Abstract
The doubled haploid system is the fastest way of hybrid variety production and plays an important role in breeding programs and developmental studies. The most commonly used methods of haploid induction, leading to haploid plants in vitro through the plant tissue/cell culture, are named as the in vitro -based (IVB) methods. These methods have been established in many of the important crops, such as barley, pepper, rapeseed, rice, sugar beet, and wheat. There are ongoing researches to optimize and improve the efficiency of these methods by focusing on factors involved in induction and regeneration phases. These factors mainly include plant genotype, the surrounding environment of parental plants, components of culture medium, the developmental stage of initial gametophytic cells, physical treatments (cold pre-treatment, heat shock) of cultured gametophytic cells, and application of different additives and plant growth regulators. Stress treatment is one of the important prerequisites for stimulation of gametophytic cells to switch towards the sporophytic pathway. However, autophagy and programmed cell death, oxidative stress, and production of reactive oxygen species (ROS) are the major limiting factors in stress-induced embryogenesis. The positive effect of different additives, such as plant growth regulators, chlormequat, polyamines (putrescine, spermidine, and spermine), stress hormones (abscisic acid, jasmonic acid, salicylic acid), DNA demethylating agents and histone deacetylase inhibitors, cellular antioxidants, cell wall remodeling agents (arabinogalactan-proteins), and compatible solutes (proline and chitosan), has been proved on the efficiency of haploid induction through IVB methods. Different mechanisms have been reported through which the aforementioned additives can enhance tolerance to embryo-inducing stresses in plants, and subsequently increase the efficiency of induction phase of IVB methods of haploid induction. Finding the best combination/interaction of inductive stresses and their corresponding chemical enhancers is crucial for successful haploid induction through IVB methods. In the present review, we highlighted recently applied additives to enhance the efficiency of the major IVB methods of haploid induction in different plants. Other potentially applicable additives, those are involved in preventing ROS accumulation, ethylene inhibitors, activating of antioxidant enzyme activity, detoxification capacity, and defense response signal pathway, which could be useful in IVB haploid induction are also discussed. The presented information could be useful to improve the efficiency of developed IVB protocols and/or to develop new protocols in recalcitrant species/genotypes.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Biotechnology has been widely used in plant breeding to accelerate and complement the conventional breeding methods. The production of F1 hybrid seeds is one of the most popular aspects of plant breeding, as has increasing importance for farmers and breeders (Ribarits et al. 2009). It is the hybrid vigor attracting plant breeders and commercial producers to create hybrid seeds. The production of inbred parental lines is the first prerequisite and one of the most difficult steps of F1 hybrid variety production, because it takes several generations of selfing/inbreeding (reviewed by Germanà 2011). The conventional plant breeding programs are tedious, time consuming, and expensive to produce inbred parental lines (Khan et al. 2017). In addition, self-incompatibility is another problem of conventional inbreeding methods to produce homozygous parents, especially in woody plants (Germanà 2006). The fastest shortcut to create inbred lines is the doubled haploidy system. In self-pollinated crops such as wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), the application of the doubled haploid (DH) methods reduces the time required for releasing a new cultivar by 3–4 years (reviewed by Shariatpanahi and Ahmadi 2016). In addition, the selection efficiency is also improved by exploiting the DH system.
The fast development of homozygous lines is the major advantage of haploid induction and subsequent DH production (reviewed by Ren et al. 2017). There are two main methods of haploid induction, including in vitro-based (IVB) and in vivo- based haploid induction systems (Fig. 1). The IVB system is based on the culture of immature male or female gametophytic cells, whereas the in vivo system is based on inter- or intraspecific hybridization and subsequent uniparental chromosome elimination (Ren et al. 2017). The IVB methods are including in vitro induced parthenogenesis (gynogenesis) and androgenesis (anther culture, isolated microspore culture, and shed microspore culture) (Fig. 1). Some valuable previous reviews addressed the principles of various methods of plant haploid induction (Forster et al. 2007; Wędzony et al. 2009; Dwivedi et al. 2015; Ren et al. 2017; Kalinowska et al. 2019).
Androgenesis is the most effective method to obtain DH plants (Wędzony et al. 2009). There are, however, some valuable genotypes of different crops, such as barley, maize (Zea mays L.), potato (Solanum tuberosum ssp. tuberosum), and rye (Secale cereale L), which are recalcitrant to this method (Maluszynski et al. 2003). Recently, different additives, including phytohormones—endogenous-produced organic compounds that are necessary for regulating plant growth and yield (Khan et al. 2020)—stress hormones, growth retardant hormones, polyamines, compatible solutes, histone deacetylase inhibitors, cellular antioxidants, and arabinogalactan-proteins, have been used to improve the efficiency of IVB methods of haploid induction, through enhancing tolerance to embryo-inducing stresses. In the present review, we first presented a general view of major IVB methods of plant haploid induction and then highlighted the recently applied chemical enhancers (additives) to improve the efficiency of IVB methods. There are some in vivo- in planta haploid induction methods, such as centromere histone H3 (CENH3), which can lead to haploidy in IVB-recalcitrant genotypes and considered as universal haploid induction systems. These methods consist of one step of pollination of a line of interest, in a target plant, with a haploid inducer line (Fig. 1). A brief summary of in vivo methods of haploid induction is provided in the last sections of this review. Advantages and disadvantages of in vivo haploid inducer line methods, compared to IVB methods, are also discussed.
Applications of doubled haploid plants
Haploid refers to a sporophytic plant with gametophytic chromosome numbers (Kasha and Maluszynski 2003). Haploids are excellent examples of cellular totipotency because they are gametophyte-originated plants containing only half of the chromosome number of a zygote and/or a somatic cell (Gilles et al. 2017; Sood and Dwivedi 2015). Haploids/doubled haploids play an important role in agricultural and developmental studies.
The completely homozygous background of DHs can be used for stable gene transformation through electroporation, microprojectile bombardment, and Agrobacterium-mediated transformation methods (Shariatpanahi and Ahmadi 2016). Gametophytic and sporophytic pathways are the two main categories for gene transformation through isolated microspore culture (Resch and Touraev 2010). In the gametophytic route, the transferred DNA can be introduced to a mature pollen or stigma before pollination or to microspores and then in vitro matured transformed pollen grains can be used for pollination, so called male germ line transformation (Fig. 2a) (Shariatpanahi and Ahmadi 2016). In the sporophytic pathway, embryogenic microspores are explants used for gene transformation, therefore genome doubling of transformed haploids can lead to completely homozygous transgenes (Fig. 2b) (Brew-Appiah et al. 2013). Microspore engineering, through genome editing methods of transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeats (CRISPR/Cas), has a great potential in plant breeding by producing doubled haploids (DHs) with desired characteristics in one season (Dwivedi et al. 2015).
Completely homozygous doubled haploid populations are worthwhile sources for molecular map constructs to analyze the quantitative trait loci (QTLs) of important quantitative agronomic traits, marker assisted selection (MAS), mutation and selection, reverse breeding (RB) (Shariatpanahi and Ahmadi 2016), and genome-wide association (GWAS) (Sanchez et al. 2018) studies.
In addition, validation of gene functions through the targeting induced local lesions in genomes (TILLING), gene overexpression, gene silencing, genome editing methods of Zinc-finger nucleases (ZFNs), TALENs, and CRISPR/Cas methods is much faster and easier by using modified homozygous genotypes that are valuable for phenotypic validation (Shen et al. 2015).
IVB methods of haploid induction
To date, two main IVB methods of haploid induction have been identified, including induced parthenogenesis and androgenesis. The induced parthenogenesis consists of in vitro rescue of parthenogenic embryos induced in situ through pollination with irradiated pollen, and gynogenesis (in vitro culture of ovaries or ovules) (Fig. 1). Female organs are the source of haploid induction in induced parthenogenesis, while the male cells/organs are responsible for haploid induction through the androgenesis pathway. The general principles of all IVB methods are discussed in the following sections.
In vitro induced parthenogenesis (gynogenesis)
In parthenogenesis, mitotic divisions of the egg cell, within the ovule, lead to the formation of an embryo that is genetically identical to the mother plant (Conner et al. 2017). Endosperm formation is required for development of the egg for the viable seed. There are two forms of induced parthenogenesis, including parthenogenesis induced by irradiated pollen and parthenogenesis induced by in vitro culture of female gametophyte (gynogenesis). In both forms, female organs are the sources of haploid induction. In the first method, pollination with defective pollen (pollen grains treated with radiation and/or chemicals) is stimulating. However, it is not well documented whether always such pollen fertilizes the egg cell, or whether it acts as not-fertilizing stimulus (pseudo-fertilization). Therefore, it is not an IVB method of haploid induction, but it is the in vitro rescue of in vivo induced embryos leading to haploid induction (Claveria et al. 2005). Although the efficiency of this category of induced parthenogenesis is lower than the androgenesis, the genetic stability of produced doubled haploids and also the absence of albino plants are the key advantages of this method (Sharma et al. 2018).
