Doubled Haploid Plants Research Articles

Protocol of European Radish (Raphanus sativus L.) Microspore Culture for Doubled Haploid Plant Production.

Here, we describe the first protocol of European radish (Raphanus sativus L. subsp. sativus convar. radicula) for obtaining doubled haploid plants through in vitro microspore culture, in which the full cycle of doubled haploid formation was successfully achieved. Using this protocol, a yield of up to eight embryoids per Petri dish can be obtained. Effectiveness of this protocol was confirmed for several genotypes of European radish.

Production of Homozygous Red Beet (Beta vulgaris L. subsp. vulgaris) Plants by Ovule Culture.

Hybrid varieties dominate the red beet market. The breeding process necessary to produce these cultivars is very difficult and time consuming. The application of in vitro gynogenesis can reduce the time needed to produce the corresponding homozygous pure lines to a few months. Our research team has developed a method to obtain red beet doubled haploid plants by gynogenesis. The best medium for gynogenesis induction is the B5 medium with the addition of 0.5mg/L IAA, 0.2mg/L BA, and 322mg/L putrescine, whereas the best medium for shoot induction from embryos proved to be the MS medium supplemented with 0.1mg/L NAA, 0.1mg/L BA, and 0.5mg/L putrescine. The shoots obtained were rooted on MS medium containing half the concentration of microelements and 3mg/L NAA, 160mg/L putrescine, and 20g/L sucrose. Ploidy evaluation of gynogenetic plants was performed by flow cytometry and homozygosity or heterozygosity was determined by two isoenzymatic systems: PGI and AAT.

Shed-Microspore Culture in Ornamental Peppers for Doubled Haploid Plant Production.

The shed-microspore culture technique is an alternative sub-method combining anther and isolated microspore culture to induce microspore embryogenesis. Recently, its effective use in different types of peppers has drawn attention, because it has a higher embryo yield potential compared to anther culture and is more practical than isolated microspore culture. In this chapter, a stepwise protocol for shed-microspore culture of ornamental pepper is described. This protocol includes the steps of donor plant growth conditions, the choice of suitable flower buds based on DAPI staining of microspores, application of a cold pretreatment to flower buds, surface sterilization of the buds, shed-microspore culture of anthers, stress treatments, regeneration of androgenic in vitro plantlets, their acclimatization and ploidy analysis, and in vivo chromosome doubling of the haploid plants.

Production of Doubled Haploid Plants in Cucumber (Cucumis sativus L.) Through Anther Culture.

As in any other economically important crop, the possibility of producing fully homozygous, doubled haploid lines in cucumber allows for faster and cheaper breeding. At present, the fastest way to doubled haploidy is the production of cucumber haploid plants and duplication of their chromosomes to make them doubled haploid. In this chapter, we describe a complete protocol to successfully produce cucumber doubled haploid plants, including the evaluation of their ploidy level by flow cytometry. Briefly, this protocol involves a first step of anther culture to induce microspores to divide and proliferate forming calli. The calli produced are isolated from anthers and transferred first to a liquid medium and then to a solid medium to induce organogenesis. Organogenic shoots will eventually give rise to entire DH plants.

Brassica rapa L. ssp. chinensis Isolated Microspore Culture Protocol.

Culture of isolated microspores is a widely used method to obtain haploid and doubled haploid plants for many crop species. This protocol describes the steps necessary to obtain a large number of microspore derived embryos for pakchoi (Brassica rapa L. ssp. chinensis) and zicaitai (Brassica rapa L. ssp. сhinensis Hanelt var. purpuraria Kitam).

In Vitro Anther Culture for Doubled Haploid Plant Production in Spelt Wheat.

Doubled haploid (DH) plant production belongs to modern biotechnology methods of plant breeding. The main advantage of DH plant production methods is the development of genetically homozygous lines in one generation, whilst in conventional breeding programmes, the development of homozygous lines requires more generations. The present chapter describes an efficient protocol for DH plant production in spelt wheat genotypes using in vitro anther culture.

Production of Doubled Haploid Sugar Beet (Beta vulgaris L.) Plants Through Gynogenesis.

Haploid and doubled haploid plant production through unpollinated ovule culture, with diverse benefits and applications, is considered among the most effective and advantageous breeding method for sugar beet (Beta vulgaris L.). It is known that sugar beet is not responsive to androgenesis, which is widely used for most plant species. Sugar beet is a recalcitrant plant in vitro due to the very low spontaneous chromosome doubling and low gynogenesis rate. Thus, a steadily increasing gynogenesis efficiency has always been an important target for an efficient sugar beet breeding program. Given the scarcity of published papers focusing on gynogenesis in sugar beet, this chapter describes haploid and doubled haploid production through ovule culture of unfertilized flowers as a practical method.

