Search Results (60)

Conventional rice breeding predominantly relies on hybridization techniques, with hybrid progenies typically requiring 8 to 10 generations of selfing to achieve genetically stable homozygous lines. In contrast, haploid breeding enables the derivation of stable doubled haploid (DH) lines from hybrid progeny in just one generation, substantially shortening the breeding cycle. Haploid breeding comprises two core steps: haploid induction and chromosome doubling, with efficient haploid induction being pivotal to the success of this technology. Currently, anther culture, due to its relatively mature and stable protocol, has become the primary method for obtaining haploids in rice haploid breeding. This review systematically summarizes the research progress in rice anther culture, focusing on the fundamental steps and applications of haploid breeding, the developmental history of anther culture, factors influencing anther culture efficiency and their underlying genetic mechanisms, current challenges and potential countermeasures, and future prospects for rice anther culture technology. Full article

Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu²⁺ ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu²⁺ and Zn²⁺ that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu²⁺ or Zn²⁺ presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu²⁺ and those treated with Zn²⁺ formed distinct groups based on DNA sequence and methylation markers. Zn²⁺ treated and control samples formed separate groups. Also, Cu²⁺ discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu²⁺ or Zn²⁺ ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye. Full article

Cannabis sativa L. is a dioecious species known to produce over 1600 chemical constituents, including more than 180 cannabinoids classified into 11 structural groups. These bioactive compounds are predominantly synthesised in the glandular trichomes of female inflorescences. However, sex determination in C. sativa is influenced by both genetic and environmental factors, often leading to the development of male flowers on female plants. This unintended fertilisation reduces cannabinoid yield and increases genetic heterogeneity and challenges in medical cannabis production. Haploid and doubled haploid (DH) technologies offer a promising solution by rapidly generating homozygous lines from gametophytic (e.g., unpollinated ovaries and ovules) or sporophytic tissues (e.g., anthers and microspores) via in vitro culture or chromosome reduction during hybridisation. In land plants, the life cycle alternates between a diploid sporophyte and a haploid gametophyte generation, both capable of mitotic division to form multicellular bodies. A single genome regulates this phase transition and encodes the molecular, genetic, and epigenetic mechanisms that precisely control the developmental processes unique to each generation. While the application of haploid technology in C. sativa remains limited, through recent progress in haploid induction (HI) and CRISPR-based genome editing, the direct modification of haploid gametes or embryos enables the creation of null homozygous lines following chromosome doubling, improving genetic uniformity. Understanding the molecular mechanisms of spontaneous chromosome doubling may further facilitate the development of elite cannabis genotypes. Ultimately, enhancing the efficiency of DH production and optimising genome editing approaches could significantly increase the speed of genetic improvement and cultivar development in Cannabis sativa. Full article

The anther culture-based breeding of rice is a plant tissue culture technique that utilizes rice pollen to rapidly obtain haploid plants. In comparison with traditional breeding methods, this technique shortens the breeding cycle and enables the quick generation of homozygous plants, which is of great significance for the development of new rice varieties and the expansion of germplasm resources. With the advancement of technologies, the use of the anther culture technique in rice breeding has matured and has been applied to the development and utilization of new varieties with high yield, multiple resistances, and superior quality, in combination with other breeding methods. This technique has gained widespread attention globally, with many countries adopting it to create new germplasm resources. This study reviews advances in the rice anther culture technique, the factors influencing anther culture efficiency, and the progress in breeding rice varieties using this technique, as well as analyzes the current challenges and future prospects of anther culture breeding. Full article