In gynogenesis, female gametophyte is responsible for haploid induction. In vitro culture of unfertilized (unpollinated) ovules or ovaries containing an unfertilized egg cell, or synergids, leads to development of haploid plants (reviewed by Bohanec 2009). This method is similar to the first form of induced parthenogenesis (induced by irradiated pollen), because female cells are the source of haploid induction but in gynogenesis, the stimulating role of the male part is not necessary. Intergeneric crossing is a useful pretreatment that can enhance gametophytic embryogenesis efficiency in the gynogenesis pathway. Piosik et al. (2016) reported the successful growth of haploid embryos in lettuce (Lactuca sativa L.) by culture of ovaries, ovules and embryo sacs after cross-pollination with Helianthus annuus and H. tuberosus. The selection of the proper flowers, containing responsive ovules, is a critical factor affecting the efficiency of gynogenesis. The genotype and surrounding environment of donor plants, physical treatments (cold pre-treatment, and heat shock), the developmental stage of female gametophyte, culture medium, plant growth regulators (PGRs), and culture conditions are the factors affecting successful haploid induction and subsequent DH production in the gynogenesis pathway (Fayos et al. 2015; Dong et al. 2016).
Androgenesis
Androgenesis is the most widely used IVB method of haploid induction (Kasha 2005). Androgenesis refers to the unusual development of cultured male reproductive units of plants, which switching their pathway from gametophytic to sporophytic (Seguí-Simarro and Nuez 2008; Makowska et al. 2017). This method can be done in three main forms, including anther culture, isolated microspore culture, and shed-microspore culture. All three methods have their own advantages and disadvantages; however, the second method (isolated microspore culture) is the most efficient one (Kasha et al. 2002; Szarejko 2003). The genotype and growth condition of donor plants are the factors that can affect the efficiency of the androgenesis pathway, similar to the gynogenesis. The developmental stage of microspores is another factor affecting androgenesis. It is mentioned that the beginning of the flowering period is the best time to harvest the anthers, while later stages will lead to the reduction of androgenesis efficiency (Dwivedi et al. 2015). The cultivation of donor plants in a controlled environmental condition can also lead to better results as compared to field cultivated plants (Datta 2005).
Anther culture
The in vitro culture of the whole immature anthers, containing microspores, is the simplest method of haploid induction through the androgenesis pathway. This method was first discovered by Guha and Maheshwari (1964).
There are many factors affect the efficiency of anther culture, including plant genotype, growth condition of donor plants, developmental stage of immature pollen grain/microspores, basal culture medium, type and concentrations of PGRs, and type and intensity of stresses (Shariatpanahi et al. 2006a; Wang et al. 2018). The thickness of the anther wall and proper bud size are the other important factors that should be considered in anther culture studies. Younger anthers have thin walls facilitating the entry of effective factors leading to a better responsiveness of its internal microspores, whereas thick walls act like a barrier (Salas et al. 2012). The position of flower buds within the inflorescence can help to isolate younger anthers before maturation, because there is a maturity gradient within the inflorescence. The identification of suitable flower buds containing microspores at the appropriate developmental stage is also very important in anther culture studies. The bud size is a selection criterion helping to better identify anthers containing microspores in a proper developmental stage. However, it is variable in different plant species (Gu et al. 2014). Wang et al. (2018) reported that callus induction in the anther culture of kiwifruit (Actinidia arguta Planch.) at the late-uninucleate stage of microspores was significantly more than those with microspores in tetrad, early-uninucleate, and binucleate stages.
Isolated microspore culture (IMC)
The mechanical isolation of microspores from anthers, using magnetic stirring/blending, and transferring them to a culture medium is the second form of androgenesis, called isolated microspore culture. IMC is the most common and efficient method of DH production (Shariatpanahi et al. 2006b). In the anther culture method, the presence of the anther wall can lead to somatic embryogenesis whereas IMC is free from this disadvantage (Ferrie and Caswell 2011). The negative effects of anther walls on microspores, production of diploid somatic callus originated from anther walls, more time-consuming and labor-intensive, preventing the availability of nutrients for developing microspores through anther walls, and limitations for tracking and studying the microspores maturation and embryo development, are the disadvantages of the anther culture method, that could be avoided in the microspore culture method (Ferrie and Caswell 2011). Rapid optimization of culture conditions, due to the direct observation of microspore embryogenesis, is another advantage of isolated microspore culture over anther culture (Lantos et al. 2009). However, external supplementing of embryogenesis factors provided by anthers, is necessary in IMC (Sood and Dwivedi 2015). Albinism is a major problem for haploid induction through androgenesis in cereals (Forster et al. 2007).
Morphological characteristics such as bud size, changes in the pattern of cytokinesis, and nuclear changes are the most common and easiest indicators for identification of the proper developmental stage of microspores (Seguí-Simarro and Nuez 2008). Nuclear changes, including decondensed chromatin pattern, forming a reticulum of small chromatin patches connected together through chromatin fibers, and chromatin condensation pattern similar to interphase nucleus of cycling cells, are the markers that can be used for early monitoring of pollen embryogenesis (Testillano et al. 2000). However, genetics also helps this process through the identification and isolation of genes and proteins involved in the embryogenesis process of isolated microspores. In rapeseed (B. napus L.), two genes, SERK1 and SERK2, were identified as the two main genes involved in microspore embryogenesis and plant regeneration, as their expression level was significantly increased during the early steps of embryo formation (Ahmadi et al. 2016). Krzewska et al. (2017) analyzed the protein profile of four winter triticale (× Triticosecale Wittm.) DH lines, using mapping of sub-proteome of anthers by two-dimensional gel electrophoresis, to understand the physiological background of microspore embryogenesis and reported that protein species of responsive DH lines (31 protein species) was significantly more than other investigated DH lines.
Shed-microspore culture (SMC)
In SMC, microspores are non-mechanically shed to a medium, after initial culture of intact anthers on a liquid (single-layer) or semi-liquid (double-layer) medium (Wędzony et al. 2009). In fact, SMC is the interstitial mode of anther and isolated microspore culture that is created by a simple modification in anther culture method. Physical damages to microspores in SMC is lower than IMC, however the efficiency of haploid induction in SMC may be less than IMC. Supena et al. (2006) developed a shed-microspore culture protocol in Indonesian hot peppers (Capsicum annuum L.) through a double-layer medium where the liquid upper layer contained zeatin and indole-3-acetic acid (IAA) of PGRs and the solid under layer contained 2% maltose plus 1% activated charcoal. They used flower buds with more than 50% late unicellular microspores and reported 76.11% haploid plants from shed-microspore culture-derived embryos. Shariatpanahi et al. (2006b) applied heat shock and starvation stresses in shed-microspore culture of two spring wheat cultivars, Falat and Rasool, and compared the results with freshly isolated microspore culture method without any stress pretreatment and reported that regeneration frequency and the percentage of green plants in freshly isolated microspore culture was significantly higher than in the SMC method. Supena and Custers (2011) reported that a reduction of incubation temperature from 28 °C to 21 °C and adding abscisic acid to the solid lower layer led to a significant improvement of their previous shed-microspore culture protocol in Indonesian hot peppers (Supena et al. 2006) so that normal-looking embryos were more than 50%.
Inductive stresses and improving the efficiency of in vitro-based methods of haploid induction
Stress treatment, applied in a variety of ways, is a common embryogenesis trigger (Shariatpanahi et al. 2006a). There are three groups of stresses, resulting in embryogenesis induction, in isolated microspore/anther cultures, including: (1) widely used stresses such as temperature (cold and heat), carbon starvation, and colchicine, (2) neglected stresses such as gamma irradiation, ethanol stress, centrifugal treatment, reduced atmospheric pressure, and abscisic acid, and (3) novel stresses such as high medium pH, carrageenan oligosaccharides, heavy metals stress, 2,4-Dichlorophenoxyacetic acid (2,4-D) pre-treatment, and chemical inducers (Shariatpanahi et al. 2006a). The positive effect of cold pretreatment (4 °C for 2 days) followed by heat shock (30 °C for 2–5 days) has been reported on microspore embryogenesis efficiency in SMC of tomato (Lycopersicon esculentum L.) (Ahmadi et al. 2015). Heidari et al. (2017) investigated the effect of cold pretreatment and heat shock on the efficiency of anther culture in sweet peppers (Capsicum annuum L.) and reported the positive effect of both cold (4 °C for 24 h) and heat (35 °C for eight days) shocks on the induction of microspore embryogenesis and regeneration of plantlets. Bhatia et al. (2016) studied the effect of two different heat shock treatments (30 and 32.5 °C) in microspore embryogenesis of cauliflower (B. oleracea var. botrytis L.) and reported the highest number of embryos per petri dish obtained by heat shock treatment at 30 °C for 24 h followed by maintenance at 25 °C.
