Mesbahuddin Ahadi ^{1, 2}*; Mohammad Gul Arabzai ^{3, 4}

1, College of Horticulture, Fujian Provincial Key Laboratory, Fujian Agriculture and Forestry University (FAFU), Fuzhou 350002, Fujian Province, China

2, Horticulture Department of Agriculture faculty, Badakhshan University (BDKU), Afghanistan

3, College of Agriculture, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China

4, Department of Agronomy, Agriculture Faculty, Paktia University, Gardiz City, Paktia, Afghanistan

 

Manuscript link
http://dx.doi.org/10.30493/DAS.2025.484402

Abstract

The method of isolated microspore culture is extensively employed in the generation of haploid and double haploid plants, facilitating genetic and biotechnological research while expediting breeding operations. Isolated microspore culture is superior than anther culture due to the propensity of anther walls to generate unwanted diploid and somatic calli in plants during the process. This paper encompasses an examination of doubled haploid plant development utilizing isolated microspore culture techniques in modern Brassica species. The review provides a systematic account of the Brassica genus haploid/doubled haploid plant production process, encompassing donor plant management, floral collection, microspore separation, culture initiation, embryo generation, chromosome duplication, and the subsequent development of haploid/doubled haploid plants. Thereafter, the recent advances in this technique will be surveyed, and the major obstacles facing its application in Brassica breeding programs will be discussed.

Keywords: Isolated microspore culture, Double haploid, Brassica, embryogenesis

Introduction

Isolated microspore culture (IMC) entails the excision of the anther to procure microspores devoid of anther tissue and somatic cells and culturing them in vitro under controlled conditions to produce haploid or doubled haploid plants. This technique expedites plant growth and diminishes both labor and financial expenditures [1]. The improvements in microspore separation methods have markedly increased the efficacy of IMC, especially in Brassicaceae species [2]. A crucial element of IMC is the capacity of a single microspore to mature into an entire plant. Microspores, originating from haploid tissues, are particularly adept in facilitating androgenic development. Furthermore, the deterioration of the anther wall, particularly the septum and tapetum layers, promotes microspore culture by establishing a more favorable environment for development [3]. Consequently, IMC offers an optimal framework for the examination of microspore embryogenesis [4].

Various parameters significantly influence embryogenesis and plant regeneration in androgenic cultures. For example, anther culture can release endogenous hormones and regulatory substances that influence embryogenic results [5]. Moreover, many stress pretreatments, including heat shock, nutrient deprivation, osmotic stress, and colchicine administration, might augment the androgenic response by redirecting microspores from the gametophytic to the sporophytic developmental pathway [6-8].

Reports showed that microspore culture can produce embryos at rates tenfold higher than alternative embryogenesis methods [9]. In the last three decades, significant advancements have occurred in microspore cultivation, particularly in Brassica species such as Brassica rapa, Brassica napus, Brassica carinata, and Brassica juncea [10]. This technique has been effectively utilized on multiple cabbage varieties, including loose and semi-loose Brassica oleracea var. costata, B. oleracea var. gongylodes, decorative cabbage, and other Brassica species [11-13]. The effective formation of microspore embryos is affected by various factors, including as the physiological condition of the donor plant, environmental conditions, and specific characteristics of the culture media [14].

In contrast to conventional anther culture, IMC presents several significant advantages: 1) it removes diploid somatic tissue and the anther wall, which could otherwise induce callus formation or undesired embryo development; 2) it is more efficient and circumvents the laborious task of anther selection; 3) it offers a more regulated and nutrient-rich environment for embryo growth; and 4) it functions as a valuable model for investigating microspore maturation and embryogenesis. While anther culture is beneficial in specific conditions, IMC outperforms it by circumventing challenges associated with tissue interactions and excessive microspore growth [15-17].

