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.     

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.     

Doubled haploid production in Flax (Linum usitatissimum L.)

There is a requirement of haploid and double haploid material and homozygous lines for cell culture studies and breeding in flax. Anther culture is currently the most successful method producing doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this review we focus on tissue and plants regeneration using anther culture, and cultivation of ovaries containing unfertilized ovules. The effect of genotype, physiological status of donor plants, donor material pre-treatment and cultivation conditions for flax anthers and ovaries is discussed here. The process of plant regeneration from anther and ovary derived calli is also in the focus of this review. Attention is paid to the ploidy level of regenerated tissue and to the use of molecular markers for determining of gametic origin of flax plants derived from anther and ovary cultures. Finally, some future prospects on the use of doubled haploids in flax biotechnology are outlined here.     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.     

First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L.

Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterranean basin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes for new superior cultivar development in many crops. Haploid-diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In the case of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often, self-incompatibility, there is no way to obtain haploidization through conventional methods. Induction of microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or cold stress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In the present work we analyzed whether microspore reprogramming could be induced in apricot cultivars by cold stress through anther culture. We report the development of an in vitro anther culture protocol in P. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media for inducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos. The effect of two culture media in the embryogenic response was also analyzed, being the responses genotype-dependent. Monitoring of the cellular changes on the microspores was performed by structural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore and the first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and DH plants, for future potential applications in breeding programmes of this economically important fruit tree.     

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.     

Doubled haploid production in fruit crops

The interest of fruit breeders in haploids and doubled haploids (DH), lies in the possibility of shortening the time needed to produce homozygous lines compared to conventional breeding. Haplo-diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In a conventional breeding programme, a pure line is developed after several generations of selfing. With fruit crops, characterized by a long reproductive cycle, a high degree of heterozygosity, large size, and, sometimes, self-incompatibility, there is no way to obtain haploidization through conventional methods. This paper reviews the current status of research on doubled haploid production in the main fruit crops: Citrus, Malus domestica, Pyrus communis, Pyrus pyrifolia, Prunus persica, Prunus avium, Prunus domestica, Prunus armeniaca, Vitis vinifera, Actinidia deliciosa, Olea europaea, Morus alba, Actinidia deliziosa, [Musa balbisiana (BB)], Carica papaya, Annona squamosa, Feijoa sellowiana, Opuntia ficus-indica, Eriobotrya japonica.     

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.     

Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

The ability to produce doubled haploid (DH) plants has found broad application in research and breeding. For major crop species such as maize (Zea mays L.) and barley (Hordeum vulgare L.), routine large-scale production of DHs has enabled the acceleration of breeding processes, for example through efficient generation of homozygous lines. However, in forage crops such as perennial ryegrass (Lolium perenne L.), low and genotype-specific responses to in vitro anther culture (AC) still limit wide-spread use of DHs. Here, we report the responses of nine bi-parental populations, segregating for microspore embryogenesis and plant regeneration capacity, to an effective AC protocol. Genotypes of exceptionally high androgenic ability, producing over 200 green plants per 100 anthers cultured, could be selected. Continuous and distinctly shaped distributions for the evaluated traits were indicative of quantitative polygenic control and the presence of different alleles in each population. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield (ρ?=?0.20), suggested that different genes influence these traits. The populations evaluated here provide a rich source of alleles needed for the introgression of high levels of androgenic capacity into recalcitrant material. Moreover, this germplasm is ideally suited for use in future genotyping and mapping studies so that the genetic control of androgenic capacity in perennial ryegrass can be elucidated. Ultimately, our results will help to realize the potential of DH induction in one of the world’s most important forage crop species.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

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.     

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl- p -toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18-h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μ M trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.     

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.     

Reverse breeding: a novel breeding approach based on engineered meiosis

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity . Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

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...     

In vitro haploid and dihaploid production via unfertilized ovule culture

Haploids and doubled haploids are very important in plant breeding, enabling the time needed to produce homozygous lines to be shortened compared with conventional breeding. In the present review, emphasis is given to haploid induction through unfertilized ovule/ovary culture. Attention is given to induction of haploid plants from female gametophyte culture through analysis of factors in the processes of gynogenesis, including genotype selection, stage of ovule development, pretreatment, and culture media containing nutritional components and phytohormones. The gynogenetic approach may be of great value in discovering novel genetic recombinations. Application of double haploids in genetics and plant breeding is also highlighted. This review also identifies some existing knowledge gaps where work may increase the efficiency of this process in different plant species.     

Novel technologies in doubled haploid line development

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.     

Stress-induced formation of haploid plants through anther culture in cork oak (Quercus suber)

Induction of haploid embryos and regeneration of plantlets have been obtained, for the first time, in cork oak ( Quercus suber L.) by combining a starvation treatment in anther culture with a mild heat shock at 33°C for 5 days, followed by culture at 25°C in a simple agar medium without growth regulators. The same conditions had been shown previously to be optimal for embryogenic induction in isolated microspore cultures of several model species such as tobacco and wheat. These results support the notion that stress, particularly sucrose starvation, a heat shock or a combination of both treatments could be the major and general signal responsible for the inhibition of normal gametophytic development of the microspores and for the induction of the alternative embryogenic pathway. A similar approach may be used for the production of haploid and doubled haploids for plant breeding in other species that, like most forest trees, are still recalcitrant in anther culture.     

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.