Despite their vital role in guiding gametophytic cells towards embryogenesis, induced stresses have an adverse effect on the metabolism and growth of the regenerated plants (Pourabdollah Najafabadi et al. 2015). In addition, these stresses can lead to recombination events in nuclear genes or chloroplast genome, resulting in low regeneration and albino plants in DH plants (Shariatpanahi et al. 2006b). The selection of the suitable stresses and their concentrations/intensity and durations are the critical points that must be addressed in any target plant. Pourabdollah Najafabadi et al. (2015) compared the effects of heat shock (30 ± 0.5 °C for 14 days) and 2,4-D treatment (159.08 µM/L for 30 min) on morphological and physiological characteristics of microspores and microspore-derived DH plants of B. napus and reported that the total protein content of treated microspores was reduced by using both 2,4-D and heat shock, whereas heat shock treatment led to an increase in concentration of chlorophyll a and b of DH plants.
Although there are some internal responses to applied inductive stresses, such as increase the accumulation of proteins responsible for cell defense against oxidative stress, like L-ascorbate peroxidase and HSP70, (Krzewska et al. 2017). However, using some components that are able to reduce stress-induced cell death during microspore embryogenesis is an applicable strategy to enhance the efficiency of IVB haploid induction. There are various strategies to improve the efficiency of IVB methods of haploid induction. One of these solutions is co-culture strategy. The co-culture of isolated microspores with immature ovaries/pistils is a strategy to enhance the efficiency of microspore embryogenesis in some plant species (Żur et al. 2015). Ovary co-culture is an efficient strategy to enhance microspore embryogenesis efficiency of both monocots and dicots (Lantos et al. 2009). It seems that ovaries release active signaling molecules, which can increase microspore-derived embryo yield and improve plant regeneration efficiency or act like nurse agents (Żur et al. 2015; Lantos et al. 2009). Ovary or microspores–conditioned medium has also been applied to enhance microspore embryogenesis. The addition of conditioned medium from actively growing isolated ovaries or microspore cultures of other responsive plant genotypes can potentially enhance the embryogenesis efficiency of isolated microspore cultures in recalcitrant genotypes. Lantos et al. (2018) investigated the effect of ovary co-culture in isolated microspore culture of spelt wheat (Triticum spelta L.) and reported the positive effect of ovary co-culture on development of embryo-like structures.
It has been reported that addition of some chemical treatments to the culture medium can improve the efficiency of IVB method of haploid induction (Chen et al. 2019). The enhancement of tolerance to embryo-inductive stresses, using different chemical enhancers, is another important strategy to improve the efficiency of IVB methods of haploid induction. Here, we focused on recently applied chemical enhancers used as additive materials in IVB methods of haploid induction in different plant species.
Additives influencing IVB haploid induction
The induction phase of IVB methods of haploid induction is more important than the regeneration phase (Wędzony et al. 2009), therefore, working on this step is crucial for successful haploid induction. As aforementioned, application of some specific additives can improve tolerance to embryo-inducing stresses and subsequent lead to a significant increase in haploid induction efficiency. The main groups of these additives are i.e. PGRs, stress hormones, polyamines, compatible solutes, cellular antioxidants, and histone deacetylase inhibitors. Some of these additives are discussed in the following sections.
PGRs: Plant growth regulators—synthetic versions of phytohormones—are key signaling molecules affecting the efficiency of in vitro studies, including IVB methods of haploid induction. These materials can affect gametophyic embryogenesis and final haploid/DH regeneration through biosynthesis, distribution, gene expression or signaling pathways steps (Żur et al. 2015). Besides regulating role in development and basic metabolism, PGRs play an important role in the acclimation of living organisms to changing environment or stresses (Malaga et al. 2020). Greater production of IAA has been reported in cucumber (Cucumis sativus L.) seedlings under chilling stress, and authors mentioned that IAA acts as a downstream signal mediating in tolerance to chilling stress in cucumber (Zhang et al. 2020). The type of PGRs and the ratio of auxin:cytokinin are the two most important points in androgenesis cultures (Abdollahi and Rashidi 2018). Although it has been reported that exogenous application of PGRs is not required for microspore embryogenesis through isolated microspore cultures (Żur et al. 2015), however, pretreatment with regulators or changing the hormone composition of the culture medium can increase the efficiency of microspore embryogenesis in recalcitrant and low-responding wheat and barley genotypes (Esteves and Belzile 2018). However, the efficiency of PGRs in IVB haploid induction depends on the plant species. Among PGRs, 2,4-D has unique ability to induce somatic embryogenesis in many species, maybe because of its allosteric/electrostatic binding effects (Phillips and Garda 2019). As the same basic cellular mechanisms, cell expansion and asymmetric cell division, regulate both somatic and gametophytic embryogenesis (Esteves and Belzile 2018), 2,4-D is important in gametophytic embryogenesis. However, 2,4-D is one of the PGRs that can lead to somaclonal variation in tissue culture studies through DNA mutation and stimulation of unorganized cell growth (Niazian et al. 2017a), therefore more care and attention are required for the application of 2,4-D in IVB haploid induction studies. The positive effect of a higher concentration of 2,4-D (45.44 µM/L) in anther cultures and haploid induction of chickpeas (Cicer arietinum L.) has been reported (Abdollahi and Rashidi 2018). The external treatment of donor plants with 2,4-D has also been reported as a positive treatment in microspore embryogenesis. Nowaczyk et al. (2016) treated six hot and sweet F1 hybrids of peppers with 4.54 µM/L of 2,4-D, by applying water solution of 2,4-D on the whole plants, one day before transferring anthers into culture and reported that the number of embryos and plantlets were increased in three of the investigated hybrids. Mishra et al. (2017) reported that an equal ratio of 2,4-D: kinetin (5 μM/L), for callus induction in anther culture of Camellia assamica, and then transfer of induced calli to Murashige and Skoog (MS) medium (Murashige and Skoog 1962), supplemented with 10 μM/L 6-Benzylaminopurine (BAP) + 3 μM/L gibberellic acid (GA3) + 5474.20 µM/L l-glutamine + 1903.13 µM/L l-serine, resulted in 85% embryogenesis. The positive effect of cytokinin thidiazuron (TDZ) in combination with auxin dicamba (DIC) has been reported in gametophytic embryogenesis of cereal species (Esteves and Belzile 2018).
Stress hormones: stress hormones are another category of additives that can enhance somatic/gametophytic embryogenesis. These are hormone-like endogenous regulators, mainly produced in response to biotic and abiotic stresses, which can manage various growth and developmental process in plants such as cell division (Ahmadi et al. 2014a). These components can increase the accumulation of nitrogen and sulfur in plant cells and subsequently maintain cell membranes and photosynthetic ability under stressful conditions (Gupta and Huang 2014). Figure 3 shows the possible pathways and mechanisms through which stress hormones can enhance the efficiency of somatic/gametophytic embryogenesis. Our results showed the positive effect of stress hormones on the microspore embryogenesis of B. napus (Ahmadi et al. 2014a). In three independent experiments, different concentrations of stress hormones, including abscisic acid (ABA) (0.75, 1.89, 3.78, 7.56, and, 18.91 µM/L), jasmonic acid (JA) (0.95, 2.37, 4.75, 9.51, and 23.77 µM/L), and salicylic acid (SA) (1.44, 3.62, 7.24, 14.48, and 36.20 µM/L) were applied in different incubation periods (6, 12, and 24 h). In final, 1.89 µM/L of ABA for 12 h, 4.75 µM/L JA for 24 h, and 1.44–3.62 µM/L of SA for 6 h, were chosen as the best concentrations and incubation durations of applied stress hormones for embryogenesis in the microspore culture of B. napus (Ahmadi et al. 2014a).
Cycocel (CCC): chlormequat chloride or cycocel (CCC) is another kind of PGRs in which significant effects on stem elongation, induction and improvement of flowering, and somatic embryogenesis have been reported (Agustí and Primo-Millo 2020; Shariatpanahi et al. 2018). CCC is a growth retardant that inhibits gibberellin metabolism by blocking cyclases copalyl-diphosphate synthase and ent-kaurene synthase (Chen and Chang 2003). The CCC-mediated disrupted metabolism of gibberellin can subsequently lead to reduced stem elongation, improvement of flowering, the enhancement of multiple buds per shoot, and the enhancement of somatic/gametophytic embryogenesis (Fig. 4). Our results also showed the positive effect of CCC in in vivo haploid induction (Ebrahimzadeh et al. 2018). Different concentrations of CCC, including 407.77, 4077.77, and 40,777.71 µM/L, were applied in the parthenogenesis pathway of cucumbers and haploid embryos were significantly increased when mother plants were sprayed with 407.77 µM/L of cycocel (Ebrahimzadeh et al. 2018).
