with 25.0% in this medium. The cultivar Dolphin was the favorable genotypes with 8 embryos and 5 plants, while no embryo and plant were obtained from cv. Flinta F1 and SU-34. A total of 2868 anthers were cultured, 195 anthers were enlarged and the growth rate was 6.8%. Finally, 37 embryos and 18 plants were obtained and the frequencies were 1.29% and 0.63%, respectively. As a result of stomatal observations, 13 plants were detected haploid and the others were double haploid.

Triticale doubled haploid plant regeneration factors linked by structural equation modeling

Triticale regeneration via anther culture faces many difficulties, e.g., a low percentage of regenerated plants and the presence of albinos. Plant regeneration may be affected by abiotic stresses and by ingredients added to the induction medium. The latter influences biochemical pathways and plant regeneration efficiency. Among such ingredients, copper and silver ions acting as cofactors for enzymatic reactions are of interest. However, their role in plant tissue cultures and relationships with biochemical pathways has not been studied yet.The study evaluated relationships between DNA methylation, changes in DNA sequence variation, and green plant regeneration efficiency influenced by copper and silver ions during triticale plant regeneration. For this purpose, a biological model based on donor plants and their regenerants, a methylation-sensitive amplified fragment length polymorphism, and structural equation modeling were employed.The green plant regeneration efficiency varied from 0.71 to 6.06 green plants per 100 plated anthers. The values for the components of tissue culture-induced variation related to cytosine methylation in a CHH sequence context (where H is A, C, or T) were 8.65% for sequence variation, 0.76% for DNA demethylation, and 0.58% for de novo methylation. The proposed model states that copper ions affect the regeneration efficiency through cytosine methylation and may induce mutations through, e.g., oxidative processes, which may interfere with the green plant regeneration efficiency. The linear regression confirms that the plant regeneration efficiency rises with increasing copper ion concentration in the absence of Ag ions in the induction medium. The least absolute shrinkage and selection operator regression shows that de novo methylation, demethylation, and copper ions may be involved in the green plant regeneration efficiency. According to structural equation modeling, copper ions play a central role in the model determining the regeneration efficiency.

Polycomb-mediated repression of paternal chromosomes maintains haploid dosage in diploid embryos of Marchantia.

Complex mechanisms regulate gene dosage throughout eukaryotic life cycles. Mechanisms controlling gene dosage have been extensively studied in animals, however it is unknown how generalizable these mechanisms are to diverse eukaryotes. Here, we use the haploid plant Marchantia polymorpha to assess gene dosage control in its short-lived diploid embryo. We show that throughout embryogenesis, paternal chromosomes are repressed resulting in functional haploidy. The paternal genome is targeted for genomic imprinting by the Polycomb mark H3K27me3 starting at fertilization, rendering the maternal genome in control of embryogenesis. Maintaining haploid gene dosage by this new form of imprinting is essential for embryonic development. Our findings illustrate how haploid-dominant species can regulate gene dosage through paternal chromosome inactivation and initiates the exploration of the link between life cycle history and gene dosage in a broader range of organisms.

Assessment of molecular diversity in doubled haploid lines of camelina (Camelina sativa(L.) Crantz), as a new emerging oil crop

AbstractCamelina (Camelina sativa(L.) Crantz), an oilseed crop, belongs to the Brassicaceae family. Two unique features of camelina in comparison with the main oil crops are an adaptation to different environments and also its unique oil composition. The development of doubled haploid plants is one of the essential methods for crop improvement. The study of genetic diversity is an important step in planning crop breeding programmes. This research was conducted to evaluate the genetic variation of 81 camelina doubled haploid lines obtained from 15 crosses by inter simple sequence repeat (ISSR) markers. The total number of amplified bands was 243, of which 239 bands (98.3%) showed polymorphism. The percentage of polymorphic bands varied between 93.75 and 100. The size of the bands ranged from 50 to 1700 base pairs. The informative ISSRs were identified by estimating marker features: polymorphism information content, effective multiplex ratio, marker index and resolving power. Three markers had higher resolving power values (9.88, 8.5 and 7.46) and were the most informative markers to identify the lines. Cluster analysis based on the complete algorithm divided the lines into five groups, indicating relatively clear configuration from the geographic distribution patterns of the parents of the doubled haploid lines. Principal coordinate analysis classified the 81 camelina doubled haploid lines into six groups. The ISSR markers detected high polymorphism to reveal the genetic variation of camelina lines. The findings of this research, along with the characterization of biochemical traits of the lines, can improve breeding programmes achieve high-yielding camelina varieties with higher and better oil content.