In vitro anther culture is a technique used to produce haploid plants when regenerating varieties with specific traits. To generate haploid plants with preferred characteristics, an anther culture technique was established for Lilium longiflorum “Show Up”. Morphological characteristics were recorded, including the flower bud length and anther color corresponding to different stages of microspore development. The effects of different flower bud lengths, various concentrations of exogenous plant growth regulators (PGRs), low-temperature pretreatment at 4 °C, and incubation under dark conditions on the induction of callus formation were studied. When the flower buds were 2.2–2.4 cm in length and the microspores were in the mononuclear development phase, callus induction reached the highest rate (15.6%). Callus was not induced when the PGRs 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KT) were added separately to the growth medium, but the highest callus induction rate occurred when anthers were cultured on the medium containing 2,4-D (0.75–1.0 mg/L) and KT (4 mg/L). The low-temperature pretreatment significantly enhanced the induction rate of anthers, but prolonged low-temperature pretreatment reduced the induction rate. The optimal period of cultivation in darkness was 6 d. After 15 days of cultivation, the number of swollen anthers was recorded, and these were transferred onto the differentiation medium Murashige and Skoog (MS) + 1-naphthaleneacetic acid (NAA) (2.0 mg/L), sucrose (30 g/L), and agar (7 g/L) at pH 5.8, whereon 100% differentiation was recorded. Overall, 14 regenerated lines were obtained by in vitro anther culture. Chromosome ploidy was determined by counting chromosomes in the root tips of ten regenerated plants, and four were found to be haploids. This study lays the foundation for anther culture in lilies to shorten the breeding cycle, improve selection efficiency, facilitate efficient genetic transformation, and enable the effective production of both haploid and double-haploid plants. Full article

Cucumber mosaic virus (CMV) is one of the most dangerous viral diseases threatening Solanaceae crops, in particular Capsicum sp. This study aims to develop double haploid (DH) pepper lines from germplasm resistant to CMV in order to speed up the breeding process. For this purpose, six genotypes previously tested for CMV resistance were used. Two induction mediums (17-2 and 17-3) with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) for anther incubation and further plant regeneration were applied. L10 was the most responsive genotype, exhibiting the highest direct embryogenesis and the most plant regenerants on both mediums. Medium-specific response was observed in genotype L9 where regenerants were observed only on 17-2. Further, eight DH lines were evaluated with two CMV isolates (L-BG and PV-0418) and checked for local and systemic presence of the pathogen in leaves and fruits for a period of 60 days by DAS-ELISA. Of the tested DH lines, four (DH2, DH6, DH7 and DH9) were resistant to both strains, two (DH5 and DH14) were resistant to L-BG, and two (DH19 and DH21) were susceptible to both isolates. Field evaluation of DH7, DH9, and DH14 for some agronomic and morphological traits divided them into two groups according to the original genotypes. Full article

Hop latent viroid (HLVd), a 256-nucleotide RNA strand with complementary base-pairing and internal stem loop structures, forms circular or rod-shaped molecules within diseased plants. RT-PCR/RT-qPCR was used to assess HLVd transmission, spread and longevity. The viroid was detected in asymptomatic stock plants and in rooted vegetative cuttings, as well as in recirculated nutrient solution sampled from propagation tables and nozzles. Plant-to-plant spread through root infection in hydroponic cultivation was demonstrated. The viroid survived for 7 days and 4 weeks, respectively, in crushed leaf extracts (sap) or dried leaves/roots at room temperature. Following stem inoculation with infectious sap, HLVd was detected in root tissues within 2–3 weeks and in the foliage within 4–6 weeks. Plants grown under a 12:12 h photoperiod to induce inflorescence development showed more rapid spread of HLVd compared to 24 h lighting. The viroid was subsequently detected in inflorescence tissues, in trichome glands, in dried cannabis flowers and in crude resinous oil extracts. Anthers and pollen from infected male plants and seeds from infected female plants contained HLVd, giving rise to up to 100% infected seedlings. Artificially inoculated tomato and tobacco plants supported viroid replication in roots and leaves. Infected cannabis leaf and root tissues treated with UV-C for 3–5 min or temperatures of 70–90 °C for 30 min contained amplifiable HLVd-RNA. Infectious plant extract treated with 5–10% bleach (0.825% NaOCl) or 1000 ppm hypochlorous acid yielded no RT-PCR bands, suggesting the RNA was degraded. Meristem tip culture from HLVd-infected plants yielded a high frequency of pathogen-free plants, depending on the genotype. Full article