Polyamines: polyamines are the another group of additives that can be used to improve gametophytic embryogenesis. Putrescine, spermidine, and spermine are the polyamines that are present in all living organisms and are considered as key modulators of plant growth and development. This category of materials acts like PGRs and therefore can be involved in the embryogenesis process, through interaction with nitric oxide (Tiburcio and Alcázar 2018). In addition to a hormonal role, these materials can act as carbon and nitrogen reserves and therefore can control many critical developmental processes. The cationic nature of putrescine, spermidine, and spermine give them the ability of interaction with phosphate groups of DNA, anionic components of phospholipids, and also cell wall components (Kakkar and Sawhney 2002; Sakhanokho et al. 2005; Tiburcio and Alcazer 2018). Polyamines are ethylene inhibitors that can improve the efficiency of anther culture experiments by delaying the senescence of cultured anthers (Sarao and Gosal 2018). Figure 5 shows the possible mechanisms of involvement of putrescine, spermidine, and spermine in somatic and gametophytic embryogenesis. Our results showed the positive effect of polyamines in androgenesis of B. napus (Ahmadi et al. 2014b) and sweet pepper (Heidari-Zefreh et al. 2018). The effect of different concentrations of putrescine, including 2.26, 5.67, 11.34, 22.68, and 56.72 µM/L, for 12, 24, and 48 h on the microspore embryogenesis of B. napus was investigated and it has been discovered that the application of 2.26 µM/L putrescine for 48 h increased microspore embryogenesis three-fold. In addition, a normal plantlet regeneration of 92% was achieved by the application of 5.67 µM/L putrescine for 48 h. In isolated microspore culture of sweet pepper, application of 6.67–11.34 µM/L putrescine, during the mannitol starvation treatment, led to the significant increase in multicellular structures, cotyledonary embryos, and haploid regenerants (Heidari-Zefreh et al. 2018). In addition to IVB methods of haploid induction, the positive effect of putrescine and spermidine in in vivo haploid induction of cucumber (irradiated pollen induced parthenogenesis), through the spraying of mother plants, has also been reported (Ebrahimzadeh et al. 2018).
Compatible solutes: compatible solutes are another category of additives that can enhance embryogenesis in IVB methods of haploid induction. These are low molecular and highly soluble organic compounds (sugar, amino acid, nitrogen and sulphur containing compounds) accumulated in large quantities when plants are exposed to stress (Ahmadi and Shariatpanahi 2015; Handa et al. 2018). These are osmoprotectant components that can interact with crucial macromolecules, with both hydrophobic and hydrophilic domains, such as plasma membranes, antioxidative enzymes, photosynthetic elements, stabilized proteins, and critical elements of both electron transport chains (Gupta and Huang 2014). This property gives compatible solutes the ability to modulate biological processes such as embryogenesis. In addition, these reactive oxygen species (ROS) scavenging materials can enhance tolerance to embryo-inducing stress in plants (Ahmadi and Shariatpanahi 2015). All possible pathways, through which compatible solutes can enhance somatic and gametophytic embryogenesis, are presented in Fig. 6. Our investigations showed the positive effect of proline and chitosan compatible solutes in the SMC of tomato (Ahmadi et al. 2015) and in the IMC of B. napus (Ahmadi and Shariatpanahi 2015). We found a positive effect of an application of 32.76 µM/L chitosan on callogenesis and shoot regeneration in the SMC of tomato (Ahmadi et al. 2015). The effects of different concentrations of proline (434.29, 868.58, 173,716, and 4342.91 µM/L) and chitosan (6.55, 13.10, 32.76, and 65.53 µM/L) were assessed on microspore embryogenesis efficiency of B. napus and the application of 868.58 µM/L of proline and 6.55 µM/L of chitosan led to significant increase in microspore embryogenesis (Ahmadi and Shariatpanahi 2015).
DNA demethylating agents and histone deacetylase inhibitors: DNA methylation and histone methylation and acetylation are the important factors that control functional status of chromatin and subsequent regulate gene expression during cell proliferation and differentiation. Epigenetic reprogramming, mainly a global DNA methylation decrease, is one of the cellular processes that happen during stress-induced embryogenesis (Testillano 2018). The positive correlation of low levels of H3K9 methylation with microspore reprogramming and initiation of embryogenesis has been proved (Testillano 2018). In B. napus, high levels of acetylated histones H3Ac and H4Ac has been reported in vacuolated microspores (Rodríguez-Sanz et al. 2014). Therefore, using DNA demethylating agents and histone deacetylase inhibitors can improve the efficiency of IVB haploid induction. Application of a DNA demethylating agent, 5-Azacytidine (AzaC) increased embryogenesis induction in microspore culture of B. napus and barley (Solís et al. 2015). Zhang et al. (2016) investigated the effect of three different histone deacetylase inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate on microspore embryogenesis of pak choi (Brassica rapa ssp. chinensis L.). They reported that the largest embryo yield and the highest frequency of plant regeneration were achieved from NLN-13 medium supplemented with 0.05–0.1 μM of suberoylanilide hydroxamic acid. Application of BIX-01294, a small molecule that inhibits H3K9 methylation, enhanced microspore reprogramming and embryogenesis initiation in B. napus and barley (Berenguer et al. 2017). Wang et al. (2019) applied epigenetic chemicals of trichostatin A, scriptaid, BIX-01294, and sodium butyrate, as histone deacetylase inhibitors, in microspore culture of wheat and reported that only trichostatin A could enhance both embryogenesis and green plant regeneration.
Cellular antioxidants: Maintaining the balance of ROS is very important for plant cell proliferation and differentiation. Antioxidants are one of the most important components to maintain ROS balance, by scavenging cellular ROS accumulation (Chen et al. 2020). The positive effect of low-molecular weight antioxidant of glutathione on microspore embryogenesis and increase the number of embryo-like structures has been reported in isolated microspore cultures of triticale (Żur et al. 2019). Other materials with antioxidant characteristics, such as L-ascorbic acid, can increase the antioxidant enzyme activities and antioxidant contents of treated cells (Chen et al. 2020). Heidari-Zefreh et al. (2018) reported that application of optimum concentrations of ascorbic acid (20 and 50 mg/L) under the mannitol starvation and heat shock treatment (32 °C) led to the significant increase in the number of cotyledonary embryos produced in isolated microspore culture of sweet pepper. In isolated microspore culture of broccoli (Brassica oleracea L. var. italica), the embryogenesis efficiency, under heat shock stress (32.5 °C for 24 h) was 1.2-fold and 2.5-fold increased by application of 10 mg/L of reduced ascorbate and 20 mg/L of reduced glutathione, respectively (Zeng et al. 2017). In microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L.), a 10.33-fold increase of frequency of embryogenesis was reported when L-ascorbic acid sodium salt (0.2 µM) was added to NLN-13 medium (Niu et al. 2019). Methylene blue is another kind of antioxidants whose positive effect has been reported in microspore embryogenesis of ornamental kale (Brassica oleracea var. acephala) (Chen et al. 2019).
Cell wall remodeling agents: Arabinogalactan-proteins (AGPs)—a type of cell surface glycoproteins enriched in arabinose and galactose residues—are a group of cell wall proteins that involved in cell growth, division and expansion, embryo pattern formation, modulation of cell wall mechanics or defense. Changes in the composition of these cell wall proteins have been reported during both microspore embryogenesis (Corral-Martínez et al. 2019) and somatic embryogenesis (Pérez-Pérez et al. 2019). It is modifications of AGPs and pectins, as two main components of cell wall, which causes remodeling of cell wall during somatic embryogenesis (Pérez-Pérez et al. 2019). Cell wall remodeling is one of the cellular processes that involved in stress-induced microspore embryogenesis. AGPs are involved in first embryogenic divisions of the microspore (Testillano 2018). The stimulating effect of AGPs, secreted from cells into the culture medium, on embryo development has been reported in microspore culture of maize (Testillano et al. 2010). Makowska et al. (2017) reported the regeneration rate of up to 2.8 times in the anther culture of barley (Hordeum vulgare L.) by adding 37.54 µM/L gum arabic, as a carrier of AGPs. In anther culture of tomato it was found that gum arabic has more important role than cold pretreatment and kinetin in callus induction and regeneration (Niazian et al. 2019).
Other applied additives: Silver nitrate, polyvinylpyrrolidone (PVP), colchicine antimitotic agent, and activated charcoal are other additives used for enhancing gametophytic embryogenesis.
Silver nitrate (AgNO3) is an effective anti-browning additive that can promote in vitro regeneration through inhibiting ethylene production and phenolic secretion (Kumar et al. 2016). Silver nitrate was reported to have positive effect in induction of morphogenic callus in anther culture of rice (Sarao and Gosal 2018). Abdollahi and Rashidi (2018) reported a positive and significant effect of silver nitrate (88.75 µM/L) on the number of regenerated embryos and final regenerated plants in the anther culture of recalcitrant chickpeas.