Recurrent haploid selection in a population of sweet corn (Zea mays convar. saccharata)

AbstractRecurrent haploid selection is based on the phenotype of haploid plants. This selection method was reported to be very effective because of the high selection pressure. The aim of this study was to quantify the effect of five generations of recurrent haploid selection on a sweet corn population developed by intercrossing 40 hybrid cultivars. We compared the initial population, five cycles of recurrent haploid selection, and mass selection after five cycles in six environments. The results indicated that recurrent haploid selection had no positive effect on yield or quality. For the traits tillering, tip fill, total yield and total number of ears, significant changes from cycle to cycle were observed but in differing directions. The performance per se of single haploid plants has probably a low correlation to their combining ability in a population. Positive mass selection of the best ears resulted in no improvement of total yield, a reduction of marketable yield and about 2‐day delay of flowering. In conclusion, recurrent haploid selection based on the phenotype of individual plants was not an efficient method to improve the sweet corn population investigated.

Controlled Induction of Parthenogenesis in Transgenic Rice via Post-translational Activation of PsASGR-BBML.

Modern plant breeding programs rely heavily on the generation of homozygous lines, with the traditional process requiring the inbreeding of a heterozygous cross for five to six generations. Doubled haploid (DH) technology, a process of generating haploid plants from an initial heterozygote, followed by chromosome doubling, reduces the process to two generations. Currently established in vitro methods of haploid induction include androgenesis and gynogenesis, while in vivo methods are based on uni-parental genome elimination. Parthenogenesis, embryogenesis from unfertilized egg cells, presents another potential method of haploid induction. PsASGR-BABY BOOM-like, an AP2 transcription factor, induces parthenogenesis in a natural apomictic species, Pennisetum squamulatum (Cenchrus squamulatus) and PsASGR-BBML transgenes promote parthenogenesis in several crop plants, including rice, maize, and pearl millet. The dominant nature of PsASGR-BBML transgenes impedes their use in DH technology. Using a glucocorticoid-based post-translational regulation system and watering with a 100 μM DEX solution before anthesis, PsASGR-BBML can be regulated at the flowering stage to promote parthenogenesis. Conditional expression presents a novel opportunity to use parthenogenetic genes in DH production technology and to elucidate the molecular mechanism underlying parthenogenetic embryogenesis.

An Overview of Haploid and Double Haploid Production Methods in Wheat

For about a century, researchers have been working on haploidy approach. Progressively, they discovered the importance and usefulness of haploids in various research fields. On the other hand, it is suggested that climate change impacts on crop production, especially wheat, requires rapid and efficient methods of plant breeding to produce new cultivars with a sufficient level of biotic and abiotic tolerance to avoid significant production loss. Haploid plants are the source for producing homozygous pure lines and genetic variability for breeding programs. It reduces the time for producing pure and stable forms of new recombination by half in plant breeding. Furthermore, haploids are source for exclusive male plants generation, induction of mutations, stress resistance and tolerance cultivars, cytogenetic studies, and doubled haploid crops. This review presents a brief overview of the haploid wheat production methods and previous successful experiments on producing haploid wheat.

Комбинационная способность линий кукурузы и генетический контроль морфометрических параметров

The article presents the results of studying the combination ability of new dihaploid maize lines created by diploidization of haploid plants obtained using a haploinducer line. The experiment involved simple hybrids (30 combinations) obtained according to the complete diallel scheme of 6 homozygous lines. The effects of GCA and variance of SCA lines, as well as the components of genetic variance in terms of the main morphometric parameters of the plant (stem length, ear height, panicle length, stem diameter, plant leaf surface area) were revealed. Lines with a high GCA score and a dispersion of SCA have been identified, which are advisable to use in the breeding process for tall stature, resistance to lodging, and manufacturability. The influence of overdominance in the manifestation of the length of the panicle and the diameter of the stem and the predominance of additive effects in the formation of the height of the cob were noted. Analysis of the components made it possible to identify the number of genes or groups of genes that influenced the manifestation of traits in 2021 (stem length – 1–2, ear height – 2–3, panicle length – 1–2, stem diameter – 2–3, area of leaf surface – 1–2). Dominance according to the studied parameters is directed towards parental forms with a greater severity of the trait. A significant influence of cultivation conditions on the manifestation of the length of the stem, the height of the cob, the length of the panicle, and the diameter of the stem was noted.

Genotype Effect as One of the Affecting Factors on The Success of Anther Culture in Eggplant (Solanum melongena L.)

