Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.     

Synthesizing double haploid hexaploid wheat populations based on a spontaneous alloploidization process

Doubled haploid (DH) populations are useful to scientists and breeders in both crop improvement and basic research. Current methods of producing DHs usually need in vitro culture for extracting haploids and chemical treatment for chromosome doubling. This report describes a simple method for synthesizing DHs (SynDH) especially for allopolyploid species by utilizing meiotic restitution genes. The method involves three steps: hybridization to induce recombination, interspecific hybridization to extract haploids, and spontaneous chromosome doubling by selfing the interspecific F1s. DHs produced in this way contain recombinant chromosomes in the genome(s) of interest in a homogeneous background. No special equipment or treatments are involved in the DH production and it can be easily applied in any breeding and/or genetic program. Triticum turgidum L. and Aegilops tauschii Coss, the two ancestral species of common wheat (Triticum aestivum L.) and molecular markers were used to demonstrate the SynDH method.     

Pathways to doubled haploidy: chromosome doubling during androgenesis

Production of doubled haploid (DH) plants through androgenesis induction is a promising and convenient alternative to conventional selfing techniques for the generation of pure lines for breeding programs. This process comprises two main steps: induction of androgenesis and duplication of the haploid genome. Such duplication is sometimes indirectly induced by the treatments used to promote androgenic development. But usually, an additional step of direct chromosome doubling must be included in the protocol. Duplication of the haploid genome of androgenic individuals has been thought to occur through three mechanisms: endoreduplication, nuclear fusion and c-mitosis. In this review we will revise and analyze the evidences supporting each of the proposed mechanisms and their relevance during androgenesis induction, embryo/callus development and plant regeneration. Special attention will be devoted to nuclear fusion, whose evidences are accumulating in the last years.     

玉米孤雌生殖单倍体诱导及加倍机理研究新进展

玉米单倍体技术的应用可以大大缩短自交系选育年限,加快育种进程,提高选育效率,尤其是孤雌生殖单倍体技术的广泛应用,能够在高效加倍的基础上快速选育稳定的纯系。本文较全面地综述了近年来关于玉米孤雌生殖诱导产生单倍体的机理及单倍体染色体加倍分子机制的研究新进展,旨在为单倍体技术更广泛的应用提供一定的理论依据。     

禾木科植物染色体消除型远缘杂交的研究进展

植物远缘杂交是作物育种中广泛应用的技术。除了核型稳定的种间杂交可以获得杂种以外,还可以利用核型不稳定的种间杂交后父本染色体消除的现象,通过胚培养和染色体加倍处理获得加倍单倍体(DH)植株。然而从小麦×玉米杂交获得的DH后代与其理论上应完全同质的遗传表现却不相符,总有2～5％的DH植株发生了形态学变异。最近的研究证明。通过小麦×玉米的受精作用,一些玉米特异DNA可以被转移到小麦DH后代的基因组中。     

植物单倍体的产生、鉴定、形成机理及应用

单倍体(Haploid)是指含有配子染色体数目的个体,对其进行基因组加倍可以快速获得纯合双单倍体(Doubled haploid, DH)。单倍体和双单倍体在植物品种选育、突变体筛选、基因功能鉴定、细胞学研究、遗传群体构建等方面具有重要作用,是近年来植物领域的一大研究热点。本文从单倍体和双单倍体的产生途径、鉴定、形成机理以及应用等方面较全面地综述了单倍体的最新研究进展,为单倍体的研究利用作一定的参考。     

l-Ascorbic acid sodium salt promotes microspore embryogenesis and chromosome doubling by colchicine in ornamental kale (Brassica oleracea var. acephala)

