Doubled Haploid Plants Research Articles

Spike morphology alternations in androgenic progeny of hexaploid triticale (\xd7 Triticosecale Wittmack) caused by nullisomy of 2R and 5R chromosomes

Induction of androgenesis, followed by chromosome doubling, is a crucial method to obtain complete homozygosity in one-generation route. However, in vitro androgenesis can result in various genetic and epigenetic changes in derived triticale plants. In this study, we evaluated chromosome alternations and we associated them with the changes of spike morphology in androgenic progeny of triticale. We karyotyped offspring plants that derived from double haploid plants using fluorescence in situ hybridization techniques. We distinguished four major groups of karyotypes: double ditelosomics, nullisomics N2R, nullisomics N5R, and triticale plants with a complete set of chromosomes. It is known that more than half of QTLs connected with androgenic response are located in R-genome of triticale but 2R, 5R, and 6R chromosomes are not included. We hypothesized that the reason why only aberrations of chromosomes 2R and 5R appear during androgenesis of triticale is that because these chromosomes are not involved in the stimulation of androgenic response and the following regeneration of plants is not disrupted. Concerning the established groups, we evaluated following quantitative traits: spike length, number of spikes per plant, number of spikelets per spike, and number of grains per spike. The nullisomy of chromosome 2R and 5R resulted in vast changes in spike architecture of triticale plants, which can be correlated with the location of major QTLs for spike morphology traits on these chromosomes. The spikes of nullisomic plants had significantly decreased spike length which correlated with the reduction of number of spikelets per spike and number of grains per spike.

Pseudo-chromosome-length genome assembly of a double haploid "Bartlett" pear (Pyrus communis L.).

BackgroundWe report an improved assembly and scaffolding of the European pear (Pyrus communis L.) genome (referred to as BartlettDHv2.0), obtained using a combination of Pacific Biosciences RSII long-read sequencing, Bionano optical mapping, chromatin interaction capture (Hi-C), and genetic mapping. The sample selected for sequencing is a double haploid derived from the same “Bartlett” reference pear that was previously sequenced. Sequencing of di-haploid plants makes assembly more tractable in highly heterozygous species such as P. communis.FindingsA total of 496.9 Mb corresponding to 97% of the estimated genome size were assembled into 494 scaffolds. Hi-C data and a high-density genetic map allowed us to anchor and orient 87% of the sequence on the 17 pear chromosomes. Approximately 50% (247 Mb) of the genome consists of repetitive sequences. Gene annotation confirmed the presence of 37,445 protein-coding genes, which is 13% fewer than previously predicted.ConclusionsWe showed that the use of a doubled-haploid plant is an effective solution to the problems presented by high levels of heterozygosity and duplication for the generation of high-quality genome assemblies. We present a high-quality chromosome-scale assembly of the European pear Pyrus communis and demostrate its high degree of synteny with the genomes of Malus x Domestica and Pyrus x bretschneideri.

The economic benefits of the production of double haploid for selection of white cabbage

An important task of any state in the world is to ensure food security. The Russian Federation is the third largest cabbage producer in the world. In the conditions of the modern market, for ensuring successful competitiveness, selection must promptly respond to consumer requests. To reduce some stages of the selection process, we used the modern biotechnological method of culture of isolated microspores in vitro. This method allows one to get homozygous lines in the first generation. In the Federal Scientific Center for Vegetable Growing, the protocol for obtaining doubled haploid cabbage cultures has been optimized and successfully applied in practice. However, for expediency of implementation of the obtained lines in the selection and seed-growing process, it is important for us to know the economic efficiency of production. The paper presents the data on the basic costs of obtaining lines of white cabbage with traditional and modern biotechnological methods of selection. The article provides a comparative assessment of their cost-effectiveness in the selection of white cabbage. It is shown that the use of the biotechnological method in combination with the classical method of selection allows to obtain pure lines with 100% homozygosity for 3 years. At the same time, economic costs of producing lines, when using doubled haploid plants of white cabbage, are reduced in 2 times.