The generation of haploid (H) and doubled haploid (DH) plants is a notable advancement in plant biotechnology. Multiple Brassica species have been effectively changed into H and DH lines utilizing both microspore and anther culture methodologies. The inaugural successful documentation of anther cultivation in Brassica species occurred in 1975 [18][19]. Subsequently, an autonomous microspore cultivation technique for B. napus was developed [20]. The doubled haploid (DH) approach holds immense value for variety development, mutagenesis, and fundamental plant research [21][22]. Among the various techniques, microspore transformation into double haploid plants is the most effective. Microspore-derived haploid embryos can directly regenerate into whole plants, whereas calli formed from microspores necessitate indirect regeneration [23-25].

Due to its simplicity and excellent efficiency, IMC remains the favored approach for genetic research and plant breeding within the Brassica genus. Nonetheless, its extensive implementation is constrained by three primary challenges: the impact of genotype on embryo induction and plant regeneration, the incidence of secondary embryogenesis, and the absence of dependable technologies for chromosome doubling [17]. This review seeks to elucidate recent advancements and approaches in isolated microspore culture (IMC) within the Brassica group, emphasizing the genetic and environmental aspects influencing its efficacy.

Fundamental protocol for isolated microspore culture

Research has consistently shown that genetic variables significantly impact the success of microspore growth in the Brassica family [26-28]. Notably, F1 hybrid lines demonstrated superior performance compared to traditional breeding lines. This enhanced response is likely due to heterosis, which improves hybrid morphogenesis. In fact, F1 lines exhibited approximately 63% embryonic differentiation and 37% cell division, highlighting their increased responsiveness [28]. Moreover, nurturing donor plants in meticulously optimized greenhouse environments (Controlled temperature, humidity, and light conditions) produces superior material suitable for microspore culture [29]. The efficacy of microspore-derived embryo development relies on three essential factors: the growing conditions of donor plants, the formulation of the culture medium, and the intrinsic responsiveness of the genotype. Furthermore, in breeding efforts that entail double haploid production, it is crucial to initially assess each genotype’s reaction across several procedures. This initial stage is crucial to guarantee successful results prior to initiating the breeding process [30]. Factors such as the developmental stage of microspore selection, sucrose concentration in the medium, and the duration of high-temperature exposure may require modification correspondingly [31].

Overall, IMC process commences with the cultivation of donor plants, succeeded by the harvesting of floral organs. Microspores are subsequently isolated, grown, and subjected to induction and chromosomal doubling. The method ultimately concludes with embryo regeneration (Fig. 1). Consequently, although the fundamental framework of the IMC protocol is universal, minor modifications and meticulous management of instruments and environments are crucial for enhancing outcomes across plant species and even varieties within the specie.

Growing of donor plants

Successful isolated microspore culture (IMC) commences with the utilization of healthy donor plants devoid of diseases and pests. Appropriate seed spacing during donor plants cultivation is crucial, since it facilitates optimal plant growth and reduce disease transmission between plants. Moreover, donor plants require regular irrigation and suitable fertilization to promote robust development. Prompt intervention for any disease or insect infestations is essential, as it mitigates the transmission of infections among cultivated plants [32]. Various studies reported the correlation between donor plant health and parameters including environmental circumstances and genetic background [33-36]. Younger plants’ microspores generally demonstrate superior androgenic potential compared to those acquired from older plants, indicating that plant age is a significant role in microspore response [37].

To achieve successful microspore generation, donor plants must be cultivated under optimal growth circumstances. This encompasses environmental conditions, appropriate culture medium composition, and the plant’s unique genotypic response. Consequently, it is imperative to evaluate the genotype’s performance under various protocols prior to initiating a breeding program focused on generating doubled haploid (DH) plants [30].

Growers may utilize both open field conditions and controlled habitats, such as greenhouses or growth chambers, while cultivating donor plants. Nonetheless, regulated ecosystems provide considerable benefits. These settings provide exact control of essential parameters including humidity, temperature, light intensity, and photoperiod. Moreover, growth chambers facilitate the management of pests and diseases, allowing for expedited and more precise treatments. Studies indicate that donor plants cultivated in controlled environments typically exhibit a reduced risk of contamination and enhanced embryogenic potential compared to those grown in open fields [38].

