Production Of Haploid Plants Research Articles

The Evaluation of Response to Isolated Microspores Culture in Some Iranian Hexaploid Wheat (Triticum aestivum L.) Cultivars

The most important method of haploid plant production is isolated microspores culture. To be successful in this method, genetic potential of cultivars is very important. In this research, ten Iranian hexaploid wheat cultivars (exclusive of MV17) on the basis of their characters such as heat, drought and salinity resistant have been selected and their responses to isolated microspore culture were evaluated. Meanwhile the effects of two pretreatments (cold and heat + chemical) and embryo induction media (NPB99 and CHB-2) on androgenic traits including embryogenesis, total plant regeneration and green plant regeneration were studied in selected cultivars. There were significant interactions between cultivars and pretreatments and also between cultivars and media in most cases. The interaction between cultivars Chamran and Navid with 21 days cold pretreatment produced the highest number of embryos and the interaction between cultivar Hamoon and 21 days cold and heat + chemical pretreatments produced the most green plant regeneration percentage. Also, the results showed that the interaction between cultivar Chamran and CHB-2 medium produced the highest number of embryos while the interaction between cultivar Hamoon and NPB99 medium had the highest green plant regeneration.

Pollen and Anther Cultures as Potential Means in Production of Haploid Kenaf (Hibiscus cannabinus L.)

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus for human needs. It is annual upright plant that produces high biomass, has good adaptability to its environment and inexpensive to be grown. Kenaf has been cultivated in many countries since long time ago mainly for fibers. In Malaysia, however, this plant is considered new and cultivated especially in the eastern parts of Peninsular to replace tobacco plantations that have been no longer supported by the government. This plant has potential to be bred to improve the quality and the adaptability. One of the potential breeding methods to improve the plant is by the production of hybrid varieties. These varieties are superior over the inbred varieties because of the heterosis effect (hybrid vigor) ofF1heterozygous. Hybrid varieties can be produced by reciprocal combinations of crosses among homozygous lines. Homozygous lines conventionally can bachieved by repeated selfing till seven or more generations that need a lot of times. The production of haploid plants in vitro followed by chromosome doubling is a ‘short-cut’ method to produce homozygous lines in relatively short time compared to conventional methods. The objective of this study is to produce haploid lines through pollen and anther cultures in vitro. The population of haploid plantlets from this research will be induced for diploidization to produce homozygous-heterogeneous population as a basis for the production of hybrid varieties. Preliminary result showed that callus can easily be induced from the anthers compared those from the pollen grains.

Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.

Comparative morphology, biology and histology of reproductive development in three lines of Manihot esculenta Crantz (Euphorbiaceae: Crotonoideae).

Cassava (Manihot esculenta), a major food staple in the tropics and subtropics, thrives even in environments undergoing threatening climate change. To satisfy the increasing demand for crop improvement and overcome the limitations of conventional breeding, the introduction of inbreeding techniques such as the production of doubled haploid lines via androgenesis or gynogenesis offers advantages. However, comprehensive studies on cassava flower bud biology or structural development are lacking and precise structural and biological information is a prerequisite to enhance the efficiency of these techniques. The floral biology of three selected cassava lines was studied, focusing on morphology, phenology and pollen biology (quantity, viability and dimorphism). Histological studies were also conducted on microsporogenesis/microgametogenesis and megasporogenesis/megagameto-genesis to generate precise developmental data for these lines. Male and female cyathia have distinct developmental phases. Pollen viability was high during immature stages of plant development; however, pollen mortality was common at later stages. Pollen trimorphism in male gametophytes towards the larger or smaller pollen size, as compared with normal size, was observed. Ten characteristic events were identified in male gametogenesis and six in female gametogenesis that were correlated with flower bud diameter. Male gametophyte diameter at different developmental stages was also determined. Results indicate that the three lines did not differ significantly, except regarding a few morphological aspects such as plant height, flower colour and number of male cyathia. Pollen grains were initially viable, but viability decreased drastically at later stages of growth. Abnormal meiosis or mitosis triggered pollen trimorphism. The demonstrated sequential events of reproductive development generated valuable information at the cellular level, which will help close the current information gap for cassava improvement via breeding programmes and doubled haploid plant production.

Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.)