Economic efficiency of obtaining carrot lines using classical and biotechnological methods

Garden carrot is a traditional food in Russia. Based on the consumption rate of 10 kg per year per person, 1,469 thousand tons of carrots per year must be produced for the population of the Russian Federation. To meet the needs of the population in fresh and processed carrot products, the development of new highly productive and resistant varieties and hybrids is necessary. The traditional production of linear material and hybrids in the breeding of garden carrots is a laborious, lengthy process due to the 2-year cycle of plant development, self-incompatibility, and inbreeding depression. On average, 16-20 years can be spent on creating a hybrid of carrots with the successful course of the selection process. Using biotechnological methods, completely homozygous lines can be obtained in 1 year and a wide variety of starting material can be created for inclusion in the breeding process. The article presents an analysis of economic efficiency and a comparative assessment of the production of garden carrot lines using classical breeding and biotechnology methods. Calculations are based on the rates being used at the Federal Scientific Center for Vegetable Growing. It is shown that the economic costs of obtaining lines when using doubled haploid plants of the garden carrot are reduced in almost five times. To increase the efficiency of the breeding process, biotechnological methods need to be embedded in classical breeding schemes.

Utilization of in Vitro Anther Culture in Spelt Wheat Breeding.

The efficiency of in vitro anther culture was screened in a full diallel population of four spelt wheat genotypes and ten F1 hybrids. Genotype dependency was observed based on the data of embryo-like structures (ELS), green-, albino plantlets. In the diallel population and ten F1 hybrids, the green plantlets production ranged from 13.75 to 85.00 and from 6.30 to 51.00, respectively. The anther culture-derived plants of F1 hybrids were grown up in the nursery. At the harvest, 436 spontaneous doubled haploid (DH) plants were identified among the 1535 anther culture-derived transplanted and grown up individual plants. The mean of spontaneous rediploidization was 28.4% which ranged from 9.76% to 54.24%. In two consecutive years, the agronomic values of ‘Tonkoly.pop1’ advanced line were compared with seven DH lines of ‘Tonkoly.pop1’ in the nursery. The DH lines achieved competitive values in comparison with ‘Tonkoly.pop1’ advanced line based on the 11 measured parameters (heading date, plant height, yield, hardness, width and length of seed, TKW, hulling yield, flour yield, protein and wet gluten content). These observations presage the efficient utilization of anther culture in spelt wheat breeding.

CRISPR/Cas9-Mediated Targeted Mutagenesis in Wheat Doubled Haploids.

CRISPR/Cas9-based genome editing technology has the potential to revolutionize agriculture, but many plant species and/or genotypes are recalcitrant to conventional transformation methods. Additionally, the long generation time of crop plants poses a significant obstacle to effective application of gene editing technology, as it takes a long time to produce modified homozygous genotypes. The haploid single-celled microspores are an attractive target for gene editing experiments, as they enable generation of homozygous doubled haploid mutants in one generation. Here, we describe optimized methods for genome editing of haploid wheat microspores and production of doubled haploid plants by microspore culture.

Integrating marker-assisted selection and doubled haploidy for rapid introgression of semi-dwarfing and blast resistance genes into a Basmati rice variety ‘Ranbir Basmati’

Ranbir Basmati, an early maturing pure line selection of traditional Basmati 370, is widely cultivated Basmati variety in the Indian state of Jammu and Kashmir. The variety is tall statured, prone to lodging and highly susceptible to rice blast. The present study was carried out for genetic improvement of Ranbir Basmati for semi dwarf stature and resistance to blast by introgressing semi-dwarfing gene, sd1, and blast resistance gene, Pi9 from an improved Basmati rice variety, Pusa Basmati 1637 (PB 1637).The selfed progenies of two superior BC2F1 recombinants of the cross Ranbir Basmati/PB 1637 showing maximum recovery of recurrent parent genome and phenome were analyzed with gene-based markers to select plants homozygous for both Pi9 and sd1. These plants were processed via anther culture to produce homozygous doubled haploid (DH) lines. All the anther culture derived DH plants were short-statured compared to Ranbir Basmati and exhibited high level of resistance to blast. The analysis of yield and its component traits revealed the presence of superior transgressive segregants for various traits namely, effective tillers, grains per panicle and yield per plant. Combined use of marker-assisted selection and anther culture ensured speedy conversion of Ranbir Basmati into semi-dwarf genotypes with in-built resistance to rice blast. The study demonstrated the utility of anther culture as an adjunct to marker-assisted backcross breeding schemes for instant elimination of the residual background heterozygosity of the selected recombinants.