Isolated microspore culture (IMC) represents a potential alternative technique in the plant breeding process, as it allows the effective production of doubled haploid (DH) homozygous lines. However, the implementation of this technique is limited by a low rate of embryogenesis, high level of embryo death, and low frequency of chromosome doubling. Thus, we investigated the effects of using different concentrations of L-ascorbic acid sodium salt (VcNa), which has never been applied for kale, to enhance the embryogenesis and regeneration by IMC. Specifically, 1 to 5 μM VcNa was added to the NLN-13 medium of four kale genotypes, while control was grown on VcNa-free medium. Overall, 1–4 μM VcNa at pH 5.84 increased embryogenesis, with 4 μM VcNa being the optimum concentration (12.92-fold increase). The proportion of embryo deaths declined when using appropriate VcNa concentrations. To increase the frequency of chromosome doubling, an artificial chromosome doubling protocol was developed for kale microspore-derived haploids. This protocol involved dipping roots of haploid plantlets in colchicine solution and adding colchicine treatment to solid Murashige and Skoog (MS) medium. Optimum chromosome doubling of haploids was achieved by dipping their roots in 750 mg/L colchicine solution for 4–6 h and 1000 mg/L colchicine solution for 2 h (doubling for nearly 50% of haploids). In conclusion, this study delineated an effective tissue culture process in promoting chromosomal ploidy of microspore-derived regenerated plants, allowing more microspores to be maintained that have excellent ornamental characteristics through crossbreeding.

     

Gametic embryogenesis and haploid technology as valuable support to plant breeding

Plant breeding is focused on continuously increasing crop production to meet the needs of an ever-growing world population, improving food quality to ensure a long and healthy life and address the problems of global warming and environment pollution, together with the challenges of developing novel sources of biofuels. The breeders’ search for novel genetic combinations, with which to select plants with improved traits to satisfy both farmers and consumers, is endless. About half of the dramatic increase in crop yield obtained in the second half of the last century has been achieved thanks to the results of genetic improvement, while the residual advance has been due to the enhanced management techniques (pest and disease control, fertilization, and irrigation). Biotechnologies provide powerful tools for plant breeding, and among these ones, tissue culture, particularly haploid and doubled haploid technology, can effectively help to select superior plants. In fact, haploids (Hs), which are plants with gametophytic chromosome number, and doubled haploids (DHs), which are haploids that have undergone chromosome duplication, represent a particularly attractive biotechnological method to accelerate plant breeding. Currently, haploid technology, making possible through gametic embryogenesis the single-step development of complete homozygous lines from heterozygous parents, has already had a huge impact on agricultural systems of many agronomically important crops, representing an integral part in their improvement programmes. The aim of this review was to provide some background, recent advances, and future prospective on the employment of haploid technology through gametic embryogenesis as a powerful tool to support plant breeding.     

Excessive homozygosity in doubled haploids — advantages and disadvantages for plant breeding and fundamental research

The utility of haploids and DH lines for breeding programmes and basic research is discussed here. High effectiveness of haploid induction from random responding gametes and from valuable donor plants is not sufficient to ensure success in breeding. DH lines often appeared inferior to conventionally obtained inbred lines. One of the causes may be a very high level of homozygosity, in this paper referred to as excessive homozygosity. Besides, colchicine treatment as well as gametoclonal and somaclonal variation could have a negative effect on the agronomic performance of DH plants. Lack of natural selection in the first stages of haploid development is another important factor, negatively influencing haploid utility. On the other hand, there is a wide range of possibilities of using haploids as model plants for basic research. Among different applications, including mapping, genetic analysis, mutations, transformation, somatic hybridisation, biochemical and physiological studies, artificial seed production and germplasm storage, the first seems to be explored most effectively and give promising results.     

Androgenesis,gynogenesis, and parthenogenesis haploids in cucurbit species

Haploids and doubled haploids are critical components of plant breeding. This review is focused on studies on haploids and double haploids inducted in cucurbits through in vitro pollination with irradiated pollen, unfertilized ovule/ovary culture, and anther/microspore culture during the last 30 years, as well as comprehensive analysis of the main factors of each process and comparison between chromosome doubling and ploidy identification methods, with special focus on the application of double haploids in plant breeding and genetics. This review identifies existing problems affecting the efficiency of androgenesis, gynogenesis, and parthenogenesis in cucurbit species. Donor plant genotypes and surrounding environments, developmental stages of explants, culture media, stress factors, and chromosome doubling and ploidy identification are compared at length and discussed as methodologies and protocols for androgenesis, gynogenesis, and parthenogenesis in haploid and double haploid production technologies.     