Protocols were developed for the generation of haploid and doubled haploid plants from isolated microspores of carrot (Daucus carota L.). Forty-seven carrot accessions, including six inbred lines, 11 cultivars, 20 F1s, two BC1F1s, four F2s, one F3, and three F4s, were screened to evaluate the genotype influence on isolated microspore embryogenesis over 4 years. Twenty-eight accessions responded by producing embryos and/or calli. A cytological analysis showed that two modes of carrot microspore embryogenesis exist: an indirect route via calli (C mode), and a direct route via embryos (E mode). Eleven accessions were in the C mode, and 17 were in both modes. The highest production rates were in 10Y25 (a European Nantes cultivar) with 27 calli and 307 embryos, and 100Q6 (a semi-Nantes F1 hybrid) with 176 calli and 114 embryos. The time period to produce embryos or calli differed significantly between 2 and 6 months. Cold and heat pretreatment generally had a negative impact on the induction of microspore embryogenesis, but a short pretreatment showed a positive influence on some accessions. Twenty-eight lines regenerated plants from the primary individual embryos or calli of three accessions were established to analyze the ploidy level. The percentage of spontaneous diploidization showed very wide differences among the accessions and lines. Differences in leaf color intensity, leaf size, and leaf dissection were found among haploid, doubled haploid, and triploid plants.

An Improved Wheat Microspore Culture Technique for the Production of Doubled Haploid Plants

ABSTRACTMicrospore culture is used to generate completely homozygous plants in a single generation, thereby reducing cost and time required for the production of doubled haploid (DH) plants for breeding and genetic studies. Many factors are known to influence green plant recovery including composition of regeneration media, donor plant genotype, microspore developmental stage, and pretreatment conditions. The objectives of this study were to: (i) develop an improved regeneration medium for wheat microspore culture and (ii) determine the optimal pretreatment conditions and regeneration media combination for increasing green plant recovery rates. Four wheat cultivars, two pretreatment methods (0.4 M mannitol at 4°C and solution B containing 0.3 M mannitol with inorganic components at room temperature), and five regeneration media were tested. Green plant recovery rates from each treatment combination were analyzed using the Proc Logistic model in SAS. Regeneration medium fortified with ascorbic acid produced the highest number of green plants across the different pretreatment conditions, regeneration media, and cultivars tested. Pretreating wheat (Triticum aestivum L.) spikes with 0.4 M mannitol at 4°C followed by embryoid induction and regeneration in a medium fortified with ascorbic acid resulted in the recovery of the highest number of green plants. This high‐efficiency method may prove useful for producing DH populations for wheat improvement efforts.

High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.)

We developed an efficient culture system for producing cotyledonary embryos from isolated microspores of hot pepper (Capsicum annuum L.) and analyzed the ploidy levels of regenerated plants. Three culture protocols were studied: liquid, double-layer, and two-step culture. In the double-layer culture, cotyledonary embryos were produced more efficiently when the same medium composition was used for the liquid upper-layer and the solid under-layer. The two-step culture system, in which microspores were first incubated on liquid medium and then subcultured on double-layer medium, was most effective for producing cotyledonary embryos. Cotyledonary embryos were produced more efficiently when the isolated microspores were cultured in liquid medium for 1 week in 60 × 15-mm plates at a density of 8–10 × 104/mL and microspore suspensions from two liquid culture plates were combined into a single 100 × 20-mm plate containing solid medium, and the culture was continued for an additional 3 weeks. When cotyledonary embryos obtained from this two-step culture were transplanted into regeneration medium, more than 95 % developed into plants. Only 31 of the 190 analyzed plants (16.3 %) generated by this method were spontaneous doubled haploids. This two-step culture system outperforms all previously reported culture protocols for isolated microspores of hot pepper, and appears to be a promising tool for the production of haploid plants for hot pepper breeding.

Development of a procedure for production of haploid plants through microspore culture of roselle (Hibiscus sabdariffa L.)

Attempts to obtain new variants of roselle through conventional breeding were unsuccessful for 358 trials of (UKMR-3×UKMR-2) and 63 trials of (UKMR-3×accession 3). Roselle is an autotetraploid plant (2n=4x=72). Therefore the objectives of this study were to obtain new variants in roselle through the establishment of a plant regeneration system via microspore culture followed by detection of the ploidy level of regenerants based on their distinctive PCR patterns. The effects of different media and hormonal combinations were evaluated on microspore culture of roselle. This method was effective for callus induction, early-stage embryo development and subsequently efficient plant regeneration. Pretreatment of microspores at 4°C and 35°C for 3 days in the dark and without pretreatment, showed significant results on percentage of callus induction. Thirty regenerants were evaluated for their ploidy level using flow cytometry combined with propidium iodide and only 1 was detected as haploid, 4 mixploids and 25 diploids. Polymerase chain reaction (PCR) analysis using M13 universal primer (5′-TTATGAAACGACGGCCAGT-3′) showed the regenerated haploid plant having four unique bands at loci M13-01, M13-03, M13-04, and M13-06, which were absent in the microspore donor plant UKMR-1. To our knowledge, this is the first report of the development of haploid plants in roselle via microspore culture.