Accelerating hybrid rice breeding through the adoption of doubled haploid technology for R-line development

Hybrid rice holds significant yield advantage over inbred lines, but its adoption rate is very slow in India. Development of cytoplasmic male sterile line (CMS) and fertility restorer line with superior grain quality is the prerequisite in three line hybrid rice breeding programme. Minimum of three to four years are required for development of useful R-line which severely delays the pace of hybrid breeding. Application of doubled haploid (DH) technology will be very useful to develop elite R-lines within short period of time. An elite inbred line LAXMI was crossed with the R-line RPR158 and anthers from the resulting F1 population was used for development of DH plants. Eight hormonal combinations and four different cold pre-treatment conditions were tried in tissue culture. Maximum callusing frequency was observed in N6 medium supplemented with 2.0 mg L−1 2, 4-diclorophenoxyacetic acid (2,4-D), 0.5 mg L−1 napthalene acetic acid (NAA), 0.5 mg L−1 benzyl amino purine(BAP) and 6% maltose with 3 days cold pre-treatment. Highest green plant regeneration frequency was found with Murashige and Skoog medium supplemented with 0.5 mg L−1 NAA, 0.5 mg L−1 kinetin, 2 mg L−1 BAP. A total of 280 DH plants were generated and evaluated under field conditions for agronomic characters and fertility restoration capacity in CMS system. Two distinct DH lines were identified as improved R-lines based on high restoration ability (94%), superior grain quality and medium slender grain type. This study successfully demonstrated that superior R-lines can be developed within two crop seasons through the application of DH technology.

Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus

Abstract Microspore embryogenesis is a model system of plant cell reprogramming, totipotency acquisition, stress response and embryogenesis initiation. This in vitro system constitutes an important biotechnological tool for haploid and doubled-haploid plant production, very useful for crop breeding. In this process, microspores (cells that produce pollen grains in planta) are reprogrammed toward embryogenesis by specific stress treatment, but many microspores die after the stress. The occurrence of cell death is a serious limiting problem that greatly reduces microspore embryogenesis yield. In animals, increasing evidence has revealed caspase proteolytic activities as essential executioners of programmed cell death (PCD) processes, however, less is known in plants. Although plant genomes do not contain caspase homologues, caspase-like proteolytic activities have been detected in many plant PCD processes. In the present study, we have analysed caspase 3-like activity and its involvement in stress-induced cell death during initial stages of microspore embryogenesis of Brassica napus. After stress treatment to induce embryogenesis, isolated microspore cultures showed high levels of cell death and caspase 3-like proteolytic activity was induced. Treatments with specific inhibitor of caspase 3-like activity reduced cell death and increased embryogenesis induction efficiency. Our findings indicate the involvement of proteases with caspase 3-like activity in the initiation and/or execution of cell death at early microspore embryogenesis in B. napus, giving new insights into the pathways of stress-induced cell death in plants and opening a new way to improve in vitro embryogenesis efficiency by using chemical modulators of cell death proteases.

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm3 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm3 kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement.

Under stress, isolated microspores are reprogrammed in vitro towards embryogenesis, producing doubled haploid plants that are useful biotechnological tools in plant breeding as a source of new genetic variability, fixed in homozygous plants in only one generation. Stress-induced cell death and low rates of cell reprogramming are major factors that reduce yield. Knowledge gained in recent years has revealed that initiation and progression of microspore embryogenesis involve a complex network of factors, whose roles are not yet well understood. Here, I review recent findings on the determinant factors underlying stress-induced microspore embryogenesis, focusing on the role of autophagy, cell death, auxin, chromatin modifications, and the cell wall. Autophagy and cell death proteases are crucial players in the response to stress, while cell reprogramming and acquisition of totipotency are regulated by hormonal and epigenetic mechanisms. Auxin biosynthesis, transport, and action are required for microspore embryogenesis. Initial stages involve DNA hypomethylation, H3K9 demethylation, and H3/H4 acetylation. Cell wall remodelling, with pectin de-methylesterification and arabinogalactan protein expression, is necessary for embryo development. Recent reports show that treatments with small modulators of autophagy, proteases, and epigenetic marks reduce cell death and enhance embryogenesis initiation in several crops, opening up new possibilities for improving in vitro embryo production in breeding programmes.

Line × tester analyses for anther culture response of bread wheat (Triticum aestivum L.)