Anther culture technique is one of the commonly used method in order to obtain haploid plants in eggplant. It is known that androgenic response in eggplant is variable and one of the most important factors determining success is the genotype effect. Low or insufficient embryogenic response limits the use of double haploid technology in breeding programs. Therefore, determining the embryogenic responses of genotypes is a step for the use of this technology in breeding studies. In this study, 61 eggplant genotypes at various breeding stages (F1, F2, and F3) in the gene pool of United Genetics Vegetable Seeds Company (Mustafakemalpaşa, Bursa, Turkey) were evaluated in terms of androgenic responses. Significant differences were determined among eggplant genotypes in terms about embryo induction and conversion rates of embryos to full plantlets. In the study, responsiveness to anther culture based on genotype ranged from 0.0%-21.58%. The conversion rate of induced embryos to plants varied between 0.0% and 100%.

Enhancement of Androgenesis and Plant Regeneration From Wheat Anther Culture by Seed Pre-Sowing Gamma Irradiation

Combination breeding and mutation breeding are widely used methods in plant breeding. Intensive studies are carried out on biotechnological methods that will allow obtaining homozygous lines in a short time in populations obtained with these two techniques. Numerous studies have been carried out by different researchers on mutation breeding in wheat. There are few studies on anther culture response in mutagen-treated genotypes. Two different bread wheat promising advanced lines were used as the material in the study, in which the possibilities of combining mutation breeding with anther culture through ionizing radiation in high quality wheat breeding were investigated. In the study, the responses of advanced bread wheat mutant lines to anther culture, to which eight different doses of gamma rays (0, 100, 150, 200, 250, 300, 350, 400 Gy) were applied, including the control, were investigated. There are significant differences between genotypes and irradiation doses for all traits studied. It has been shown that it is possible to decrease albinism and increase the response of anther culture with dose-dependent gamma irradiation depending on bread wheat varieties. In the multiple comparison test to classify the difference between doses; statistically, 150 gray dose 5.60 is in the first statistical class and in the first place with the number of transferred green plants. After that, it ranks second in the same class with the number of green plants transferred with a 300 gray dose of 5.21. In the total number of regenerated green plants excluding controls (888), 635 unit (71.5%) and 205 unit (23.1%) haploids and spontaneous double haploid plants were obtained, respectively. In the study integrated into the bread wheat breeding program, a total of 205 spontaneous double haploid mutant lines were produced. According to the data obtained, it was shown that the gamma ray doses of 150 and 200 Gy had a significant stimulation effect on all parameters studied and ultimately the success index of anther culture in bread wheat compared to control.

High throughput anther culture response in an upland rice cross ‘Khandagiri x Dular’

Aim: The experiment was conducted to maximize anther culture response in an upland indica rice cross amenable for drought tolerance breeding. Methodology: The cold pre-treated anthers of a cross Khandagiri (drought sensitive) x Dular (drought tolerant) were cultured on N6, SK3, MS and CIM media at varying concentration of hormones (2,4-D, Kn and NAA) to assess callusing response. Embryogenic calli were placed on regeneration medium (RM) with varying concentration of Kn, BAP and NAA for plant regeneration. The plantlets were acclimatized in half-strength MS basal liquid medium for one week before transferring to pot mixture (peat moss: perlite 2:1, v/v) for plant establishment in glasshouse under partial shade. Finally, the plantlets were checked for doubled haploid status by cytological study of root tips. Results: Anther culture response was found to be media and genotype-specific. F1-progenies responded better than either of the parents. A modified MS callus induction medium (CIM) with 1.5mg/l 2,4-D + 0.5mg/l Kn resulted impressively higher callusing response (30.2%) with nodular calli than SK3>MS>N6. High frequency (12.8%) of albino-free green plant regeneration with well developed rooting was achieved in RM medium (a modified MS) containing 2mg/l BAP and 0.5mg/l NAA. A total 129 doubled haploid (DH) plants (each with 12 pairs of chromosomes) were recovered which maintained normal growth, set seeds and resulted in true breeding DH lines. Interpretation: The high throughput regeneration system is amenable for doubled haploid production from indica crosses. Besides, the present doubled haploid stock can serve as an ideal mapping population and as such targeted for marker aided selection for early development of drought tolerant rice varieties.