Another anti-browning agent with antioxidant activity is PVP (Phillips and Garda 2019). The positive effect of PVP on somatic embryogenesis has been reported previously (Parthibhan et al. 2018). Callus browning was reduced, and subsequently the androgenic response was significantly enhanced, when PVP (2 mg/L) was added to culture media in anther culture of borage (Borago officinalis L.) (Abdoallahi et al. 2017).
The positive effect of the antimitotic agent, colchicine, in the anther culture of borage (Borago officinalis L.) has been reported (Hoveida et al. 2017), however, its application was not effective in inducing embryo-like structures and green plants in the anther culture of winter and spring triticale hybrids (Ślusarkiewicz-Jarzina et al. 2017).
One of the signaling molecules involved in stress response and activation of the embryogenic program is calcium (Ca2+). Rivas-Sendra et al. (2017a) reported a dramatic increase in Ca2+ level during in vitro-induced microspore embryogenesis of B. napus and progressively decrease in its level after the heat shock-based inductive treatment. These results reflect the relationship between changes in Ca2+ level and subcellular distribution, and microspore embryogenesis (Rivas-Sendra et al. 2017a). Application of calcium in culture medium can improve the efficiency of microspore embryogenesis (Ahmadi et al. 2018).
In addition to applied chemical enhancers, there are other additives that participate in biotic and abiotic stress tolerance. Materials that involve in preventing accumulation of ROS, ethylene and gibberellin inhibitors, and activators of antioxidant enzyme activity, detoxification capacity, and defense response signal pathway, could potentially be used against applied embryo-inducing stresses in IVB haploid induction (Fig. 7). In addition to aforementioned ROS scavenging materials, another component that can decrease the stress caused by ROS is boron (H3BO3), as its positive effect has been reported in microspore embryogenesis of B. napus (Mahasuk et al. 2017).
Besides aforementioned phytohormones, including auxins, cytokinins, abscisic acid, salicylic acid, polyamines, and jasmonates, other phytohormones such as brassinosteroids and strigolactones are involved in imparting stress tolerance in plants (Khan et al. 2020). The positive effect of hydrogen sulfide and brassinosteroids has been reported during chilling stress (Fang et al. 2019; Zhang et al. 2020).
Other gibberellin inhibitors, such as triiodobenzoic acid, benzothiazole-2-oxyacetate, and N-dimethylamino succinamic acid (Agustí and Primo-Millo 2020), are applicable in IVB haploid induction.
Some recent IVB haploid induction studies where different additives have been used, in response to applied inductive stresses, to enhance the regeneration and final haploid induction efficiency in different plant species are presented in Table 1.
In vivo haploid induction methods
The alternative methods of haploid induction, which avoid in vitro procedures, are in vivo methods of haploid induction (Watts et al. 2018). Irradiated pollen induced parthenogenesis, intra and interspecific hybridization, and haploid inducer line methods, are the main in vivo approaches of haploid induction (Fig. 1). One of the recently highlighted in vivo haploid inducer line methods is known as “targeted centromere manipulation (CENH3) method”. The centromeric loss of CENH3 protein is responsible for centromere inactivity and uniparental elimination of H. bulbosum (Sanei et al. 2011). The targeted manipulation of CENH3 can lead to a defective spindle attachment and subsequently haploid inducer line in different crops (Ishii et al. 2016), similar to the inducer line in maize. Haploid induction through CENH3 method has been reported in maize (Kelliher et al. 2016), carrot (Daucus carota) (Dunemann et al. 2018), tomato, rice, cucumber, and melon (Kalinowska et al. 2019). State-of-the-art and novel developments of in vivo haploid induction methods has been discussed in detail by Kalinowska et al. (2019) and Watts et al. (2018).
IVB versus in planta CENH3 method
IVB methods of haploid induction have their own specific complexities. Huge numbers of factors are involved in induction phase of these methods. In addition, optimization of in vitro regeneration is a complicated and difficult step. These are highly technical, labor-intensive, time and costly consuming procedures, on top of their species and genotype dependency and low rate of haploid induction (Dwivedi et al. 2015).
The efficiency of the in vivo CENH3 method can be divided into two steps. The first step is to develop a haploid inducer line and the second step is the cross of the haploid inducer line with a wild-type parent. Haploid inducer line either must be found in TILLING population or created through genetic engineering methods. Both pathways have their own difficulties. In vitro regeneration and gene transformation need to be optimized for second pathway of haploid inducer line production (genetic engineering). For gene transformation, Agrobacterium-mediated is the most effective method, which can be done in both in vitro and in planta forms (Niazian et al. 2017b).The second step of haploid induction through haploid inducer line methods is also critical. The cross of produced haploid inducer and wild-type plants is important. In the CENH3 method, it is suggested that the maternal haploid inducer and paternal wild-type can lead to better results (Ravi and Chan 2010).In both in vitro and in vivo methods of haploid induction we faced a complex and multifactorial situation (Żur et al. 2019). However, there are some automated and predictive methods that can help us to a better management of multivariable researches and increase the efficiency of in vitro studies, including image processing, algorithm genetics, and artificial neural networks (Abdipour et al. 2018; Hesami et al. 2019; Niazian et al. 2018a, b, c). A coupled image processing-artificial neural network system has been used to find the best developmental stage of donor plants and most effective factor on callus induction and regeneration in anther culture of tomato (Niazian et al. 2019).
Some advantages and disadvantages of the IVB methods versus in vivo-in planta haploid inducer line methods are presented in Table 2.
Conclusions
The completely homozygous genome of DHs is a valuable platform for many research areas, including hybrid varieties production, genetic transformation, RB, linkage maps in QTL, GWAS, and marker assisted selection studies. In addition, the homozygous DH lines are suitable substrates for new methods of plant gene function programs including TILLING, TALENs, ZFNs, and CRISPR/Cas. There are two main categories of plant haploid induction including in vitro and in vivo methods. IVB methods of haploid induction consist of induction and regeneration phases. Application of inductive stresses is crucial for haploid induction. However, cell death, oxidative stress and production of ROS are the consequences of inductive stress for gametophytic embryogenesis. Autophagy, cell death, epigenetic modifications, and cell wall remodeling are different cellular processes involved in stress-induced embryogenesis. Treatment with some additives, whose effects are modulators of the involved cellular processes, can regulate and increase the efficiency of gametophytic embryogenesis. Application of some components that are able to reduce stress-induced cell death during microspore embryogenesis, such as autophagy and/or protease modulators, DNA demethylating agents and histone deacetylase inhibitors, and cell wall remodeling agents can enhance the efficiency of IVB haploid induction. In addition, using some chemical additives can enhance tolerance to inductive stress and improve the efficiency of IVB methods of haploid induction. In recent years, the positive effect of some additives (chemical enhancers), including PGRs, stress hormones, polyamines, compatible solutes, cellular antioxidants, silver nitrate, colchicine, ethylene inhibitors, and active charcoal, has been reported in IVB haploid induction of different plant species. Knowledge about mechanisms through which these additives increase tolerance to inductive stresses can help researchers choose other potentially applicable additives and enhance efficiency of IVB-recalcitrant genotypes of important crops. Selection of proper embryo-inducing stress and its corresponding additive(s) is crucial for improving the efficiency of IVB methods. It is difficult to find the proper combination of inductive stress-additive(s), because of multifactorial nature of IVB studies. However, some modeling methods, such as artificial neural network, can predict and facilitate this process.