A crucial aspect affecting IMC effectiveness is the temperature sensitivity of donor plants. For Brassica sp., donor plants should be cultivated at temperatures between 15°C and 20°C, with a reduction to 5°C–10°C immediately before blooming to augment embryogenic potential [27]. Furthermore, sustaining culture temperatures between 32°C and 35°C for a duration of up to 34 days has been correlated with effective embryogenesis in Brassica napus, B. carinata, and B. juncea [39][40]. Temperature treatments can provide different effects based on the species’ ploidy level. For example, embryogenesis in B. oleracea, a diploid (2n) species, is more adversely affected by temperature changes than in tetraploid (4n) species [41].

Harvesting floral organs and microspore vitality evaluation

The second stage of isolated microspore culture is the preparation of floral organs from donor plants [42]. The selection of microspores at the optimal developmental stage is a crucial aspect in IMC, as it directly affects the quality and efficiency of embryo formation. Consequently, pre-treating anthers before microspore extraction, and subsequently incubating them in an appropriate culture medium, markedly improves the embryonic development from microspores [21]. A preculturing phase in a saturated humid atmosphere at controlled temperatures for 2-3 days can significantly enhance IMC yield from excised anthers [21]. Nonetheless, such treatment necessitates precise optimization based on the species and diversity of the cultures.

The floral fluorescence approach is important in evaluating the composition and vitality of microspores. A minimal volume of microspore suspension remaining in centrifuge tubes is utilized for this evaluation. The suspension is centrifuged in 1.5 ml microcentrifuge tubes at 4000 rpm for 2 to 4 minutes. Following the removal of the supernatant, a droplet of DAPI (4′,6-diamidino-2-phenylindole) solution is introduced to the residual pellet to achieve a final volume of 10 µl. This formulated solution enables researchers to analyze microspores at different embryonic stages by detecting and quantifying those in the uninucleate and binucleate phases [43]. The vitality of microspores is also evaluated utilizing a 0.2 mg/ml fluorescein diacetate (FDA) staining solution. The concentration and distribution of the fluorescent dye within the microspores signify their viability condition [44]. The percentage of viable microspores is measured by counting stained cells relative to the total number.

Microspore isolation

Within the Brassicaceae family, three primary techniques are often employed for the isolation of microspores. The initial procedure involves the conventional grinding and minification technique, wherein sterilized flower buds are placed into a mortar containing NLN13 nutritional media and crashed with a sterile pestle. The second method, referred to as the magnetized stirrer technique, is immersing sterilized buds in sterile plates that contain a magnetic stir bar and NLN13 media. Finally, a contemporary method involves meticulously bisecting sterilized buds along their midline with a sharp blade, resulting in two symmetrical halves [20][44].

Subsequent to these preliminary procedures, a specialist instrument is generally employed to extract anthers, which are next separated utilizing a blender. This technique effectively separates microspores from adjacent somatic tissues, yielding a refined microspore culture. It is crucial to acknowledge that effectively separating anthers necessitates both expertise and time, as the procedure can be labor-intensive and technically challenging [42]. Employing a homogenizer during microspore isolation can produce superior embryogenic outcomes in comparison to conventional instruments such as glass rods and mechanical shakers. Nonetheless, the success rate of regeneration using these other approaches is often somewhat lower, and they may result in an increased occurrence of albino plantlets. In Brassica napus, the application of polytetrafluoroethylene rods to extrude anthers through sieves proved to be highly effective for anther collection [20].