Identification of BABY BOOM and LEAFY COTYLEDON genes in sweet pepper (Capsicum annuum L.) genome by their partial gene sequences

Haploid plant production via anther culture is a valuable tool to rapidly create pure lines for plant breeding programs. Some sweet pepper (Capsicum annuum L.) varieties produce embryogenic anther culture readily, while others do not respond at all. The switching of microspore developmental program from gametophyte- to sporophyte-type is regulated by transcription factors like BABY BOOM (BBM) and LEAFY COTYLEDON (LEC). Genes encoding these transcription factors have not been found in sweet pepper yet. The sequences of BBM and LEC from other plants, annotated in National Center for Biotechnology Information (NCBI), were used to design degenerative primers for conservative regions in these genes and to amplify BBM- and LEC-like sequences from genomic DNA of sweet pepper by PCR. The PCR products were cloned and sequenced. Sequence comparison revealed high similarity between the isolated sequences and those annotated in NCBI for BBM and LEC1. The obtained sequences were subjected to bioinformatics analyses to determine the exon–intron structure. Exons were translated in protein products in silico. The protein blast of the pepper BBM in the NCBI database revealed presence of an AP2-type functional domain, which is typical for the BBM family. The pepper LEC sequence was found to carry CBFD-NFYB-HMF motif, typical for the LEAFY COTYLEDON gene product family. RT-PCR confirmed high levels of expression of the BBM and LEC genes at the early stages of direct embryos development in anther culture. Expression of the two genes was not detected either in mature pepper plants or in non-embryogenic anthers. The two genes were detected in genomes of 14 pepper varieties, some of which do not respond with formation of direct embryos. We assume that differences in responses between varieties should be attributed not to presence/absence of two genes, but to their interaction with other genes’ products and different factor like micro-RNAs involved in the regulation of their expression.

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.

English

In cell culture, the maintenance of proper growing conditions is a key approach for improving the formation of embryos, and is useful in the production of doubled haploid (DH) plants. Optimal nutritional and environmental conditions for the microspore culture of Brassica oleracea L. var italica were determined in order to reduce time and effort in breeding. The optimal conditions for microspore embryo formation differed depending on genotype. Microspore-derived embryos (MDE) formation was influenced by the strength of the NLN medium, the microelement and sugar concentration, and the heat shock temperature and period. The 0.5XNLN liquid medium was the most favorable for MDE formation. The most efficient formation of MDE was observed in the 0.5X NLN liquid medium, without the addition of microelements. When 13 or 15% sucrose was added to the 0.5X NLN liquid medium, the amount of normal MDE formation increased. The optimum heat shock temperature and period for MDE formation was 32.5°C and 24 h, respectively. A polyploidy test indicated that 30% of the microspore derived plants were diploid throughout the embryogenesis process.

English

  Various factors affecting microspore culture were studied in Zantedeschia aethiopica. Starting microspores at late-unicellular stage, NLN as culture medium and treatment of culture at 32°C for 2 days prior to incubation at 25°C, gave the best early sporophytic response. A comparison of different carbohydrates applied in culture medium showed that glucose and fructose favored more occurrences of early sporophytic divisions than sucrose and maltose. However, multicellular structures stopped growing in continues cultivation with each carbohydrate. The development of multicellular structures was sustained and calli were formed by culturing microspores in the medium containing glucose or fructose for initial 7 days, subsequently changing to the medium containing sucrose. Thus, this studyrevealed an essential and novel role of monosaccharides in sporophytic development of Z. aethiopica microspores, whereas, it is known that monosaccharides are less effective for many other crops. In total, 24 calli were obtained, of which, 18 produced plants. Chromosome number counting of 18 plants indicated that 8 were haploid, 6 were diploid and the rest were other numeric chromosomal constitutions.   Key words: Calla lily, haploid plant production, isolated microspore culture, glucose, fructose.