The aim of this study was to investigate the gene effects that play a role in the inheritance of the anther culture response of the F1 and F2 populations obtained from the hybridization between the seven doubled haploid (DH) wheat line and four bread wheat cultivar using line × tester method as well as, to determine suitable parents and promising hybrids. Eleven parents and their twenty-eight hybrid combinations were examined for androgenic properties related to anther culture response such as the number of callus, green, albino, haploid, spontaneous DH and fertile plants. The obtained data were evaluated by the combining ability analysis calculated using the line × tester method and by estimating the heritability degrees. According to the results, for all traits examined, the non-additive gene effects were found as significant and narrow sense heritability at a low level. Significant combining ability effects have been identified for some parents and hybrids regarding traits examined. DH19, DH22, and Harmankaya-99 have been identified as suitable parents to develop the androgenic traits, while crosses formed between them have been determined as promising. A total of 444 plants (142 F1, 302 F2) were transferred to the greenhouse from the plants obtained from the anther culture, and 205 (68 in F1, 137 in F2) were fertile. Ninety-three of the fertile plants were obtained from the parents. The seeds of 112 DH plants obtained from hybrids were replicated to be used in breeding programs.

Anther Culture in Potato (Solanum tuberosum L.) in vitro

Double haploid (DH) plants Production is a valuable tool in plant breeding programs. Since conventional breeding takes long time for the development of newly improved cultivars, this technique reduces the time needed for this purpose. Practically, application of (DH) technology in potato breeding through androgenesis in Santana cultivar is possible by considering number of factors that influence androgenesis. Culture medium has to be considered in respect of constituents to fulfill the needs of culture target where N6 medium significantly showed better results (14.61%) if compared to MS medium (8.78%) anthers produced embryos. Auxin/cytokinin represented in this study by 2,4-D/BA showed better results in induction of embryogenesis when combined together at (2.0 mg/l 2,4-D and 0.5 mg/l BA) which resulted in (35.67%) anthers produced embryos rather than each one of them alone. Enhanced results were obtained after floral buds pretreatment with thermal chock with three different degrees (4°C, 25°C, 30°C) for two different exposure time (48 h and 72 h). N6 medium showed the highest percentage of anthers produced embryos (44%) when supplemented with 2 mg/l 2,4-D+0.5 mg/l BA, while pretreatment with 4°C for 72 h favored the embryos production (16.67%) followed by (14.00%) and (12.00%) at 25°C and 4°C for 48 h respectively with no significant difference among them. Pretreatment with 32°C for (48 h and 72 h) resulted in (4.67%) and (5.33%) respectively differed significantly form the low temperature. Silver nitrate has shown to be potent at 2 mg/l in inhibiting ethylene, increasing embryogenesis (60.0 %) and reducing the non-responsive anthers (6.67 %).

State-of-the-art and novel developments of in vivo haploid technologies

The ability to generate (doubled) haploid plants significantly accelerates the crop breeding process. Haploids have been induced mainly through the generation of plants from cultivated gametophic (haploid) cells and tissues, i.e., in vitro haploid technologies, or through the selective loss of a parental chromosome set upon inter- or intraspecific hybridization. Here, we focus our review on the mechanisms responsible for the in vivo formation of haploids in the context of inter- and intraspecific hybridization. The application of a modified CENH3 for uniparental genome elimination, the IG1 system used for paternal as well as the BBM-like and the patatin-like phospholipase essential for maternal haploidy induction are discussed in detail.

CYTOLOGICAL ASSESSMENT OF DOUBLED HAPLOIDS IN SUMMER SQUASH (CUCURBITA PEPO L.)

147 new forms were obtained only from nine responsive unpollinated ovules of summer squash in 2015-2017. The cytological analysis is used to estimate the level of ploidy in regenerated plants R0. The small size of mitotic chromosomes and large number in Cucurbita genus make it difficult to count them even though they are well separated. As a result of analysis the optimal method of chromosome staining has been chosen with the use of modified propiono- lacmoid cytological technique. The rootlet tips and apical meristems were used to make smear preparations. As it was shown the summer squash was a difficult species as a cytological object because of low mitosis frequency and low number of metaphase plates with well scattered chromosomes. The photos of chromosomes in squash C. pepo subsp. brevicaulis var. giraumons Duch; distant hybrid (breeding accession N37) between variety ‘Cornishon’ and winter squash C. pepo subsp. longicaulis Greb. var. pepo and doubled haploid plants produced from them were made. In spite of the small size 2 μm the chromosomes observed were clearly seen. Nearly all regenerated plants that had been analyzed passed well the adaptation in vivo and occurred to be a doubled haploids (2n = 2x=40). The seed progeny was then obtained through self-pollination. About 20% of plants R0 analyzed were mixploids and supposedly only 7% were haploids.

