Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.).

BACKGROUND: Isolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration. RESULTS: Low-temperature pre-treatment at 4 â„ƒ for 10-15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61-211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency. CONCLUSIONS: This study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment.

Induced mutations are important for genetic research and breeding. Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations. However, mutations must be fixed prior to phenotyping or field trials, which requires additional rounds of self-pollination. Microspore culture is an effective method to produce double-haploid (DH) plants that are fixed homozygotes. In this study, we conducted ethyl methanesulfonate (EMS)-induced mutagenesis of microspore cultures of barley (Hordeum vulgare) cultivar 'Hua30' and landrace 'HTX'. The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration. The two genotypes showed different regeneration efficiencies. The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations, revealed by whole-genome sequencing, highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants. Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture (somaclonal variation) rather than EMS-induced mutagenesis. In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials, this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations (mainly by fine-tuning the mutagen dosage).

Rapid Generation of Barley Homozygous Transgenic Lines Based on Microspore Culture: HvPR1 Overexpression as an Example.

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T2) from the same DH0 line (T1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

Au-courant and novel technologies for efficient doubled haploid development in barley (Hordeum vulgare L.).

Bounteous modern and innovative biotechnological tools have resulted in progressive development in the barley breeding program. Doubled haploids developed (homozygous lines) in a single generation is significant. Since the first discovery of haploid plants in 1920 and, in particular, after discovering in vitro androgenesis in 1964 by Guha and Maheshwari, the doubled haploidy techniques have been progressively developed and constantly improved. It has shortened the cultivar development time and has been extensively used in: genetic studies, gene mapping, marker/trait association, and QTL studies. In barley, the haploid occurrence developed gradually from being a sporadic and random process (spontaneous) to haploid development by in vivo method of modified pollination or by in vitro culture of immature male or female gametophytes. Although significant improvement in DH induction protocols has been made, challenges still exist for improvement in areas such as: low efficiency, albinism, genotypic specificity etc. Here, the paper focuses on: haploidization via different in vitro, in vivo techniques, the recent advances technologies like centromere-mediated haploidization, hap induction gene, and Doubled haploid CRISPR. The au-courant work of different researchers in barley using these technologies is reviewed. Studies on different factors affecting haploid induction and work on genome doubling of barley haploids to produce DH lines via spontaneous and induced technologies has also been highlighted.

Suberoylanilide hydroxamic acid induced microspore embryogenesis and promoted plantlet regeneration in ornamental kale (Brassica oleracea var. acephala).

Isolated Microspore Culture (IMC) is an efficient method to obtain the homozygous strain; however, it is difficult to apply in ornamental kale due to its low rate of microspore embryogenesis. Histone acetylation is an important epigenetic mechanism and may affect the changes of the microspore development pathway, promoting microspore embryogenesis. Here, microspores from three cut-flower ornamental kales, namely Crane Feather Queen (CFQ), Crane Pink (CP), and Crane Bicolor (CB), were treated with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) to induce embryogenesis. The haploid 'CFQ' microspore plantlets were doubled with colchicine. The results for 'CFQ' revealed that, the appropriate concentration of SAHA was 0.03 µM and obtained 17.27 embryos per bud. For 'CP,' the appropriate concentration of SAHA was 0.045 µM and obtained 11.19 embryos per bud. For 'CB,' the appropriate concentration of SAHA was 0.045 µM and obtained 6.10 embryos per bud. Haploid 'CFQ' microspore plantlets were treated with 75 mg/L colchicine for 7 d and the doubling rate was 41.7%. Haploid 'CFQ' plantlets were treated with 1000 mg/L colchicine by root-soaking for 4 h and the doubling rate was 64.3%. SAHA could promote microspore embryogenesis, and colchicine root soaking was more effective than adding colchicine to the medium for haploid plantlet doubling in cut-flower ornamental kale.

Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea).

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage '01-88' under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment.

Effects of genotype and culture conditions on microspore embryogenesis in radish (Raphanus sativus L.).