Anther Culture in Sweet Pepper (Capsicum annuum L.).

Peppers have a prominent role in traditional cuisine of many different countries all around the world. This is why pepper is one of the most important crops worldwide. Production of doubled haploid (DH) pepper plants has been assessed by different approaches, but at present, the most efficient and universal method is by far anther culture, based on the use of the Dumas de Vaulx et al. protocol published in 1981, and adapted to the particularities of each specific pepper background. In this chapter, we present a method to produce pepper DHs by anther culture, based on the Dumas de Vaulx et al. protocol, but including a number of modifications which, in our experience, allow for a more efficient production DH plants in different pepper genotypes.

The Double-Layer Method to the Genesis of Androgenic Plants in Anemone coronaria.

Homozygous lines occur for plant breeding programs and for studies about gene expression and genetic mapping and they can be derived from anther culture. In this chapter, the method to obtain androgenic plants from an ornamental cut flower, Anemone coronaria belonging to the Ranunculaceae family, is described. In this species, androgenic plants were obtained culturing anthers with responsive microspores in Petri dishes containing a double layer of substrate with specific composition. Moreover, thermic treatment has been applied to induce the switch from pollen development program to embryo development program. The method allows to produce both double-haploid plants from diploid mothers (2n) and di-haploid plants from tetraploid mothers (4n).

Production of doubled-haploid(DH) selfed-progenies in ‘Banpeiyu’ pummelo [Citrus maxima (Burm.) Merr.] and its genetic analysis with simple sequence repeat markers

The genetic analysis of five haploid plants (BX1-BX5) of ‘Banpeiyu’ pummelo (BP), [Citrus maxima (Burm.) Merr.] using 32 simple sequence repeat (SSR) markers carried out for the purpose of confirming their gynogenetic origin and genetic differences. As the results, all the haploid plants possessed only either one of the BP alleles, confirming that they are gynogenetic origin. We also confirmed that BX1 and its doubled haploid (BX1-DH) have exactly the same SSR alleles. Interestingly, all the five haploids had common alleles on the linkage group (LG) 6 in the case of three markers. Then, we performed self-pollination of DH plants and reciprocal crosses between DH and BP, which has strong self-incompatibility. The seeds obtained by these crosses germinated in vitro or ex vitro normally and developed into diploid seedlings, except for one tetraploid seedling. Genetic analysis of these seedlings with 32 SSR markers revealed that segregation ratios of the 32 loci in the 30 BP inbred, 30 BP x DH and 30 DH x BP progenies mostly fitted to the expected ratios. However, significantly distorted segregation ratios were found in one, five and three SSR markers for BP inbred, BP x DH and DH x BP progenies, respectively. All of the 30 DH inbred progenies showed exactly the same SSR alleles as the original DH. This is the first example in which self and back cross progenies of DH in fruit crops were genetically analyzed using SSR markers.

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species.

In F1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer–protector model.

Effect of Chemical Composition of Nutritive Medium and Explant Size Over Androgenetic Response in Microspore Culture of Brassica oleracea L.

The dimension of the bud is a key factor for the orientation of microspore culture and the success of obtaining double haploid plants as it is a strong correlation between bud size and the developmental stages of microspores, and it is specific for each plant species and genotype. Our study was focused to determine the correlation between morphological characteristics, namely floral bud size and specific microspore developmental stages in order to determine the proper size, suitable for a successful protocol of obtaining double haploid plants in Brassica oleracea var. italica. Thus, we tested four bud sizes ranging from 2.0 to 4.0 mm measured from the base to the tip of the bud. After the statistical analysis of the results it can be emphasized that the best results were obtained in the case of using as a source of microspores the flower buds with the size between 3.1-3.5 mm. At this dimension, the share of microspores in the uninucleate stage, predominantly in the late uninucleate stage, is 90%, thus ensuring a homogeneous population of microspores in the optimum stage of development. Their evolution is predominantly embryogenic, the percentage of microspores following the gametophytic pathway is reduced, by only 9.12%.

Trichostatin A Affects Developmental Reprogramming of Bread Wheat Microspores towards an Embryogenic Route.