Anther culture in pepper (Capsicum annuum L.) in vitro

Pepper (Capsicum annuum L.) is an important vegetable crop that can be improved using plant tissue culture and biotechnology. However, it is difficult to develop appropriate breeding material by in vitro cultivation in this species. Haploid plant production is useful in the breeding programs to facilitate recovery of recessive mutations and unique genetic recombinations. In embryogenesis, haploid formation from pollen in anther culture is a scientifically advanced, but controversial system. Various techniques for haploid plant regeneration are used to establish an efficient double haploid production method. The purpose of this article is to summarize, through comparison, results in pepper anther culture, problems associated with work in this field, and the influence of critical factors for successful embryo formation and plantlet development.

Isolated microspore culture of chickpea (Cicer arietinum L.): induction of androgenesis and cytological analysis of early haploid divisions

Routine production of haploid plants has not been reported for any legume, despite the major role these species play in sustainable farming systems and human nutrition. It is within this context that we report a protocol for the induction of haploid development in chickpea (Cicer arietinum L.) using isolated microspore culture. The cultivars “Rupali”, “Narayen”, and “Kimberley Large” were identified as responsive to isolated microspore culture. Flower bud length and microspore developmental stage were correlated for these cultivars. Depending on the cultivar, buds 2.85–3.5 mm in length contained uninucleate microspores. Microspores from donor plants grown in winter and spring were more responsive than those grown in summer. A cold treatment (4°C) of between 24 and 48 h enhanced microspore response in winter- and spring-grown material but was not effective in summer-grown material. A medium developed by the authors was effective for microspore induction and early-stage embryo development. The addition of hormones to this medium was promotive of microspore induction in winter- and spring-grown material, but not in summer-grown material. The initial haploid division predominantly occurred via symmetrical division of the vegetative nucleus. Further research is under way to convert pro-embryos into plants.

Improvement in efficiency of microspore culture to produce doubled haploid canola (Brassica napus L.) by flow cytometry

The efficiency of production of doubled haploid plants in canola (Brassica napus L.) breeding programmes is reduced when large numbers of haploid and infertile plants survive until flowering. We used flow cytometry to determine ploidy status and predict subsequent fertility of microspore-derived plantlets from three canola genotypes, with or without colchicine treatment of microspore suspensions. Young leaf tissue was sampled from microspore-derived plantlets within 1 week of transfer to soil, and processed immediately by flow cytometry. The process was repeated on the same plants 3–5 weeks later. Of the 519 plants transferred to soil, 57.2% were consistently haploid at both sample times, 33.5% were consistently diploid at both sample times, and the remainder (9.2%) were uncertain or inconsistent in ploidy status across sampling times. Of the 518 plants that survived to flowering, 32.4% were diploid at both times of sampling and fertile (set seed) and 46.3% were haploid at both sampling times and infertile. Another 10.8% were haploid at both sampling times and fertile, but had low pollen viability and seed set, and some were triploid or of uncertain ploidy level. Colchicine treatment of microspore suspensions significantly increased the proportion of diploid plants from 9.7 to 69.7%, with significant variation among genotypes. Evidence from simple sequence repeat marker loci indicated that diploid and fertile plants from the control treatment (no colchicine) were derived from spontaneously doubled haploid gametes, rather than unreduced gametes or somatic tissue. Flow cytometry at the first sample time was very efficient in detecting diploid plants of which 94.2% were subsequently fertile.

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

An isolated microspore culture provides an excellent system for the study of microspore induction and embryogenesis, provides a platform for an ever-increasing array of molecular studies, and can produce doubled haploid (DH) plants, which are used to accelerate plant-breeding programs. Moreover, isolated microspore cultures have several advantages over anther culture, wherein presence of the anther walls can lead to the development of diploid, somatic calli and plants. Although protocols for isolated microspore culture vary from laboratory to laboratory, the basic steps of growing donor plants, harvesting floral organs, isolating microspores, culturing and inducing microspores, regenerating embryos, and doubling the chromosomes, remain the same. Over the past few years, a large proportion of the research reports on isolated microspore culture have focused on cereal and Brassica species. For some of these species, isolated microspore culture protocols are well established and routinely used in laboratories around the world for developing new varieties, as well as for basic research in areas such as genomics, gene expression, and genetic mapping. Although these species are considered highly responsive to microspore culture, improvements in efficiency are still being made. However, with many species, isolated microspore culture is simply not yet efficient enough at producing DH plants to be cost-effective for breeding programs. There has been a recent resurgence of haploidy research with response being reported in some species once considered recalcitrant. Future research programs aimed at elucidating pathways involved in microspore induction and embryogenesis will be of benefit, as will novel approaches to improve the efficiency of microspore culture for DH production. With many species, anther culture has proven to be more effective than isolated microspore culture, necessitating more research to clarify the contribution of the anther wall to embryogenesis. The development of molecular markers for use in determining the gametic origin of regenerated plants, irrespective of their ploidy, would also be beneficial. In this review, we aim to provide an overview of the basic isolated microspore culture protocol with an emphasis on recent progress in several crop species.