Abbreviations
- AGPs:
-
Arabinogalactan-proteins
- BAP:
-
6-Benzylaminopurine
- CCC:
-
Chlormequat chloride
- CENH3:
-
Centromere histone H3
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- Cas:
-
CRISPR-associated
- DH:
-
Doubled haploid
- GA3 :
-
Gibberellic acid
- IAA:
-
Indole-3-acetic acid
- IMC:
-
Isolated microspore culture
- IVB:
-
In vitro-based
- GWAS:
-
Genome-wide association
- MS:
-
Murashige and Skoog medium
- NAA:
-
1-Naphthaleneacetic acid
- PGR:
-
Plant growth regulator
- QTL:
-
Quantitative trait loci
- RB:
-
Reverse breeding
- ROS:
-
Reactive oxygen species
- SMC:
-
Shed microspore culture
- TALENs:
-
Transcription activator-like effector nucleases
- TDZ:
-
Thidiazuron
- TILLING:
-
Targeting induced local lesions in genomes
- ZFNs:
-
Zinc-finger nucleases
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
References
Abdipour M, Ramazani SHR, Younessi-Hmazekhanlu M, Niazian M (2018) Modeling oil content of sesame (Sesamum indicum L.) using artificial neural network and multiple linear regression approaches. J Am Oil Chem’ Soc 95:283–297
Abdollahi MR, Rashidi S (2018) Production and conversion of haploid embryos in chickpea (Cicer arietinum L.) anther cultures using high 2,4-D and silver nitrate containing media. Plant Cell Tissue Organ Cult 133(1):39–49
Abdollahi MR, Eshaghi ZC, Majdi M (2017) Improvement in androgenic response of borage (Borago officinalis L.) cultured anthers using antibrowning agents and picloram. Turk J Biol 41(2):354–363
Agustí M, Primo-Millo E (2020) Flowering and fruit set. In: Talon M, Caruso M, Gmitter FG (eds) The genus citrus. Woodhead Publishing, Cambridge, pp 219–244
Ahmadi B, Shariatpanahi ME (2015) Proline and chitosan enhanced efficiency of microspore embryogenesis induction and plantlet regeneration in Brassica napus L. Plant Cell Tissue Organ Cult 123(1):57–65
Ahmadi B, Shariatpanahi ME, da Silva JAT (2014a) Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tissue Organ Cult 116(3):343–351
Ahmadi B, Shariatpanahi ME, Ojaghkandi MA, Heydari AA (2014b) Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaxime and vancomycin in Brassica napus L. Plant Cell Tissue Organ Cult 118(3):497–505
Ahmadi B, Shariatpanahi ME, Asghari-Zakaria R, Zare N, Azadi P (2015) Efficient microspore embryogenesis induction in tomato (Lycopersicon esculentum Mill) using shed microspore culture. J Pure Appl Microbiol 9(2):21–29
Ahmadi B, Masoomi-Aladizgeh F, Shariatpanahi ME, Azadi P, Keshavarz-Alizadeh M (2016) Molecular characterization and expression analysis of SERK1 and SERK2 in Brassica napus L: implication for microspore embryogenesis and plant regeneration. Plant Cell Rep 35(1):185–193
Ahmadi B, Ahmadi M, da Silva JAT (2018) Microspore embryogenesis in Brassica: calcium signaling, epigenetic modification, and programmed cell death. Planta 248(6):1339–1350
Amirian R, Hojati Z, Azadi P (2020) Male flower induction significantly affects androgenesis in cucumber (Cucumis sativus L.). J Hortic Sci Biotech 95(2):183–191
Begheyn RF, Roulund N, Vangsgaard K, Kopecký D, Studer B (2017) Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (Lolium perenne L.). Plant Cell Tissue Organ Cult 130(3):667–679
Berenguer E, Solís MT; Pérez-Pérez Y, Minina Y, Risueño MC, Bozhkov P, Testillano PS (2017) Metacaspases and autophagy are induced in microspore embryogenesis of Brassica napus. In: Proceedings 2nd meeting WG3 transautophagy cost action CA15138, Madrid, 23–24 Mar 2017
Bhatia R, Dey SS, Sood S, Sharma K, Sharma VK, Parkash C, Kumar R (2016) Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B oleracea var botrytis L) in India. Euphytica 212(3):439–454
Bohanec B (2009) Doubled haploids via gynogenesis. In: Touraev A, Forster BP, Jain SM (eds) Advances in haploid production in higher plants. Springer, Dordrecht
Brew-Appiah RA, Ankrah N, Liu W, Konzak CF, von Wettstein D, Rustgi S (2013) Generation of doubled haploid transgenic wheat lines by microspore transformation. PLoS One 8(11):e80155
Chen JT, Chang WC (2003) Effects of GA3, ancymidol, cycocel and paclobutrazol on direct somatic embryogenesis of Oncidium in vitro. Plant Cell Tissue Organ Cult 72(1):105–108
Chen W, Zhang Y, Ren J, Ma Y, Liu Z, Hui F (2019) Effects of methylene blue on microspore embryogenesis and plant regeneration in ornamental kale (Brassica oleracea var. acephala). Sci Hortic 248:1–7
Chen H, Hao H, Han C, Wang H, Wang Q, Chen M, Juan J, Feng Z, Zhang J (2020) Exogenous l-ascorbic acid regulates the antioxidant system to increase the regeneration of damaged mycelia and induce the development of fruiting bodies in Hypsizygus marmoreus. Fungal Biol. 1:1. https://doi.org/10.1016/j.funbio.2020.02.010
Claveria E, Garcia-Mas J, Dolcet-Sanjuan R (2005) Optimization of cucumber doubled haploid line production using in vitro rescue of in vivo induced parthenogenic embryos. J Am Soc Hortic Sci 130(4):555–560
Conner JA, Podio M, Ozias-Akins P (2017) Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes. Plant Reprod 30(1):41–52
Corral-Martínez P, Driouich A, Seguí-Simarro JM (2019) Dynamic changes in arabinogalactan-protein, pectin, xyloglucan and xylan composition of the cell wall during microspore embryogenesis in Brassica napus. Front Plant Sci 10:332
Datta SK (2005) Androgenic haploids: factors controlling development and its application in crop improvement. Curr Sci 89:1870–1878
Dong YQ, Zhao WX, Li XH, Liu XC, Gao NN, Huang JH, Wang WY, Xu XL, Tang ZH (2016) Androgenesis, gynogenesis, and parthenogenesis haploids in cucurbit species. Plant Cell Rep 35(10):1991–2019
Dunemann F, Unkel K, Sprink T (2018) Using CRISPR/Cas9 to produce haploid inducers of carrot through targeted mutations of centromeric histone H3 (CENH3). In: Grzebelus D, Barański R (eds) II international symposium on carrot and other apiaceae. ISHS, Leuven, pp 211–220
Dwivedi SL, Britt AB, Tripathi L, Sharma S, Upadhyaya HD, Ortiz R (2015) Haploids: constraints and opportunities in plant breeding. Biotechnol Adv 33(6):812–829
Ebrahimzadeh H, Shariatpanahi ME, Ahmadi B, Soltanloo H, Lotfi M, Zarifi E (2018) Efficient parthenogenesis induction and in vitro haploid plant regeneration in cucumber (Cucumis sativus L.) using putrescine, spermidine, and cycocel. J Plant Growth Regul 37(4):1127–1134
Esteves P, Belzile FJ (2018) TDZ in cereal gametic embryogenesis. In: Ahmad N, Faisal M (eds) Thidiazuron: from urea derivative to plant growth regulator. Springer, Singapore
Fang P, Yan M, Chi C, Wang M, Zhou Y, Zhou J, Shi K, Xia X, Foyer CH, Yu J (2019) Brassinosteroids act as a positive regulator of photoprotection in response to chilling stress. Plant Physiol 180:2061–2076
Fayos O, Vallés MP, Garcés-Claver A, Mallor C, Castillo AM (2015) Doubled haploid production from Spanish onion (Allium cepa L.) germplasm: embryogenesis induction, plant regeneration and chromosome doubling. Front Plant Sci 6:384. https://doi.org/10.3389/fpls.2015.00384
Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104(3):301–309
Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12(8):368–375
Germanà MA (2006) Doubled haploid production in fruit crops. Plant Cell Tissue Organ Cult 86(2):131–146
Germanà MA (2011) Anther culture for haploid and doubled haploid production. Plant Cell Tissue Organ Cult 104(3):283–300
Gilles LM, Martinant JP, Rogowsky PM, Widiez T (2017) Haploid induction in plants. Curr Biol 27(20):1095–1097
Gu H, Sheng X, Zhao Z, Yu H, Wang J (2014) Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra. Vitro Cell Dev Biol Plant 50(5):534–540
Guha S, Maheshwari SC (1964) In vitro production of embryos from anthers of Datura. Nature 204:497–498
Gupta B, Huang B (2014) Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. Int J Genomics 1:1–18
Handa N, Kohli SK, Kaur R, Sharma A, Kumar V, Thukral AK, Arora S, Bhardwaj R (2018) Role of compatible solutes in enhancing antioxidative defense in plants exposed to metal toxicity. In: Hasanuzzaman M, Nahar K, Fujita M (eds) Plants under metal and metalloid stress. Springer, Singapore
Heidari AA, Shariatpanahi ME, Mousavi A, Kalatejari S (2017) Efficient androgenic embryo induction and plant regeneration in different genotypes of sweet pepper via anther culture. J Pure Appl Microbio 11(1):23–29
Heidari-Zefreh AA, Shariatpanahi ME, Mousavi A, Kalatejari S (2018) Enhancement of microspore embryogenesis induction and plantlet regeneration of sweet pepper (Capsicum annuum L.) using putrescine and ascorbic acid. Protoplasma 256(1):13–24