A major obstacle in microspore culture is the mortality of multicellular microspores, frequently caused by the secretion of inhibitory chemicals from anthers. These chemicals can inhibit sporophytic growth and obstruct embryogenesis [45]. Consequently, it is imperative to eradicate all somatic tissue from the culture environment, so insuring the retention of only viable microspores. In this context, a filtration phase utilizing 63µm nylon mesh is implemented, succeeded by centrifugation at 46 g for five minutes. The purified microspores are subsequently suspended in a solution with 30% sucrose to facilitate their growth [46]. Therefore, the successful creation of a clean and functional microspore culture devoid of debris and inhibitory components depends on rigorous procedural control and meticulous attention to details during the isolation process [47].

Culture and microspore induction

Optimal ambient conditions, along with a nutrient-rich culture medium, are crucial for facilitating microspore formation during isolation processes [25]. Similar to other tissue culture systems, microspores necessitate a balanced solution comprising macro- and micronutrients, carbohydrates, and vital vitamins to promote optimal growth [42]. When contamination is a problem, antibiotics like cefotaxime may be included into the medium as a precautionary step [48]. However, studies indicate that even moderate levels of cefotaxime (approximately 50 mg/L) can impede microspore formation in Brassica napus and Brassica oleracea, implying that its application requires meticulous assessment [49]. Consequently, evaluating the impact of culture medium on microspore formation is essential for optimizing in vitro settings [25].

The efficacy of microspore embryogenesis in various plant species is predominantly influenced by the culture conditions and the unique constituents of the medium, especially the type and concentration of carbohydrate sources [50][51]. Sucrose functions as both an osmotic agent and a nutritional source in the majority of microspore embryogenesis methods. In this regard, studies show that sucrose concentrations ranging from 8% to 17% are ideal for Brassica sp. [52][53].

The totipotency of plant cells in vitro enables microspores and other isolated cells to differentiate into embryos and ultimately into full plants. This metamorphosis necessitates particular environmental factors. Various stress conditions are significant triggers for microspores to deviate from their gametophytic growth and commence the embryogenic, sporophytic route [54-56]. Consequently, stress induction is essential for the proper generation of double haploid plants. Three principal stressors have been recognized as notably impactful: severe temperatures (both high and low), carbon deficiency, and colchicine inadequacy [8]. Moreover, interventions such as osmo-shock and supplementary physical or chemical stressors are frequently utilized to augment microspore reprogramming into embryonic cells. Although heat therapy and carbon deprivation have demonstrated efficacy across various species, colchicine is utilized less often due to its restricted significance relative to other stressors. Moreover, specific stress therapies may have specialized technical apparatus that is not consistently accessible, thereby constraining their practical use. Novel techniques such as pH changes, heavy metals, chemical inducers, and carrageenan oligosaccharides have been devised to facilitate microspore embryogenesis [57].

Embryo regeneration

Heat-stressed microspores undergo development in induction media, with this advancement significantly affected by the type and concentration of plant growth regulators present. The administration of 2,4-D generally enhances callus formation, whereas Naphthaleneacetic acid (NAA) more directly induces embryogenesis and promotes fetal development. The trajectory of microspore development, whether towards callus or embryo, is predominantly influenced by these hormonal conditions. In instances of indirect embryogenesis, haploid embryos are generated after a sequence of disordered and asynchronous cell divisions that ultimately result in organogenesis. The direct embryogenic pathway emulates the natural sequence of zygotic embryo development, progressing through certain stages: spherical, heart, torpedo, and ultimately, the cotyledon stage [22][58][59].

Microspore-derived embryogenesis exemplifies the phenomenon of cellular totipotency [60]. Investigations concerning Brassica campestris, Brassica napus, and Brassica oleracea have validated the development of both haploid and diploid embryos during regeneration [3]. Embryonic structures generally become discernible under culture conditions between 10 to 12 days post-induction [61][62]. Nonetheless, early-stage embryos frequently experience developmental halt, leading to the creation of vacuoles and callus-like cells rather than viable embryos. In the initial phases of embryogenesis, every cell has the capacity for transformation; however, this capability becomes restricted to the outermost cells in subsequent stages. While numerous embryos develop correctly, some do not progress beyond the globular stage in advanced development. Nonetheless, specific Brassica species demonstrate the capacity to regenerate these aborted embryos. In these instances, the utilization of hormone-enriched medium facilitates sustained cell division, even in partially aborted embryos. This hormonal equilibrium allows researchers to develop viable embryos from embryogenic calluses that would otherwise be removed. It is crucial to acknowledge that a considerable fraction of cellular structures is frequently compromised during these recovery endeavors, potentially affecting overall regeneration efficacy [63].