Barley Anther Culture.

The production of doubled haploid (DH) barley plants through anther culture is a very useful yet simple in vitro technique. DH plants derive from divisions of haploid microspores that have undergone a developmental switch under the appropriate conditions. The successive divisions lead to the formation of an embryo or callus rather than the formation of mature pollen grains. Plants that regenerate from these embryos are often either haploid, in which case their chromosome set can be doubled by treatment with colchicine, or spontaneous double haploids. The efficiency of DH plant production is highly variable depending on the genotype of the source material. Despite this limitation, DH plants have been widely used in breeding and research programs. Compared to conventional approaches, breeding strategies that makes use of DH plants achieve a homozygous state, allowing transgene or mutation stabilization in the genome, within a considerably shorter time, thus accelerating workflow or reducing work volume.

Isolated Microspore Culture in Barley.

Isolated microspore culture (IMC) is the most efficient way to produce large numbers of doubled-haploid (DH) barley plants in a short time. Yet, while IMC is more cost-efficient and less labor-intensive than anther culture, it is technically more complex and requires more experienced personnel if it is to yield its full potential. In part, this is because of multiple and important interactions that exist between factors at its many different phases, including genotype effects as well. When every phase is fine-tuned, the protocol that is presented below yields a useful number of DHs with almost all genotypes and can allow the production of up to 300 DH plants from a single F1 plant in just a few months.

Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.

Improved chromosome doubling of parthenogenetic haploid plants of cucumber (Cucumis sativus L.) using colchicine, trifluralin, and oryzalin

An effective chromosome doubling protocol was established in essential garden crop of cucumber (Cucumis sativus L.) Cv. Hi Power. The different concentrations of colchicine (0, 250, 500, 750, and 1500 mg/L), oryzalin (0, 5, 15, 25, 50, 75, and 150 mg/L) and trifluralin (0, 5, 15, 25, 50, 75, and 150 mg/L) were applied on parthenogenesis-induced haploid nodal and shoot tip explants of cucumber for 18 and 38 h in three independent factorial experiments. Increasing concentrations of applied antimitotic agents led to the significant reduction in the survival rate of both shoot tip and nodal explants, especially in longer exposure duration. Three ploidy levels including haploid, mixoploid, and doubled haploid were regenerated form both explant types treated with colchicine, oryzalin, and trifluralin. Flow cytometry analysis proved successful chromosome doubling of haploid plants. Based on the results obtained, the highest number of regenerated doubled haploid plants (92.31%) and fruit set (86.21%) were related to immersion of nodal explants in 50 mg/L oryzalin for 18 h. The highest doubled haploid regeneration for colchicine and trifluralin antimitotic agents were 58.33 and 83.33%, respectively. The leaf size of doubled haploid plants was larger than their correspond haploids. The optimized chromosome doubling protocol would be applicable for doubled haploid production in garden crops of Cucurbitaceae family, which is recalcitrant to the spontaneous doubling, and also for in vitro polyploidy induction studies.

Regeneration of highland papaya (Vasconcellea pubescens) from anther culture.

Premise of the Study Vasconcellea pubescens is an important Caricaceae species cultivated in several countries of South America. The objective of this study was to investigate different media compositions and plant growth regulators to induce plant regeneration.MethodsAnthers were cultured in Murashige and Skoog medium with varying concentrations of naphthalene acetic acid (NAA) and 2,4‐dichlorophenoxyacetic acid (2,4‐D) plus a cytokinin (N‐(2‐chloro‐4‐pyridyl)‐N′‐phenylurea). The effect of the basal medium supplemented with auxins and cytokinins on shoot regeneration from the induced calli was also evaluated. Addition of maltose to the basal medium was also tested.ResultsThe combination of 0.54 μM NAA and 22.66 μM 2,4‐D induced the highest rate of calli formation. Regeneration via organogenesis was obtained in Murashige and Skoog and Woody Plant Medium supplemented with maltose and containing 8.88 μM 6‐benzylaminopurine, 5.71 μM indoleacetic acid, and 2.28 μM zeatin.DiscussionThe plant regeneration protocol reported here permits the development of haploid and double haploid plants that can be useful for propagation purposes, allow a better molecular understanding of the species, and facilitate the production of new cultivars.