Radish (Raphanus sativus L.), an important annual or biennial root vegetable crop, is widely cultivated in the world for its high nutritive value. Isolated microspore culture (IMC) is one of the most effective methods for rapid development of homozygous lines. Due to imperfection of the IMC technology system, it is particularly important to establish an efficient IMC system in radish. In this study, the effects of different factors on radish microspore embryogenesis were investigated with 23 genotypes. Buds with the largest population of late-uninucleate-stage microspores were most suitable for embryogenesis, with a ratio of petal length to anther length (P/A) in buds of about 3/4 ~ 1. Cold pretreatment was found to be genotype specific, and the highest microspore-derived embryoid (MDE) yield occurred for treatment of the heat shock of 48 h. In addition, the supplement of 0.75 g/L activated charcoal (AC) could increase the yield of embryoids. It was found that genotypes, bud size, as well as temperature treatments had significant effects on microspore embryogenesis. Furthermore, somatic embryogenesis-related kinase (SERK) genes were profiled by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which indicated that they are involved in the process of MDE formation and plantlet regeneration. The ploidy of microspore-derived plants was identified by chromosome counting and flow cytometry, and the microspore-derived plants were further proved as homozygous plants through expressed sequence tags-simple sequence repeats (EST-SSR) and genetic-SSR markers. The results would facilitate generating the large-scale double haploid (DH) from various genotypes, and promoting further highly efficient genetic improvement in radish. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01312-w.

Methodological approaches for producing doubled haploids in sugar beet and red beet (Beta vulgaris L.).

The in vitro production of doubled haploids is a biotechnological path of an accelerated development of parental lines in F1-hybrid breeding programs. Unlike the traditional inbreeding method requiring 5 to 6 generations to reach a suf-f icient homozygosity of lines, the number of generations to produce pure lines of beet by haploid technologies is reduced to 2. The production of doubled haploids by gynogenesis is the most common biotechnological approach in sugar and red beets. Protocols for the production of doubled haploids for B. vulgaris species are few and have been developed mainly for sugar beets. There are no protocols for the production of doubled haploids for red beet (B. vulgaris convar. esculenta Salisb.), and the protocols developed for sugar beet (B. vulgaris convar. saccharifera Alef.) are ineffective for red beet, even though these two crops belong to the same species. The greatest success has been achieved in the production of doubled haploids by gynogenesis through isolated ovule culture, especially in sugar beet. Studies on the production of doubled haploids by androgenesis were actively carried out in the 1970s and 1980s and did not lead to the production of regenerated plants. However, at present, there is renewed interest among researchers in this approach, and scientists in different countries are conducting studies of Beta vulgaris androgenesis through isolated microspore culture. This article provides an overview of studies devoted to the production of doubled haploids, addressing the main problems of doubled haploid technologies, and methods to increase the frequency of embryogenesis and doubled haploid plant formation in B. vulgaris crops.

Occurrence of albinism during wheat androgenesis is correlated with repression of the key genes required for proper chloroplast biogenesis.

MAIN CONCLUSION: The phenomenon of albinism in wheat androgenesis is linked to the transcriptional repression of specific genes involved in chloroplast biogenesis during the first weeks of in vitro culture. Isolated microspore culture is widely used to accelerate breeding programs and produce new cultivars. However, in cereals and particularly in wheat, the use of this technique is limited due to the high proportion of regenerated albino plantlets. The causes and mechanisms leading to the formation of albino plantlets in wheat remain largely unknown and, to date, no concrete solution has been found to make it possible to overcome this barrier. We performed a molecular study of proplastid-to-chloroplast differentiation within wheat microspore cultures by analyzing the expression of 20 genes specifically involved in chloroplast biogenesis. Their expression levels were compared between two wheat genotypes that exhibit differential capacities to regenerate green plantlets, i.e., Pavon and Paledor, which produce high and low rates of green plants, respectively. We observed that chloroplast biogenesis within wheat microspores was affected as of the very early stages of the androgenesis process. A successful transition from a NEP- to a PEP-dependent transcription during early plastid development was found to be strongly correlated with the formation of green plantlets, while failure of this transition was strongly correlated with the regeneration of albino plantlets. The very low expression of plastid-encoded 16S and 23S rRNAs within plastids of the recalcitrant genotype Paledor suggests a low translation activity in albino plastids. Furthermore, a delay in the activation of the transcription of nuclear encoded key genes like GLK1 related to chloroplast biogenesis was observed in multicellular structures and pro-embryos of the genotype Paledor. These data help to understand the phenomenon of albinism in wheat androgenesis, which appears to be linked to the transcriptional activation of specific genes involved in the initial steps of chloroplast biogenesis that occurs between days 7 and 21 of in vitro culture.