In vitro production of double haploid plants from two rice species (Oryza sativa L. and Oryza glaberrima Steudt.) for the rapid development of new breeding material

Eight genotypes of two rice species (Oryza sativa and Oryza glaberrima) were studied for their response to anther culture in terms of callus induction and frequency of plant regeneration. N6 medium (Chu et al., 1975) was used for callus induction, and MS medium (Murashige and Skoog, 1962) with 1 mg/l BAP and 0.5 mg/l NAA for plant regeneration. Generally, O. glaberrima genotypes produced more callus than O. sativa genotypes. All O. glaberrima genotypes regenerated plants. Among O. sativa genotypes only japonica variety IKP produced plants. Other O. sativa genotypes showed low frequency of callus induction without plant regeneration. Many albino plants were obtained from the culture. Only one O. glaberrima genotype (6202 Tog) produced green plants. In this experiment, a total of 93 plants were regenerated with 14 green plants and 79 albino plants. Anther culture response is largely species and genotype dependent. Key words: Rice species, Oryza sativa (L.), Oryza glaberrima (Steudt.), anther culture, medium, callus induction, plant regeneration.

High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos

Transgenic doubled haploid rapeseed (Brassica napus L. cvs. Global and PF(704)) plants were obtained from microspore-derived embryo (MDE) hypocotyls using the microprojectile bombardment. The binary vector pCAMBIA3301 containing the gus and bar genes under control of CaMV 35S promoter was used for bombardment experiments. Transformed plantlets were selected and continuously maintained on selective medium containing 10 mg l(-1) phosphinothricin (PPT) and transgenic plants were obtained by selecting transformed secondary embryos. The presence, copy numbers and expression of the transgenes were confirmed by PCR, Southern blot, RT-PCR and histochemical GUS analyses. In progeny test, three out of four primary transformants for bar gene produced homozygous lines. The ploidy level of transformed plants was confirmed by flow cytometery analysis before colchicine treatment. All of the regenerated plants were haploid except one that was spontaneous diploid. High frequency of transgenic doubled haploid rapeseeds (about 15.55% for bar gene and 11.11% for gus gene) were considerably produced after colchicines treatment of the haploid plantlets. This result show a remarkable increase in production of transgenic doubled haploid rapeseed plants compared to previous studies.

Haploid plants produced by centromere-mediated genome elimination

Production of haploid plants that inherit chromosomes from only one parent can greatly accelerate plant breeding. Haploids generated from a heterozygous individual and converted to diploid create instant homozygous lines, bypassing generations of inbreeding. Two methods are generally used to produce haploids. First, cultured gametophyte cells may be regenerated into haploid plants, but many species and genotypes are recalcitrant to this process. Second, haploids can be induced from rare interspecific crosses, in which one parental genome is eliminated after fertilization. The molecular basis for genome elimination is not understood, but one theory posits that centromeres from the two parent species interact unequally with the mitotic spindle, causing selective chromosome loss. Here we show that haploid Arabidopsis thaliana plants can be easily generated through seeds by manipulating a single centromere protein, the centromere-specific histone CENH3 (called CENP-A in human). When cenh3 null mutants expressing altered CENH3 proteins are crossed to wild type, chromosomes from the mutant are eliminated, producing haploid progeny. Haploids are spontaneously converted into fertile diploids through meiotic non-reduction, allowing their genotype to be perpetuated. Maternal and paternal haploids can be generated through reciprocal crosses. We have also exploited centromere-mediated genome elimination to convert a natural tetraploid Arabidopsis into a diploid, reducing its ploidy to simplify breeding. As CENH3 is universal in eukaryotes, our method may be extended to produce haploids in any plant species.