Chromosome doubling

Plant breeders and genetic researchers have traditionally utilized conventional chromosomal doubling techniques to produce double haploid plants. In recent years, chromosomal doubling approaches have garnered increased interest because to their efficacy in generating totally homozygous lines. The ideal time for triggering chromosome doubling occurs during the initial stage of gametic cell division, which is essential for the successful development of haploid and double haploid (DH) plants. In Brassica napus, brief episodes of elevated temperature, referred to as thermal shocks, are routinely administered post-microspore harvesting to induce embryogenesis. As a result, this approach has demonstrated a chromosomal doubling success rate of approximately 5 to 10 percent. Furthermore, chemical treatments like colchicine and trifluralin have demonstrated markedly higher efficacy, achieving success rates of up to 90% in the production of DH plants. Conversely, other agents such as amiprophos-methyl, butanol, and oryzalin exhibit relatively lower efficacy in facilitating chromosomal doubling [64-66].

Research indicates that polyploidy-induced chromosomal doubling results in changes in chromosome counts among several plant species, including those in the Brassica genus. These alterations frequently entail chromosomal fusion or fission events. Consequently, similar gene sequences are often preserved and disseminated over extensive taxonomic groups, indicating common evolutionary lineages. Consequently, comparative genomic methodologies, especially those employing orthologous gene mapping, have become essential instruments in genome sequencing and structural analysis in DH plants. The advent of sequence-associated amplified polymorphism (SAAP) has established a cost-efficient, dependable, and uncomplicated PCR-based marker system for evaluating genetic variation in Brassica oleracea. This method has increased researchers’ ability to investigate genomic organization and variation both within and among species [67][68].

Progress in microspore isolation and double haploid Brassica plant production

A plethora of scholarly works illustrate the swift progress in microspore separation techniques, pollen culture, and methodologies for generating double haploid (DH) plants. The inaugural successful isolated microspore cultivation in Brassica napus was documented in 1982, signifying a prominent advancement in this sector [20]. This seminal discovery facilitated the adaptation and optimization of the approach across several crop species.

In this context, researchers determined that the ideal sucrose content for microspore culture in Brassica species generally ranges from 8% to 17% [52][53]. Furthermore, research indicates that brief heat shock treatments at 32°C markedly improve microspore viability, cellular division, and embryogenesis in Brassica campestris ssp [69]. The integration of plant growth regulators and temperature shock treatments has demonstrated efficacy in enhancing microspore culture for turnip (Brassica rapa subsp. rapa) [70]. Moreover, the application of cold treatments to flower buds, alongside moderately acidic circumstances, has been shown to enhance embryonic induction in Brassica species [71][72]. An increase in success generation of DH plants was observed when primary embryos experience secondary embryogenesis, implying that numerous cycles of embryogenesis can augment DH yield. Nonetheless, each species, as well as distinct genotypes within a single species, has a unique response to identical culture density levels. For instance, Cabbage genotypes KTCBH-2085, KTCBH-6621, and KTCBH-6045 exhibit optimal performance at a density of 4×10⁶ cells/ml, whereas the variety ‘Golden Acre’ necessitates a higher density of 6×10⁶ cells/ml for optimal outcomes [73][74].

Kozar’s research team advanced the field by devising an innovative microspore separation technique for the Brassicaceae [11]. This procedure involves slicing buds crosswise with a knife and dividing them into halves; one half is then placed in a sterile nutrition tube and shaken on a rotary shaker for 10-60 seconds. This approach boosted embryonic development, especially when embryos were positioned on dry filter paper, which facilitated their regeneration into whole plants [75].