Androgenesis of Red Cabbage in Isolated Microspore Culture In Vitro.

Red cabbage belongs to the economically important group of vegetable crops of the Brassicaceae family. A unique feature of this vegetable crop that distinguishes it from other members of the family is its unique biochemical composition characterized by high anthocyanin content, which gives it antioxidant properties. The production mainly uses F1 hybrids, which require constant parental lines, requiring 6-7 generations of inbreeding. Culture of isolated microspores in vitro is currently one of the promising methods for the accelerated production of pure lines with 100% homozygosity. The aim of this study is to investigate the factors and select optimal parameters for successful induction of red cabbage embryogenesis in isolated microspore culture in vitro and subsequent regeneration of DH plants. As a result of research, for the first time, it was possible to carry out the full cycle of obtaining DH plants of red cabbage from the induction of embryogenesis to their inclusion in the breeding process. The size of buds containing predominantly microspores at the late vacuolated stage and pollen at the early bi-cellular stage has to be selected individually for each genotype, because the embryoid yield will be determined by the interaction of these two factors. In the six samples studied, the maximum embryoid yield was obtained from buds 4.1-4.4 mm and 4.5-5.0 mm long, depending on the genotype. Cultivation of microspores was carried out on liquid NLN culture medium with 13% sucrose. The maximum number of embryoids (173.5 ± 7.5 pcs./Petri dish) was obtained on culture medium with pH 5.8 and heat shock at 32 °C for 48 h. Successful embryoid development and plant regeneration by direct germination from shoot apical meristem were achieved on MS culture medium with 2% sucrose and 0.7% agar, supplemented with 6-benzylaminopurine at a concentration of 1 mg/L. Analysis of the obtained regenerated plants, which successfully passed the stage of adaptation to ex vitro conditions by flow cytometry, showed that most of them were doubled haploids (up to 90.9%). A low number of seeds produced by self-fertilization in DH plants was observed.

Doubled Haploids in Eggplant.

Eggplant is a solanaceous crop cultivated worldwide for its edible fruit. Eggplant breeding programs are mainly aimed to the generation of F1 hybrids by crossing two highly homozygous, pure lines, which are traditionally obtained upon several self crossing generations, which is an expensive and time consuming process. Alternatively, fully homozygous, doubled haploid (DH) individuals can be induced from haploid cells of the germ line in a single generation. Several attempts have been made to develop protocols to produce eggplant DHs principally using anther culture and isolated microspore culture. Eggplant could be considered a moderately recalcitrant species in terms of ability for DH production. Anther culture stands nowadays as the most valuable technology to obtain eggplant DHs. However, the theoretical possibility of having plants regenerated from somatic tissues of the anther walls cannot be ruled out. For this reason, the use of isolated microspores is recommended when possible. This approach still has room for improvement, but it is largely genotype-dependent. In this review, we compile the most relevant advances made in DH production in eggplant, their application to breeding programs, and the future perspectives for the development of other, less genotype-dependent, DH technologies.

Doubled Haploidy for Fennel (Foeniculum vulgare Mill.) and Dill (Anethum graveolens L.).

Doubled haploidy technology is a powerful tool to accelerate the breeding of new crop varieties. Protocols are not universal, as even species within the same family require a specific process. Here we describe methods for developing doubled haploids for fennel and dill, both Apiaceae species which are used for food, flavorings, and medicine.