Hesami M, Naderi R, Tohidfar M (2019) Modeling and optimizing in vitro sterilization of chrysanthemum via multilayer perceptron-non-dominated sorting genetic algorithm-II (MLP-NSGAII). Front Plant Sci 10:282
Hoveida ZS, Abdollahi MR, Mirzaie-Asl A, Moosavi SS, Seguí-Simarro JM (2017) Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress. Plant Cell Tissue Organ Cult 130(2):369–378
Ishii T, Karimi-Ashtiyani R, Houben A (2016) Haploidization via chromosome elimination: means and mechanisms. Annu Rev Plant Biol 67:421–438
Jiang F, Ryabova D, Diedhiou J, Hucl P, Randhawa H, Marillia EF, Foroud NA, Eudes F, Kathiria P (2017) Trichostatin A increases embryo and green plant regeneration in wheat. Plant Cell Rep 36(11):1701–1706
Kakkar RK, Sawhney VK (2002) Polyamine research in plants—a changing perspective. Physiol Plant 116(3):281–292
Kalinowska K, Chamas S, Unkel K, Demidov D, Lermontova I, Dresselhaus T, Kumlehn J, Dunemann F, Houben A (2019) State-of-the-art and novel developments of in vivo haploid technologies. Theor Appl Genet 132:593–605
Kasha KJ (2005) Chromosome doubling and recovery of doubled haploid plants. In: Don Palmer C, Keller WA, Kasha KJ (eds) Haploids in crop improvement II: biotechnology in agriculture and forestry, vol 56. Springer, Berlin
Kasha KJ, Maluszynski M (2003) Production of doubled haploids in crop plants: an introduction. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Springer, Dordrecht
Kasha KJ, Simion E, Oro R, Yao QA, Hu TC, Carlson AR (2002) An improved in vitro technique for isolated microspore culture of barley. In: Maluszynski M, Kasha KJ (eds) Mutations, in vitro and molecular techniques for environmentally sustainable crop improvement. Springer, Dordrecht
Kästner U, Kittler J, Marthe F (2016) Comparison of in vitro haploid induction in balm (Melissa officinalis). Plant Cell Tissue Organ Cult 126(3):561–566
Kelliher T, Starr D, Wang W, McCuiston J, Zhong H, Nuccio ML, Martin B (2016) Maternal haploids are preferentially induced by CENH3-tailswap transgenic complementation in maize. Front Plant Sci 7:414
Khan H, Bhardwaj SC, Gangwar OP, Prasad P, Rathore R (2017) Efficiency of double haploid production in wheat through wide hybridization and embryo rescue. Indian J Genet Plant Breed 77(3):428–430
Khan N, Bano A, Ali S, Babar MA (2020) Crosstalk amongst phytohormones from planta and PGPR under biotic and abiotic stresses. Plant Growth Regul 1:1–15
Krzewska M, Gołębiowska-Pikania G, Dubas E, Gawin M, Żur I (2017) Identification of proteins related to microspore embryogenesis responsiveness in anther cultures of winter triticale (× Triticosecale Wittm.). Euphytica 213(8):192
Kumar GP, Sivakumar S, Siva G, Vigneswaran M, Kumar TS, Jayabalan N (2016) Silver nitrate promotes high-frequency multiple shoot regeneration in cotton (Gossypium hirsutum L.) by inhibiting ethylene production and phenolic secretion. Vitro Cell Dev Biol Plant 52(4):408–418
Kurtar ES, Balkaya A, Ozer MO (2018) Production of callus mediated gynogenic haploids in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.). Czech J Genet Plant Breed 54(1):9–16
Lantos C, Juhász AG, Somogyi G, Ötvös K, Vági P, Mihály R, Kristóf Z, Somogyi N, Pauk J (2009) Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Plant Cell Tissue Organ Cult 97(3):285–293
Lantos C, Bóna L, Nagy É, Békés F, Pauk J (2018) Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (Triticum spelta L.) genotypes. Plant Cell Tissue Organ Cult 133(3):385–393
Li Q, Shi Y, Wang Y, Liu L, Zhang X, Chen X, Zhang L, Su Y, Zhang T (2020) Breeding of cabbage lines resistant to both head splitting and fusarium wilt via an isolated microspore culture system and marker-assisted selection. Euphytica 216(2):34
Mahasuk P, Kullik AS, Iqbal MC, Möllers C (2017) Effect of boron on microspore embryogenesis and direct embryo to plant conversion in Brassica napus (L.). Plant Cell Tissue Organ Cult 130(2):443–447
Makowska K, Kałużniak M, Oleszczuk S, Zimny J, Czaplicki A, Konieczny R (2017) Arabinogalactan proteins improve plant regeneration in barley (Hordeum vulgare L.) anther culture. Plant Cell Tissue Organ Cult 131(2):247–257
Malaga S, Janeczko A, Janowiak F, Waligórski P, Oklestkova J, Dubas E, Krzewska M, Nowicka A, Surówka E, Rapacz M, Wójcik-Jagła M (2020) Involvement of homocastasterone, salicylic and abscisic acids in the regulation of drought and freezing tolerance in doubled haploid lines of winter barley. Plant Growth Regul 90(1):173–188
Maluszynski M, Kasha KJ, Forster BP, Szarejko I (2003) Doubled haploid production in crop plants. Springer, Dordrecht
Mishra VK, Bajpai R, Chaturvedi R (2017) An efficient and reproducible method for development of androgenic haploid plants from in vitro anther cultures of Camellia assamica ssp assamica (Masters). Vitro Cell Dev Biol Plant 53(3):239–248
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497
Naik N, Rout P, Umakanta N, Verma RL, Katara JL, Sahoo KK, Singh ON, Samantaray S (2017) Development of doubled haploids from an elite indica rice hybrid (BS6444G) using anther culture. Plant Cell Tissue Organ Cult 128(3):679–689
Niazian M, Sadat Noori SA, Galuszka P, Tohidfar M, Mortazavian SMM (2017a) Genetic stability of regenerated plants via indirect somatic embryogenesis and indirect shoot regeneration of Carum copticum L. Ind Crop Prod 97:330–337
Niazian M, Sadat Noori SA, Galuszka P, Mortazavian SMM (2017b) Tissue culture-based Agrobacterium-mediated and in planta transformation methods. Czech J. Genet. Plant Breed 53(4):133–143
Niazian M, Sadat-Noori SA, Abdipour M, Tohidfar M, Mortazavian SMM (2018a) Image processing and artificial neural network-based models to measure and predict physical properties of embryogenic callus and number of somatic embryos in ajowan (Trachyspermum ammi (L.) Sprague). Vitro Cell Dev Biol Plant 54:54–68
Niazian M, Sadat-Noori SA, Abdipour M (2018b) Modeling the seed yield of Ajowan (Trachyspermum ammi L.) using artificial neural network and multiple linear regression models. Ind Crop Prod 117:224–234
Niazian M, Sadat-Noori SA, Abdipour M (2018c) Artificial neural network and multiple regression analysis models to predict essential oil content of ajowan (Carum copticum L.). J Appl Res Med Aroma Plants 9:124–131
Niazian M, Shariatpanahi ME, Abdipour M, Oroojloo M (2019) Modeling callus induction and regeneration in anther culture of tomato (Lycopersicon esculentum L.) using image processing and artificial neural network method. Protoplasma 56(5):1317–1332
Niu L, Shi F, Feng H, Zhang Y (2019) Efficient doubled haploid production in microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee). Sci Hortic 245:57–64
Nowaczyk L, Nowaczyk P, Olszewska D (2016) Treating donor plants with 2, 4-dichlorophenoxyacetic acid can increase the effectiveness of induced androgenesis in Capsicum spp. Sci Hortic 205:1–6
Parthibhan S, Rao MV, Da Silva JT, Kumar TS (2018) Somatic embryogenesis from stem thin cell layers of Dendrobium aqueum. Biol Plantarum 62(3):439–450
Pazuki A, Aflaki F, Gürel E, Ergül A, Gürel S (2018a) Gynogenesis induction in sugar beet (Beta vulgaris) improved by 6-benzylaminopurine (BAP) and synergized with cold pretreatment. Sugar Tech 20(1):69–77
Pazuki A, Aflaki F, Gürel S, Ergül A, Gürel E (2018b) Production of doubled haploids in sugar beet (Beta vulgaris): an efficient method by a multivariate experiment. Plant Cell Tissue Organ Cult 132(1):85–97
Pérez-Pérez Y, Carneros E, Berenguer E, Solís MT, Bárány I, Pintos B, Gómez-Garay A, Risueño MC, Testillano PS (2019) Pectin de-methylesterification and AGP increase promote cell wall remodeling and are required during somatic embryogenesis of Quercus suber. Front Plant Sci 9:1915
Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. Vitro Cell Dev Biol Plant 55(3):242–257
Piosik L, Zenkteler E, Zenkteler M (2016) Development of haploid embryos and plants of Lactuca sativa induced by distant pollination with Helianthus annuus and H. tuberosus. Euphytica 208(3):439–451
Popova T, Grozeva S, Todorova V, Stankova G, Anachkov N, Rodeva V (2016) Effects of low temperature, genotype and culture media on in vitro androgenic answer of pepper (Capsicum annuum L.). Acta Physiol Plant 38:273