Microspores can be developmentally reprogrammed by the application of different stress treatments to initiate an embryogenic pathway leading to the production of doubled haploid (DH) plants. Epigenetic modifications are involved in cell reprogramming and totipotency in response to stress. To increase microspore embryogenesis (ME) efficiency in bread wheat, the effect of the histone deacetylase inhibitor trichostatin A (TSA) has been examined in two cultivars of wheat with different microspore embryogenesis response. Diverse strategies were assayed using 0–0.4 µM TSA as a single induction treatment and after or simultaneously with cold or mannitol stresses. The highest efficiency was achieved when 0.4 µM TSA was applied to anthers for 5 days simultaneously with a 0.7 M mannitol treatment, producing a four times greater number of green DH plants than mannitol. Ultrastructural studies by transmission electron microscopy indicated that mannitol with TSA and mannitol treatments induced similar morphological changes in early stages of microspore reprogramming, although TSA increased the number of microspores with ’star-like’ morphology and symmetric divisions. The effect of TSA on the transcript level of four ME marker genes indicated that the early signaling pathways in ME, involving the TaTDP1 and TAA1b genes, may be mediated by changes in acetylation patterns of histones and/or other proteins.

Meiotic crossover reduction by virus-induced gene silencing enables the efficient generation of chromosome substitution lines and reverse breeding in Arabidopsis thaliana.

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F1 in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A.thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies.

Generation of doubled haploid lines from winter wheat (Triticum aestivum L.) breeding material using in vitro anther culture

We investigated the anther culture (AC) efficiency of thirteen F<sub>4</sub> combinations of winter wheat (Triticum aestivum L.). The genotype dependency was assessed during the induction of the androgenic entities, i.e. embryo-like structures (ELS), regenerated-, green-, albino-, and transplanted plantlets. The number of green plantlets per 100 anthers (GP/100A) varied from 0.36 to 24.74 GP/100A with a mean of 8.31 GP/100A. Albino plantlets (AP) occurred in each combination, ranging from 0.20 to 22.80 AP/100A with an average value of 5.59 AP/100A. Between 25–87.76 doubled haploid (DH) plants per 100 acclimatised plantlets (DH/100ADP), depending on the combination, with a mean of 59.74% were recovered. We have found the highest DH production in the combinations Béres/Midas, Kalász/Tacitus, Béres/Pamier, and Premio/5009. This improves remarkably the choice of basic genetic material in subsequent crossing programmes. These observations emphasise the usability and efficiency of in vitro AC in producing a large number of DH lines for breeding and the applied researches of winter wheat. Although albinism was found in each combination, it was mitigated by the in vitro AC application.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Haploid Culture and Double Haploid Induction in Medicago sativa L. cv. XinJiangDaYe

Background: Alfalfa (Medicago Sativa), a perennial cross-pollinated plant, is one of the most important forage crops in the world with commercial value and ecological significance. However, due to the complexity of its genome, varietal improvement is difficult. Therefore, generating genetically homozygous materials have greater significance for breeding. In the current study, we aimed to identify the best tissue culture conditions to obtain haploid plants and double haploid plants.Methods: In this study, the haploid plants of alfalfa were obtained by combining tissue culture regeneration system with Flow cytometry. Different concentrations of colchicine were applied to the haploid plants using solid and liquid cultivation methods to determine the optimum conditions to obtain double haploid plants of Medicago Sativa L. cv. ‘XinJiangDaYe’. Result: Among the two colchicine cultivation methods tested, the doubling rate of regenerated plants obtained by liquid cultivation method was higher and the leaves developed under this system have the best doubling effect among the three explants tested. Optimal doubling conditions for alfalfa haploid (Medicago Sativa L. cv. ‘XinJiangDaYe’) were identified. The double haploid plant material generated from the current study could serve as a genetic resource for developing the hybrid combinations and for analyzing genetic linkage in alfalfa improvement programs.

Production of doubled haploid plants ofCucurbitaceaefamily crops through unpollinated ovule culture in vitro

Production of doubled haploid plants of<i>Cucurbitaceae</i>family crops through unpollinated ovule culture in vitro

An Overview of Doubled Haploid Plant Production in Cucurbita Species

F1 cultivars are currently used in vegetable production because of their many superior agronomic advantages (high yield and quality, resistance/tolerance to biotic and/or abiotic stress factors, wide adaptability, etc.). It is not possible to produce hybrid vegetables without homozygous pure parental lines with defined traits and high combining ability. However, the production of homozygous pure parents needs a long time, great efforts, and high production cost. Moreover obtaining an F1 hybrid variety takes 8-10 years with traditional breeding methods, especially in a highly open-pollinated species such as Cucurbita spp. Doubled haploid (DH) technologies (parthenogenesis, androgenesis, and gynogenesis), called biotechnological breeding methods, have become an alternative to classical breeding methods and provide to obtain pure lines within 1-2 years by adapting to these technologies in F1 hybrid breeding programs. Although the DH technologies considered a meaningful approach to enhance the production of valuable double haploid plants, the implementation of these techniques needs labor-intensive efforts, high experiences, and advanced technologies. The main objective of this review to provide a summary of DH techniques applied in the Cucurbita species in the current progress.

Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.

The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.