Morphological and molecular evidences for DNA introgression in haploid induction via a high oil inducer CAUHOI in maize

The phenomenon of maternal haploid induction in maize was first described many years ago, but the underlying mechanism is still unclear. In this study, the Stock-6-derived, haploid-inducing line CAUHOI with high kernel oil content (KOC), was used as the pollinator to produce maternal haploids from the maize hybrid ZD958 with low KOC. CAUHOI is homozygous for the dominant marker gene R1-nj. Haploids were identified by morphological and cytological investigations. The frequency of haploid induction from this cross was 2.21%. Unexpectedly, many haploid kernels had weakly pigmented purple color on the embryo, and some haploid kernels had high KOC. Simple sequence repeat (SSR) analysis showed that 43.18% of the haploids carried segments from CAUHOI, and a small proportion (average 1.84%) of the genome of CAUHOI was introgressed into haploids. Haploid kernels with high KOC had a higher frequency of segment introgression from CAUHOI (2.92%) than that in haploid kernels with low KOC (1.79%), showing that the marker gene R1-nj and high-oil genes from CAUHOI were expressed during the development of some haploid embryos, and confirmed that the DNA introgression from the inducer parent occurred during maternal haploid induction. Together, these results suggested that the chromosome elimination was probably responsible for haploid induction in maize, and late somatic elimination might occur. Several possible mechanisms underlying haploid formation are discussed. Liang Li and Xiaowei Xu contributed equally to this work.     

RFLP Variations of Common Wheat Doubled Haploid Progenies from Wheat×Maize Crosses

Wheat ( Triticum aestivum L. ) and maize ( Zea mays L. ) crosses (the chromosome elimination system) can be used to produce frequently a large number of doubled haploid (DH) wheat lines by embryo rescue and doubling treatment. The resulting DH lines are genetically homogeneous. Significant RFLP variations were detected in common wheat DH progenies from wheat and maize crosses by using wheat rDNA clone pta71 and two maize DNA clones (MR13 and MRSO) homologous to wheat genome as probes. The results revealed that the copy number and restriction fragment length of rDNA in some wheat DH progenies was changed, and also that deletion was detected in several DH plants when probed with MR13 and MR5O. In particular, the RFLP pattern of DH line No. 18 was greatly changed using MR13 as a probe. In this line, three new bands, 40.0 kb, 2.5 kb and 2.0 kb emerged while a 4.3 kb intense band from the parental common wheat genome disappeared. This change may be related to a quite large DNA rearrangement within the wheat genomic DNA or an insertion by alien maize DNA fragment.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Synthesizing double haploid hexaploid wheat populations based on a spontaneous alloploidization process

Doubled haploid(DH) populations are useful to scientists and breeders in both crop improvement and basic research.Current methods of producing DHs usually need in vitro culture for extracting haploids and chemical treatment for chromosome doubling.This report describes a simple method for synthesizing DHs(SynDH) especially for allopolyploid species by utilizing meiotic restitution genes.The method involves three steps:hybridization to induce recombination,interspecific hybridization to extract haploids,a...     

Fertilization and Uniparental Chromosome Elimination during Crosses with Maize Haploid Inducers