Pourabdollah Najafabadi F, Shariatpanahi ME, Ahmadi B, Sima NKK, Alizadeh B, Oroojloo M (2015) Effects of heat shock and 2,4-D treatment on morphological and physiological characteristics of microspores and microspore-derived doubled haploid plants in Brassica napus L. Iran J Biotechnol 13(2):31–38
Ravi M, Chan SW (2010) Haploid plants produced by centromere-mediated genome elimination. Nature 464(7288):615–618
Ren J, Wu P, Trampe B, Tian X, Lübberstedt T, Chen S (2017) Novel technologies in doubled haploid line development. Plant Biotechnol J 15(11):1361–1370
Resch T, Touraev A (2010) Pollen transformation technologies. In: Stewart N, Touraev A, Citovsky V, Tzfira T (eds) Plant Transformation Technologies. Wiley, New York, pp 83–91
Ribarits A, Mamun ANK, Li S, Resch T, Fiers M, Heberle-Bors E, Liu CM, Touraev A (2009) A Novel and reversible male sterility system using targeted inactivation of glutamine synthetase and doubled haploidy. In: Touraev A, Forster BP, Jain SM (eds) Advances in Haploid Production in Higher Plants. Springer, Dordrecht
Rivas-Sendra A, Calabuig-Serna A, Seguí-Simarro JM (2017a) Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena. Front Plant Sci 8:1177
Rivas-Sendra A, Campos-Vega M, Calabuig-Serna A, Seguí-Simarro JM (2017b) Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica 213(4):89
Rodríguez-Sanz H, Moreno-Romero J, Solís MT, Kohler C, Risueño MC, Testillano PS (2014) Changes in histone methylation and acetylation during microspore reprogramming to embryo-genesis occur concomitantly with Bn HKMT and Bn HAT expression and are associated with cell totipotency, proliferation, and differentiation in Brassica napus. Cytogenet Genome Res 143:209–218
Sakhanokho HF, Ozias-Akins P, May OL, Chee PW (2005) Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton. Plant Cell Tissue Organ Cult 81(1):91–95
Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184(2):235–250
Sanchez DL, Liu S, Ibrahim R, Blanco M, Lübberstedt T (2018) Genome-wide association studies of doubled haploid exotic introgression lines for root system architecture traits in maize (Zea mays L.). Plant Sci 268:30–38
Sanei M, Pickering R, Kumke K, Nasuda S, Houben A (2011) Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. PNAS 108(33):E498–E505
Sarao NK, Gosal SS (2018) In vitro androgenesis for accelerated breeding in rice. In: Gosal S, Wani S (eds) Biotechnologies of Crop Improvement, vol 1. Springer, Cham
Seguí-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol Plant 134(1):1–12
Shariatpanahi ME, Ahmadi B (2016) Isolated microspore culture and its applications in plant breeding and genetics. In: Anis M, Ahmad N (eds) Plant tissue culture: propagation, conservation and crop improvement. Springer, Singapore, pp 487–507
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006a) Stresses applied for the re-programming of plant microspores towards in vitro embryogenesis. Physiol Plant 127(4):519–534
Shariatpanahi ME, Belogradova K, Hessamvaziri L, Heberle-Bors E, Touraev A (2006b) Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment. Plant Cell Rep 25(12):1294–1299
Shariatpanahi ME, Ebrahimzadeh H, Niazian M, Eskandari A, Ahmadi B (2018) Amino acids and cycocel application to enhance cucumber haploid embryogenesis with gamma irradiated pollen. FAO/IAEA International Symposium on Plant Mutation Breeding and Biotechnology; Vienna (Austria); IAEA-CN–263-93
Sharma S, Satardekar KV, Barve SS (2018) Genetic improvement of medicinal and aromatic plants through haploid and double haploid development. In: Kumar N (ed) Biotechnological Approaches for Medicinal and Aromatic Plants. Springer, Singapore, pp 523–556
Shen Y, Pan G, Lübberstedt T (2015) Haploid strategies for functional validation of plant genes. Trends Biotechnol 33(10):611–620
Ślusarkiewicz-Jarzina A, Pudelska H, Woźna J, Pniewski T (2017) Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants. J Appl Genet 58(3):287–295
Solís MT, El-Tantawy AA, Cano V, Risueño MC, Testillano PS (2015) 5-Azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley. Front Plant Sci 6:472
Sood S, Dwivedi S (2015) Doubled haploid platform: an accelerated breeding approach for crop improvement. In: Bahadur B, Venkat Rajam M, Sahijram L, Krishnamurthy K (eds) Plant Biology and Biotechnology, Volume II: Plant Genomicsand Biotechnology. Springer, New Delhi, pp 89–111
Supena EDJ, Custers JBM (2011) Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper (Capsicum annuum L.). Sc Hortic 130(4):769–774
Supena EDJ, Suharsono S, Jacobsen E, Custers JBM (2006) Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25(1):1–10
Szarejko I (2003) Anther culture for doubled haploid production in barley (Hordeum vulgare L.). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled Haploid Production in Crop Plants. Springer, Dordrecht
Testillano PS (2018) Stress-induced microspore embryogenesis in crop plants: cell totipotency acquisition and embryo development. In: Cánovas F, Lüttge U, Leuschner C, Risueño MC (eds) Progress in Botany, vol 81. Springer, Cham
Testillano PS, Coronado MJ, Seguı JM, Domenech J, González-Melendi P, Raška I, Risueño MC (2000) Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis. J Struct Biol 129(2–3):223–232
Testillano PS, Coronado MJ, Thierry AM, Matthys-Rochon E, Risueño MC (2010) In situ detection of Esr proteins secretion during maize microspore embryogenesis and their secretion blockage show effects on the culture progression. Funct Plant Biol 37:985–994
Tiburcio AF, Alcázar R (2018) Potential applications of polyamines in agriculture and plant biotechnology. In: Alcázar R, Tiburcio A (eds) Polyamines: Methods in molecular biology, vol 1694. Humana Press. New York, NY, pp 489–508
Uskutoğlu T, Uskutoğlu D, Turgut K (2019) Effects on pre-treatment and different tissue culture media for androgenesis in Stevia rebaudiana Bertoni. Sugar Tech 21(6):1016–1023
Wang GF, Qin HY, Sun D, Fan ST, Yang YM, Wang ZX, Xu PL, Zhao Y, Liu YX, Ai J (2018) Haploid plant regeneration from hardy kiwifruit (Actinidia arguta Planch.) anther culture. Plant Cell Tissue Organ Cult 134(1):15–28
Wang HM, Enns JL, Nelson KL, Brost JM, Orr TD, Ferrie AMR (2019) Improving the efficiency of wheat microspore culture methodology: evaluation of pretreatments, gradients, and epigenetic chemicals. Plant Cell Tissue Organ Cult 139(3):589–599
Watts A, Kumar V, Raipuria RK, Bhattacharya RC (2018) In vivo haploid production in crop plants: methods and challenges. Plant Mol Biol Rep 36(5–6):685–694
Wędzony M, Forster BP, Żur I, Golemiec E, Szechyńska-Hebda M, Dubas E, Gotębiowska G (2009) Progress in doubled haploid technology in higher plants. In: Touraev A, Forster BP, Jain SM (eds) Advances in haploid production in higher plants. Springer, Dordrecht
Yerzhebayeva RS, Abekova AM, Ainebekova BA, Urazaliyev KR, Bazylova TA, Daniyarova AK, Bersimbayeva GK (2017) Influence of different concentrations of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale. Cytol Genet 51(6):448–454
Zeng A, Song L, Cui Y, Yan J (2017) Reduced ascorbate and reduced glutathione improve embryogenesis in broccoli microspore culture. S Afr J Bot 109:275–280
Zhang L, Zhang Y, Gao Y, Jiang X, Zhang M, Wu H, Liu Z, Feng H (2016) Effects of histone deacetylase inhibitors on microspore embryogenesis and plant regeneration in Pakchoi (Brassica rapa ssp. chinensis L.). Sci Hortic 209:61–66
Zhang XW, Liu FJ, Zhai J, Bi HG, Ai XZ (2020) Auxin acts as a downstream signaling molecule involved in hydrogen sulfide-induced chilling tolerance in cucumber. Planta 251:69
Żur I, Dubas E, Krzewska M, Janowiak F (2015) Current insights into hormonal regulation of microspore embryogenesis. Front Plant Sci 6:424
Żur I, Dubas E, Krzewska M, Zieliński K, Fodor J, Janowiak F (2019) Glutathione provides antioxidative defence and promotes microspore-derived embryo development in isolated microspore cultures of triticale (× Triticosecale Wittm). Plant Cell Rep 38(2):195–209
Acknowledgements
The authors are thankful to Piama Svoboda for her kind assistance in the English language editing of the manuscript.
Author information
Authors and Affiliations
Contributions
MES conceived the idea and corrected the whole body of manuscript, M N wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Niazian, M., Shariatpanahi, M.E. In vitro-based doubled haploid production: recent improvements. Euphytica 216, 69 (2020). https://doi.org/10.1007/s10681-020-02609-7
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s10681-020-02609-7