Lonicera japonica Thunb has significant edible and medicinal value, possessing heat clearing, detoxification, antibacterial, and blood pressure reduction properties. Currently, its quality is constrained by factors such as climate, environment, flowering period, and germplasm degradation. The strategy of using bioreactors and abiotic inducers to produce bioactive metabolites has not yet been implemented. This study reports, for the first time, the induction of an embryogenic callus from L. japonica anthers, the identification of tissue morphological structures, and the effects of light induction on the callus morphology, metabolite accumulation, and antioxidant activity. The results showed that the MS medium, supplemented with 1.0 mg·L⁻¹ 6-BA, 1.5 mg·L⁻¹ NAA, 1.5 mg·L⁻¹ 2,4-D, and 0.2 mg·L⁻¹ KT, induced 89% embryogenic callus formation. Uniform callus lines were obtained using 2.0 mg·L⁻¹ 6-BA, 0.5 mg·L⁻¹ NAA, and 0.2 mg·L⁻¹ KT in each subcultivation. Embryogenic cells were observed to have closely arranged spherical protruding granules on their surface, along with visible nuclei and numerous starch grains. After 15 days of blue light induction, active metabolites and antioxidant activities peaked. This experimental system not only provides support for germplasm innovation but also indicates that abiotic inducers can be utilized as a means to achieve higher yields of metabolic products. Full article

Historically, traditional crossbreeding schemes have predominated in oat breeding. In vitro culture techniques seek to expedite the breeding process and enhance selection efficiency. Maximum yields are achieved from hybrid plants produced by crossing pure (homozygous) lines with the desired traits. Homozygous lines can be produced through conventional breeding methods, which are time-consuming and costly. Alternatively, the production of homozygous lines can be accelerated by producing doubled haploid (DH) plants derived from (haploid) male gametophytes or their microspores (androgenesis). This method condenses the various stages required for producing homozygous lines in a single generation, resulting in significant time and cost savings. These and other advantages render androgenic DHs the preferred choice in numerous important crops where any of the various in vitro experimental techniques (anthers culture or isolated microspores culture) are well-established. However, in the case of oat (Avena sativa L.), an efficient plant regeneration method remains not very effective compared to the most common cereals, possibly due to the known recalcitrance of this cereal to in vitro culture. This review presents the methods through anther and microspore cultures utilized in the production of oat DHs revealing the crucial factors influencing the efficiency of this method in oat (Avena sativa L.). Full article

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid–liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines. Full article

As one of the most important staple crops in the world, rice plays a pivotal role in world food security. The creation of doubled haploids based on anther culture is an important technology for rice breeding. However, at present, rice anther culture technology still faces many problems, such as genotype dependency, especially genotypes of indica rice. In this study, fifteen rice genotypes, including twelve japonica rice genotypes and three indica rice genotypes, were randomly selected and used to study anther culture by using a modified M8 medium. The results showed that the total callus induction rates of these different rice genotypes ranged from 0.81 to 13.95%, with an average of 6.64%, while the callus induction rates calculated for the top ten highest callus inductions for each rice genotype ranged from 2.75 to 17.00%, with an average of 10.56%. There were varying gaps between the total callus induction rates and the callus induction rates in these different rice genotypes. The fact that the gaps for some rice genotypes were relatively large indicated that standard tiller or anther collection was not applicable to all rice genotypes and that there was still a lot of room for improvement in the callus induction rate of some rice genotypes through optimization of the sampling method. The plantlet regeneration rates ranged from 12.55 to 456.54%, with an average of 200.10%. Although there were many albinos from anther culture for some rice genotypes, these would still meet the requirement if the rice genotypes had higher callus induction rates or regeneration rates. The percentages of seed setting of regenerated green seedlings ranged from 14% to 84%, with an average of 48.73%. Genetic diversity analysis showed that the genetic background of these different rice genotypes was representative, and the phylogenetic tree and Principal Component Analysis (PCA) divided them into indica and japonica types. Therefore, in this study, an anther culture method suitable for both indica and japonica rice genotypes was established, which could improve doubled haploid breeding in rice. Full article