Producing maternal haploids via a male inducer can greatly accelerate maize (Zea mays) breeding process. However, the mechanism underlying haploid formation remains unclear. In this study, we constructed two inducer lines containing cytogenetic marker B chromosome or alien centromeric histone H3 variant-yellow fluorescent protein vector to investigate the mechanism. The two inducer lines as the pollinators were crossed with a hybrid ZhengDan958. B chromosomes were detected in F1 haploids at a low frequency, which was direct evidence to support the occurrence of selective chromosome elimination during haploid formation. We found that most of the inducer chromosomes were eliminated in haploid embryonic cells during the first week after pollination. The gradual elimination of chromosomes was also detected in the endosperm of defective kernels, although it occurred only in some endosperm cells as late as 15 d after pollination. We also performed a genome-wide identification of single nucleotide polymorphism markers in the inducers, noninducer inbred lines, and 42 derived haploids using a 50K single nucleotide polymorphism array. We found that an approximately 44-Mb heterozygous fragment from the male parent was detected in a single haploid, which further supported the occurrence of paternal introgression. Our results suggest that selective elimination of uniparental chromosomes leads to the formation of haploid and possible defective kernels in maize as well, which is accompanied with unusual paternal introgression in haploid cells.Doubled haploid (DH) technology is widely used in maize (Zea mays) breeding. Although the life cycle of most sexually reproducing plant species alternates between a diploid sporophytic phase and a highly reduced gametophytic haploid phase, it is possible to obtain haploid plants containing the same number of chromosomes in their somatic cells as do the normal gametes of the species (Dunwell, 2010). Haploids can be obtained by in vitro or in vivo approaches. Anther and microspore culture are the most commonly used in vitro approaches; however, many species and genotypes are recalcitrant to these processes (Forster et al., 2007). Interspecies cross, which is an in vivo approach, induces haploids via chromosome elimination (Forster et al., 2007). For example, the cross between Hordeum vulgare and Hordeum bulbosum produces haploids in barley (Kasha and Kao, 1970; Sanei et al., 2011); maize and pearl millet (Pennisetum glaucum) are used as pollinators to produce haploids in wheat (Triticum aestivum; Laurie and Bennett, 1988; Gernand et al., 2005). There are two approaches for in vivo haploid induction in maize. One is using an ig mutant to generate both maternal and paternal haploids (Kermicle, 1969; Evans, 2007), and the other is using Stock6-derived inducers to produce maternal haploids only. The application of Stock6-derived inducers has become the foundation for modern DH technology (Prigge and Melchinger, 2012; Xu et al., 2013). The haploid induction rate (HIR) of inducer line Stock6 is 1% to 2% (Coe, 1959), which is 10 to 20 times higher than the spontaneous HIR in maize. The haploid-inducing capacity of inducers can be improved by selection (Sarkar et al., 1972). New inducers with improved HIRs have been developed, such as WS14 (2%–5% induction rate; Lashermes et al., 1988), RWS (Röber et al., 2005), and CAU5 (Xu et al., 2013).The first haploid inducer line Stock6 was discovered 50 years ago, and DH technology based on in vivo induction of maternal haploids has been widely used in maize breeding (Geiger, 2009). The mechanism underlying haploid formation remains unclear. Two hypotheses have been proposed for the mechanism. The first hypothesis is that one of the two sperm cells fails to fuse with an egg cell, but instead triggers haploid embryogenesis. In the second hypothesis, the two sperm cells fuse with an egg cell and a central cell, and the chromosomes from the inducer degenerate and are eliminated stepwise in the primordial cells during subsequent cell divisions.Evidence supporting the first hypothesis was reported by Bylich and Chalyk (1996). They found that 6.3% of the pollen grains of the ZMS haploid inducer line have two sperms showing different morphology. Thus, they proposed that the morphological defects of one of the sperms interfere in the sperm’s function, causing single fertilization. Another abnormality of inducer lines was described by Chalyk et al. (2003). They found that the frequency of aneuploid microsporocytes is much higher in haploid inducers than that in normal maize.Wedzony et al. (2002) studied the ovaries of inducer line RWS during the first 20 d after self-pollination. They found that about 10% of the embryos contain micronuclei with various sizes in the cytoplasm of every cell of shoot primordium. Their result supports the second hypothesis, which is selective chromosome elimination. Moreover, Fischer (2004) speculated that maternal haploids might possess small fractions of inducer genome. Consistently, previous studies (Zhang et al., 2008; Li et al., 2009) demonstrated morphological and molecular evidence for paternal DNA introgression in haploids, indicating the mechanism of chromosome elimination.Robust evidence to support either hypothesis is still missing due to lack of cytogenetic makers to trace chromosomes from inducers. In this study, we used B chromosomes and centromeric histone H3 variant (CENH3)-yellow fluorescent protein (YFP) as cytogenetic markers to discover direct evidence supporting selective chromosome elimination in both embryo and endosperm during haploid formation. In addition, we performed a genome-wide identification of single nucleotide polymorphism (SNP) markers in inducers, noninducer inbred lines, and 42 haploids using a 50K SNP array and detected unusual DNA introgression from inducer lines during haploid formation.     