Recent research endeavors have advanced the comprehension of the genetic and molecular foundations of microspore embryogenesis [76]. Researchers identified fifteen proteins in Brassica that are analogous to Divergent Male Pollen (DMP) proteins. BoC03 and DMP9 demonstrated significant sequence similarity to the ZmDMP protein present in Zea mays [77]. Furthermore, haploid-inducing genes in dicotyledons was found to exhibit significant similarity to ZmDMP [78][79].

These developments have improved technical ability for double haploid induction via isolated microspores and anther culture, while also enriching the overall comprehension of the molecular and cytological pathways that govern embryological development. The key accomplishments and milestones in Brassica DH plant production through microspore culture are defined in (Table 1).

Future perspectives of isolated microspore culture

The future of isolated microspore culture technology for Brassica species presents significant possibilities for plant breeding and crop enhancement [101]. As research advances, scientists are discovering improved methods to maximize genotypic responses in microspore culture, hence enhancing the genetic variety accessible for the production of double haploid (DH) plants [102]. Furthermore, the comprehension of the molecular mechanisms behind microspore embryogenesis enhances, new opportunities for augmenting the efficiency and success rates of DH formation are created [103]. Simultaneously, innovations in chromosome doubling methodologies, encompassing enhanced chemical and physical treatments, are expected to expedite and improve the reliability of the process. This will thus minimize the time and resources required to generate fully homozygous plants [104]. Furthermore, the incorporation of high-throughput techniques with robotics, artificial intelligence, and automation has the potential to transform microspore culture, facilitating extensive, commercial production of doubled haploid plants [105]. A vital area of emphasis is the identification of appropriate stressors to promote stress-induced embryogenesis, as this significantly enhances microspore formation. Nevertheless, comprehensive study remains essential to optimize plant regeneration and production yield in this domain [106].

Notwithstanding these promising developments, numerous obstacles persist until microspore culture can be established as a broadly effective instrument for plant breeding [103]. A significant difficulty is that various Brassica genotypes have disparate responses to microspore culture; some do not develop optimally, resulting in inconsistency across plant species [107]. Moreover, contamination of somatic tissue can disrupt culture growth, resulting in undesirable callus formation and the emergence of albino plants, hence diminishing the overall success rate [108]. Another obstacle is the intricate equilibrium necessary for stress-induced development. Inadequate stress levels can lead to cellular damage or mortality, necessitating the identification of optimal circumstances for each plant species [109]. On the other hand, chromosome doubling is a major issue, especially when employing heat shock techniques, which exhibit poor success rates. Identifying more effective and dependable methodologies necessitates additional investigation [110][111]. The extensive commercial application of microspore culture is hindered by the necessity for specialized equipment and stringent regulation of growing conditions, complicating mass manufacturing. Consequently, in order to render isolated microspore culture and doubled haploid plant production viable for agriculture, these challenges must be addressed [112][113].

Conclusion

The isolated microspore culture is a crucial research and breeding technique that particularly advantages Brassicaceae species. This approach allows researchers to grow double haploid plants more rapidly, enhancing breeding program efficiency and the development of homozygous lines. Research on microspore embryogenesis persists in advancing, but persistent problems related to genotype limitations and the inability to double chromosomes. The methodologies for microspore cultivation and stress interventions, together with the understanding of developmental mechanisms in microspores, have advanced considerably. Advancements in microspore isolation techniques and the identification of embryonic proteins will enhance the development of this technology. The technique of isolated microspore culture serves as an essential approach for the expedited enhancement of superior Brassicaceae plant varieties and the advancement of plant biotechnology research.

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Cite this article:

Ahadi, M., Arabzai, M. G. Isolated microspore culture in Brassica breeding: The technique, progress, and future perspectives. DYSONA – Applied Science, 2025;6(2): 361-377. doi: 10.30493/das.2025.484402