The efficient doubled haploid (DH) plant production methods play a key role in accelerating the breeding of new varieties and hybrids in cultivated plants. Consequently, DH plant production methods are continuously improving for barley (Hordeum vulgare L.) breeding and research programs. Two plant regeneration (FHGR and K4NB) and three rooting media (MSr, N6I and ½N6I + Ca) were compared with four F₁ barley cross-combinations to clarify the effect of medium on the regeneration of green and albino plantlets and acclimatization. The plant regeneration efficiency was higher using K4NB medium (74.53 green plantlets/100 anthers and 30.85 albino/100 anthers) compared to FHGR (55.77 green plantlets/100anthers and 21.32 albino/100 anthers). The percentage of acclimatization was highest when the K4NB regeneration medium was combined with the MSr rooting medium. Altogether, 61.83% of the anther culture-derived plantlets of 8 cross-combinations acclimatized to the greenhouse conditions, and 1403 acclimatized plantlets were produced from the F₁ cross-combinations. Haploid (22.52%), diploid (69.37%) and tetraploid (8.11%) plantlets were identified among the 111 tested green plantlets by flow cytometric analyses. The tetraploid lines can be explored to offer new scopes for future barley research and breeding directions. Nearly one thousand DH plants have been integrated into our barley breeding program. Full article

This study aimed to evaluate the effects of dimethyl sulfoxide (DMSO) on microspore embryogenesis and green plant regeneration in wheat anther culture. Five culture media, as well as the inclusion of 1% DMSO in the surface disinfection solution, were investigated in three winter wheat genotypes. Our results showed that the Altındane genotype produced the highest number of embryoids, 215 per 100 anthers cultured in CHB-3 medium, whereas the Dariel and Pehlivan genotypes produced 6.6 and 0 embryoids, respectively, from 100 anthers cultured. On the other hand, the addition of 1% DMSO to the same medium adversely affected embryoid production compared to the medium without DMSO. A 70% ethanol solution with 1% DMSO for the surface disinfection of spikes was effective in increasing the embryoids from approximately 0 to 17.8% and from 1 to 48.4% in CHB-3 + 1%DMSO and CHB-3 medium, respectively. Furthermore, the Altındane genotype produced 22.2 plantlets/100 anthers (17.7 albino and 4.4 green plants) and 17.7 albino plantlets per 100 anthers in CHB-3 and CHB-3 + DMSO, respectively. Our results suggested that the inclusion of 1% DMSO in the disinfection solution increased the number of embryoids without supporting the production of green plants. Full article

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP—9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar. Full article

Anther culture (AC) is a valuable technique in rice breeding. However, the genetic mechanisms underlying anther culturability remain elusive, which has hindered its widespread adoption in rice breeding programs. During AC, microspores carrying favorable alleles for AC are selectively regenerated, leading to segregation distortion (SD) of chromosomal regions linked to these alleles in the doubled haploid (DH) population. Using the AC method, a DH population was generated from the japonica hybrid rice Shenyou 26. A genetic map consisting of 470 SNPs was constructed using this DH population, and SD analysis was performed at both the single- and two-locus levels to dissect the genetic basis underlying anther culturability. Five segregation distortion loci (SDLs) potentially linked to anther culturability were identified. Among these, SDL5 exhibited an overrepresentation of alleles from the female parent, while SDL1.1, SDL1.2, SDL2, and SDL7 displayed an overrepresentation of alleles from the male parent. Furthermore, six pairs of epistatic interactions (EPIs) that influenced two-locus SDs in the DH population were discovered. A cluster of genetic loci, associated with EPI-1, EPI-3, EPI-4, and EPI-5, overlapped with SDL1.1, indicating that the SDL1.1 locus may play a role in regulating anther culturability via both additive and epistatic mechanisms. These findings provide valuable insights into the genetic control of anther culturability in rice and lay the foundation for future research focused on identifying the causal genes associated with anther culturability. Full article