Phylogeographic structure ofSyntrichia caninervisMitt, a xerophytic moss, highlights the expanded during glacial period

AbstractThe rapid uplift of the Qinghai–Tibet Plateau and its aridification has significantly affected the distribution and community structure of the plants in these regions. However, most of the studies have focussed on vascular plants, and it has been unclear whether bryophytes, which are haploid plants, had similar historical population dynamics to other vascular plants during the dramatic geological and climatic environment changes of the Quaternary. This study used Syntrichia caninervis Mitt as the research object and investigated its genetic variation, differentiation and population dynamic history in China. We genotyped 27 populations throughout the distributional range of S. caninervis using two chloroplast DNA regions and 19 nuclear microsatellite loci and supplemented these data with ecological niche modelling of the potential distribution areas from the last interglacial period. The results showed that genetic data consistently identified three clades: the Qinghai–Tibet Plateau, Pamir Plateau–TienShan and Central Asia. The genetic variation of Syntrichia caninervis mainly occurred within populations and in the populations within a specific region. However, there was a significant gene exchange between the different regions. S. caninervis may have expanded during the glacial period and shrank during the interglacial period. This study provides new evidence for the dynamic population history of drought-tolerant bryophytes in response to severe environmental changes during the Quaternary glacial and interglacial cycles.

The mysterious sex chromosomes of haploid plants

The mysterious sex chromosomes of haploid plants

Genome-Wide Survey and Development of the First Microsatellite Markers Database (AnCorDB) in Anemone coronaria L.

Anemone coronaria L. (2n = 2x = 16) is a perennial, allogamous, highly heterozygous plant marketed as a cut flower or in gardens. Due to its large genome size, limited efforts have been made in order to develop species-specific molecular markers. We obtained the first draft genome of the species by Illumina sequencing an androgenetic haploid plant of the commercial line “MISTRAL® Magenta”. The genome assembly was obtained by applying the MEGAHIT pipeline and consisted of 2 × 106 scaffolds. The SciRoKo SSR (Simple Sequence Repeats)-search module identified 401.822 perfect and 188.987 imperfect microsatellites motifs. Following, we developed a user-friendly “Anemone coronaria Microsatellite DataBase” (AnCorDB), which incorporates the Primer3 script, making it possible to design couples of primers for downstream application of the identified SSR markers. Eight genotypes belonging to eight cultivars were used to validate 62 SSRs and a subset of markers was applied for fingerprinting each cultivar, as well as to assess their intra-cultivar variability. The newly developed microsatellite markers will find application in Breeding Rights disputes, developing genetic maps, marker assisted breeding (MAS) strategies, as well as phylogenetic studies.

Overexpression of HVA1 Enhances Drought and Heat Stress Tolerance in Triticum aestivum Doubled Haploid Plants.

Plant responses to multiple environmental stresses include various signaling pathways that allow plant acclimation and survival. Amongst different stresses, drought and heat stress severely affect growth and productivity of wheat. HVA1, a member of the group 3 LEA protein, has been well known to provide protection against drought stress. However, its mechanism of action and its role in other stresses such as heat remain unexplored. In this study, doubled haploid (DH) wheat plants overexpressing the HVA1 gene were analyzed and found to be both drought-and heat stress-tolerant. The transcriptome analysis revealed the upregulation of transcription factors such as DREB and HsfA6 under drought and heat stress, respectively, which contribute toward the tolerance mechanism. Particularly under heat stress conditions, the transgenic plants had a lower oxidative load and showed enhanced yield. The overexpression lines were found to be ABA-sensitive, therefore suggesting the role of HsfA6 in providing heat tolerance via the ABA-mediated pathway. Thus, apart from its known involvement in drought stress, this study highlights the potential role of HVA1 in the heat stress signaling pathway. This can further facilitate the engineering of multiple stress tolerance in crop plants, such as wheat.

Genome-wide characterization of Histone gene family and expression profiling during microspore development in radish (Raphanus sativus L.).

Histone, a predominant protein component of chromatin, participates in DNA packaging and transcriptional regulation. However, the available information of Histone gene family is limited in radish. In this study, a total of 42 Histone gene family members were identified from the radish genome. Sequence alignment and phylogenetic analyses classified the Histone family into three groups (H2A, H2B and H3). Motif analysis showed that the functions of some motifs shared by H3 subfamily genes were related to chromosome regulation and cell development activities, such as motif 5 containing Cks1 and PPR region. Analysis of intron/exon structure indicated that RsCENH3 (RsHistone 18) has the characteristics of variant Histone. Furthermore, several motifs, including the LTR, G-box and TC-elements, were found in the promoters of RsHistone genes, which involved in cell development or various abiotic stresses responses. Transcriptome analysis indicated that the RsHistone genes exhibited higher expression level in floral buds than in roots and leaves. Subcellular localization showed that the RsCENH3 was localized on the nucleus, and it was highly expressed in the floral bud of 3.0-4.0mm in radish. These findings would provide valuable information for characterization and potential utilization of Histone genes, and facilitate the efficient induction of double haploid plants in radish.