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).

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.

Isolation of Staged and Viable Maize Microspores for DH Production.

Isolated microspore culture systems have been designed in maize by several groups, mainly from the late 1980s to early 2000s. However, even with optimized protocols, microspore embryogenesis induction has remained very dependent on the genotype in maize, with elite germplasm generally displaying no response or very low response. Yet, these last few years, significant progress has been accomplished in understanding and controlling microspore embryogenesis induction in model dicot and monocot species. This knowledge may be transferred to maize, and isolated microspore culture may gain new interest in this crop, at least for embryogenesis research. The methods we hereby present in detail permit the purification of 3-12 × 105 viable microspores per maize tassel, at the favorable stage for microspore embryogenesis. When cultured in appropriate liquid media, microspores from responsive genotypes give rise to androgenic embryos, which can then be regenerated into fertile doubled haploid plants.

Species with Haploid or Doubled Haploid Protocols.

In this chapter, we present a list of species (and few interspecific hybrids) where haploids and/or doubled haploids have been published, including the method by which they were obtained and the corresponding references. This list is an update of the compilation work of Maluszynski et al. published in 2003, including new species for which protocols were not available at that time, and also novel methodologies developed during these years. The list includes 383 different backgrounds. In this book, we present full protocols to produce DHs in 43 of the species included in this list. In addition, this book includes a chapter for one species not included in the list. This makes a total of 384 species where haploids and/or DHs have been reported up to date.

Fractional Factorial Designs: An Underutilized Tool for Rapid In Vitro System Development.

Anther and microspore culture for producing haploid plants are very complex systems and include general effects where the specific effects must be identified and optimized to develop culture systems capable of producing the large numbers of haploids required by breeding programs. These general effects include genotype, physiological state of the source plant, age of the anthers and microspores, preculture treatments, culture conditions, and culture media. Design of experiments (DoE) is an experimental approach specifically designed to identify and optimize the multiple factors that make up complex systems, and is ideally suited for developing in vitro systems to produce haploids. The basic DoE strategy starts by screening multiple factors thought to affect the responses being measured. Screening identifies factors with large and small effects. Factors with large effects are used to manipulate the system, and are moved to the DoE optimization phase such as response surface methodology. Factors with small or trivial effects are eliminated from further consideration, and this simplifies the system. The basic concepts of fractional factorial designs and how to use them are explained. Fractional factorials are the most important DoE screening tool and are the first experiments run before DoE optimization experiments. To illustrate the unique properties of fractional factorials, a detailed example is provided that includes all of the calculations so that no statistical software is required.

Applications of Impedance Flow Cytometry in Doubled Haploid Technology.

Efficient doubled haploid (DH) plant production is of great interest in the plant breeding industry and research because homozygous lines are obtained within a single generation shortening the breeding cycle substantially. DH protocol development can be a time- and resource-consuming process due to numerous factors affecting its success and efficiency. Here we present concepts and examples about how critical success factors can be identified throughout a DH protocol and an early microspore response monitored by simple impedance flow cytometry (IFC) measurements, which will help to optimize each step of an androgenesis-based DH protocol.

Microspore Embryogenesis in Citrus.

This chapter deals with microspore embryogenesis in Citrus. Microspore embryogenesis allows to induce immature gametes (microspores) and to deviate them, in this case, the male one, from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos and plants).

Production of Haploid and Doubled Haploid Lines in Nut Crops: Persian Walnut, Almond, and Hazelnut.

This chapter deals with induction of haploidy via parthenogenesis in Persian walnut and via microspore embryogenesis in almond and hazelnut. Haploid induction through in situ parthenogenesis using pollination with irradiated pollen to stimulate the embryogenic development of the egg cell, followed by in vitro culture of the immature haploid embryos. Microspore embryogenesis allows the induction of immature pollen grains (microspores), to move away from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos in this case). Unlike other fruit crops (such as Citrus), regeneration of entire plants has not yet been obtained in our studied nut crops; however, it gives the methodology should be used to continue the roadmap.