The agronomic performance of anther culture derived plants of barley produced via pollen embryogenesis

Anther culture was used to generate microspore-derived doubled haploid (DH) plants from four spring barley crosses. The culture medium used contained maltose as the sole carbohydrate source and the mode of plantlet regeneration was mainly via pollen embryogenesis. Both haploid and spontaneously doubled regenerants were produced and the doubled haploids were compared to recom-binant inbred lines generated by several rounds of selfing (single seed descent). Parental, DH and single seed descent (SSD) lines were grown in randomised, replicated field trials and the samples were scored for a range of agronomic traits. The mean performance and phenotypic distribution of the DH and SSD samples were similar and there was little evidence to support the conclusion that anther culture derived lines exhibit a reduction in vigour. Where significant differences were detected between groups these were mainly confined to crosses which were segregating for the denso dwarfing gene. The differential transmission of particular regions of the barley genome may therefore influence and confound the expression of agronomic traits in DH populations. This is the first report of the agronomic performance of anther culture lines produced via pollen embryogenesis and the results are discussed in relation to the exploitation of anther culture technology in barley breeding.     

The auxins centrophenoxine and 2,4-D differ in their effects on non-directly induced chromosome doubling in anther culture of wheat (T. aestivum L.)

Doubled haploid technologies have become key tools for plant breeding. Using these techniques, the speed and efficiency of plant improvement processes can be significantly enhanced. Anther culture-based technologies have the potential to regenerate large numbers of doubled haploid plants without colchicine treatment. In an attempt to elucidate the influence of phytohormones on non-directly induced chromosome doubling, two synthetic auxins, 2,4-D and centrophenoxine, were tested in a wheat anther culture approach. Whereas the induction of androgenic embryo-like structures (ELSs) was efficient for both auxins, we observed a significantly higher frequency of chromosome doubling when using 2,4-D than when using centrophenoxine. When 2,4-D was added to the induction medium, a positive correlation between the size of ELSs and their ploidy level was detected by flow cytometry. The morphological selection of ELSs, a process that was included in routine operations of the method without significantly extending the input of time and effort, facilitates the production of fertile DH plants with a frequency of 60 %. Our findings may contribute to a more efficient production of doubled haploid wheat plants using a colchicine-free anther culture approach.     

New insights into the genetics of in vivo induction of maternal haploids, the backbone of doubled haploid technology in maize

Haploids and doubled haploid (DH) inbred lines have become an invaluable tool for maize genetic research and hybrid breeding, but the genetic basis of in vivo induction of maternal haploids is still unknown. This is the first study reporting comparative quantitative trait locus (QTL) analyses of this trait in maize. We determined haploid induction rates (HIR) in testcrosses of a total of 1061 progenies of four segregating populations involving two temperate haploid inducers, UH400 (HIR = 8%) and CAUHOI (HIR = 2%), one temperate and two tropical inbreds with HIR = 0%, and up to three generations per population. Mean HIR of the populations ranged from 0.6 to 5.2% and strongly deviated from the midparent values. One QTL (qhir1) explaining up to p = 66% of the genetic variance was detected in bin 1.04 in the three populations involving a noninducer parent and the HIR-enhancing allele was contributed by UH400. Segregation ratios of loci in bin 1.04 were highly distorted against the UH400 allele in these three populations, suggesting that transmission failure of the inducer gamete and haploid induction ability are related phenomena. In the CAUHOI × UH400 population, seven QTL were identified on five chromosomes, with qhir8 on chromosome 9 having p > 20% in three generations of this cross. The large-effect QTL qhir1 and qhir8 will likely become fixed quickly during inducer development due to strong selection pressure applied for high HIR. Hence, marker-based pyramiding of small-effect and/or modifier QTL influencing qhir1 and qhir8 may help to further increase HIR in maize. We propose a conceptual genetic framework for inheritance of haploid induction ability, which is also applicable to other dichotomous traits requiring progeny testing, and discuss the implications of our results for haploid inducer development.     

Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

植物单倍体技术及其应用的研究进展

该文较全面地综述了获得植物单倍体的相关途径及其在基础科学研究和植物育种方面的重要应用,着重介绍了一种基于着丝粒改造的染色体消除法诱导单倍体的策略及植物单倍体在基因组学研究中潜在的应用价值,旨在促进植物单倍体技术的完善并开拓其应用领域。