Triticale haploidy <i>in vitro</i> (literature review)

Triticale (× Triticosecale Wittmack) is a hybridized grain crop developed from wheat and rye crossings. Today, triticale is a multipurpose commercial grain crop with great potential as a human food and animal feed. The sown area of the new grain crop in the world reached 4 million hectares in 2018, grain production was about 14 million tons. The current climate change, the rapid evolution of pathogens, as well as the requirements of the modern market dictate the necessity for accelerated development of varieties while reducing the cost of their development. The production of double haploids makes it possible to reduce the time required for the development of homozygous lines by an average of 5–7 years. For the mass production of haploid triticale plants in vitro, there are used two methods, namely anther/microspore culture and distant hybridization followed by selective chromosome elimination of the pollinator. The most critical factors for the success of developing haploids in anther culture are a genotype, growing conditions of donor plants, a microspore development stage, stress effects on heads or anthers, and a nutrient media. Among the unresolved problems of the method are a genotypic dependence, a high incidence of albinism and a presence of aneuploids in the androgenic plant progeny. The rye genome is more often involved in chromosomal transformations than the wheat genome. Most aneuploids are nullisomics, most often on the 2R and 5R chromosomes. Nullisomic plants for 2R and 5R chromosomes have fewer number of spikelets per head and fewer number of kernels per head. In order to develop haploids by the method of selective chromosome elimination during distant hybridization, there have been successfully used such grain crops whose pollen is insensitive to Kr-genes, as maize (Zea mays L.) and wild cereal grass ‘Imperata Cylindrical’ (Imperata cylindrical L.). The advantages of the method are less genotypic dependence, absence of albino plants, genetic stability of regenerants, and reduced costs for developing haploid plants. The length of flowering period of ‘Imperata Cylindrical’ and the absence of the need to combine the timing of flowering period of the parents ensure the economy and efficiency of using this species when developing haploid triticale plants. The purpose of the current review was to characterize the methods of mass development of haploid triticale plants, to describe their advantages and disadvantages when being used in the breeding process.

Double Haploids for Vegetable Crop Improvement: A Review

Vegetables are the richest source of vitamins and minerals and play a major role in nutritional security. The primary objective of vegetable breeding is enhancing production and productivity. Breeders use various conventional and non-conventional breeding approaches for genetic advance. Recent advances in biotechnology helps to enhance the efficiency and shorten the time required to achieve the fixed goals in a breeding program, as well as to address the economic and ecological goals. Among these, haploid (H) and doubled haploid (DH) production through gametic embryogenesis has long been recognized as a valuable tool to help crop improvement. Haploid plants are the sporophytes with a gametophytic chromosome number and doubled haploids (DH) are haploids that have undergone chromosome duplication. Haploids and double haploids occur spontaneously in nature. Haploids can also be induced artificially by various techniques such as wide hybridization, pollination with irradiated pollens androgenesis, gynogenesis and finally further chromosome doubling results in double haploids. Production of double haploids in vegetables will shorten the breeding cycle and also DH technology is useful in reverse breeding, CMS line production, gene stacking and various other genetic studies.

Effects of genotype and nutrient medium on obtaining haploid plants through ovary culture in cucumber.

Cucumber is a species that breeding studies for variety development are carried out intensively. However, double haploidy technology, which aims to shorten the breeding process, has not yet reached the desired level. Three induction (M1: Murashige and Skoog (MS), 0.04mg L-1 Thidiazuron (TDZ); M2: MS, 0.15mg L-1 2,4-Dichlorophenoxyacetic acid (2,4-D), 1.5mg L-1 Kinetin; M3: MS, 0.1mg L-1 2,4-D, 1mg L-1 6-Benzylaminopurine (BAP) and one regeneration (MS, 0.2mg L-1 BAP, 0.05mg L-1 1-Naphthaleneacetic acid (NAA) media and 31 cucumber genotypes were used. At the end of study, in terms of embryo formation, M3 (33.41 embryos per 100 cultured ovaria, 99.61 embryos per 100 developed ovaria) and M2 (30.70 embryos per 100 cultured ovaria, 122.05 embryos per 100 developed ovaria) were found to be better than M1 (17.54 embryos per 100 cultured ovaria, 68.34 embryos per 100 developed ovaria). For plant formation, M1 (13.23 plants per 100 embryos) and M2 were found to be more succesful than M3. Ploidy analyses performed on 72 of 98 plants through flow cytrometry showed that obtained plants were various ploidy level (34.72% haploid, 37.5% diploid, 22.22% mixoploid, and 5.55% tetraploid). According to the results of the research, 2,4-D added to the nutrient media seems to be successful in induction of ovary culture in cucumber. In plants determined as diploid according to ploidy analysis, doubled haploid situation should be checked by molecular analysis.