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Article

Comparative Analyses of Green Plantlet Regeneration in Barley (Hordeum vulgare L.) Anther Culture

by
Csaba Lantos
*,
Ferenc Markó
,
Róbert Mihály
and
János Pauk
Cereal Research Non-Profit Ltd., P.O. Box 391, H-6701 Szeged, Hungary
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(9), 1440; https://doi.org/10.3390/agriculture14091440
Submission received: 18 July 2024 / Revised: 7 August 2024 / Accepted: 22 August 2024 / Published: 24 August 2024
(This article belongs to the Section Crop Genetics, Genomics and Breeding)
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Abstract

:
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.

1. Introduction

Barley (Hordeum vulgare L) is one of the four important cereal species used for food, feed and beverage production. The changing climate and the demands of industries and consumers present continuous new opportunities to the participants of the sector. The breeding of competitive varieties and hybrids and the development of breeding technologies are therefore essential for breeding companies.
Genetic purity is an essential criterion in the breeding process of crop plants. Homogenous lines can be produced in one generation using different biotechnology tools. Well-established DH plant production methods are available for breeding and research programs of some crop plants, for example, barley, wheat, spelt wheat, triticale, maize and rice [1,2,3,4,5,6,7,8]. In barley, chromosome elimination technique crossing with Hordeum bulbosum L., haploid gene inducer (the hap gene), ovary culture, anther- and isolated microspore culture are the most known methods for DH plant production [6,7].
The methods of in vitro androgenesis (anther- and isolated microspore culture) are frequently applied in barley breeding and research programs. Barley is considered a model cereal crop for DH plant production by androgenesis (anther- and isolated microspore culture) due to responsive genotypes with high production of embryo-like structures (ELSs) and green plantlets [6,7,8]. Furthermore, these methods have been efficiently combined by several other biotechnology methods like marker-assisted selection [9], induced mutations [10,11], genetic transformation [12,13] or genome editing [14,15].
Many factors affect the efficiency of in vitro androgenesis, such as genotype, growing conditions of donor plants, the developmental stage of microspores, pre-treatment, composition of induction and regeneration medium. Stress pre-treatments are essential for reprogramming of gametophytic pathway of microspores to the sporophytic one, for induction of in vitro androgenesis. Various stresses can be used for androgenesis induction in barley anther culture, such as long-term (28 days) cold (at 4 °C) pre-treatment [16], or osmotic and starvation stress [17,18,19,20]. The application of the Ficoll-400, and using maltose instead of sucrose, significantly enhanced green plantlet production in barley anther culture [21,22,23,24]. Makowska et al. reported that the plant regeneration rate was increased by adding gum Arabic [25,26]. Growth regulators also have a significant role during androgenesis induction. 6-Benzylaminopurine is generally applied in induction media [6,19,20], while some researchers have used combinations of hormones (2,4-D, 2-Naphthaleneacetic acid (NAA) and kinetin) for induction of androgenesis in barley [27,28,29,30]. The relationship between Cu(2+), AG2+ and a pattern of DNA methylation was studied during the induction of barley anther culture to optimize the culture conditions and the efficiency of green plant production [31,32]. However, the improvement of the plant regeneration phase of barley in vitro anther culture has received less attention. Increasing the green plantlet production, and reducing albinism and genotype dependency are key issues.
In this study, the main goal was the assessment of green plantlet production in the anther culture of barley (Hordeum vulgare L.). Different plant regeneration (FHGR and K4NB) and rooting media (MSr, N6I and ½N6I + Ca) were compared in four F1 cross-combinations to test the number of regenerated plantlets (green and albino) and check the percentage of acclimatization after the different plant regeneration methods were used. Haploid, diploid and tetraploid plants were identified among the anther culture-derived plantlets tested by flow cytometric analysis.

2. Materials and Methods

2.1. Plant Materials

Five 6-row winter barley (‘GK Árpád×Quadriga’, ‘GKH-36-20×GKH-88-19’, ‘SU Ellen×GKH-88-19’, ‘GKH-88-19×GKH-39-18’, ‘GW3801×GKH-36-20’) and three 2-row winter barley (‘Leopard×GK Judy’, ‘Monroe×Leopard’, ‘GK Stramm×Leopard’) F1 cross-combinations were selected for DH plant production. Cross-combinations were prepared for breeding purposes (6-row genotypes) and research programs (2-row genotypes) in 2021.
Two 6-row winter barley (‘SU Ellen×GKH-88-19’, ‘GKH-88-19×GKH-39-18’) and two 2-row winter barley (‘Leopard×GK Judy’, ‘GK Stramm×Leopard’) F1 cross-combinations were used to examine the effect of different plant regeneration and rooting media, and the best treatment was validated with the each selected F1 combinations.
The seeds of the cross-combinations were sown in the nursery of Cereal Research Non-Profit Ltd., and the plants were grown according to a European winter cereal nursery protocol. The donor plants were fertilized using NPK artificial fertilizer (1:1:1) in fall (12 g/m2), 18 g/m2 ammonium nitrate was applied in April, and the plants were protected by insecticide treatments twice. Before sowing, weeds were reduced by mechanical treatment and chemical treatment (Granstar SuperStar herbicide, FMC-AGRO HUNGARY Ltd., Budapest, Hungary), and manual weeding was applied within the growing season.

2.2. Preparation of In Vitro Anther Cultures

The donor tillers containing anthers with microspores in early- and mid-uninucleated stages were collected to perform the experiments of in vitro anther culture. The developmental stages of microspores were checked by Olympus CK-2 inverted microscope (Olympus Ltd., Southend-on-Sea, UK). The surface of spikes was sterilized in 300 mL 2% NaClO solution and one drop of Tween-80 for 20 min on a shaker, and then the donor materials were rinsed three times with distilled water for a few seconds in laminar airflow.
From the spikes, 100 anthers were isolated in each 55 mm-diameter plastic Petri dish (Biolab Zrt., Budapest, Hungary) containing 0.7 M mannitol pre-treatment medium containing 5.88 g/L CaCl2 and solidified with 4 g/L agar [19]. During the pre-treatment period (three days), the Petri dishes were incubated at 24 °C in a dark thermostat to induce in vitro androgenesis. After this period, the anthers were transferred to 55 mm-diameter plastic Petri dishes (100 anthers/Petri dish) containing 5 mL FHG induction medium (Supplementary Table S1) [19]. The anther cultures were grown at 24 °C for 2 months in darkness.

2.3. Plant Regeneration and Acclimatization of Anther Culture-Derived Green Plants

The well-developed ELSs 1–2 mm in size were used to carry out the plant regeneration experiments, and the same number of ELSs (30 ELSs/Petri dishes) were placed in 90 mm-diameter plastic Petri dishes (Biolab Zrt., Budapest, Hungary) containing plant regeneration media. The effects of FHGR [19] and K4NB [33] plant regeneration media on the number of regenerated (green and albino) plantlets were examined (Supplementary Table S2). This K4NB medium is an improved version of earlier reported media by Kumlehn et al. [34].
The albino plantlets were discarded. The well-shooted green plantlets were transferred individually to glass tubes that contained different rooting media. The green plantlets regenerated on FHGR media were rooted on MSr medium, while the green plantlets derived from K4NB medium were applied to test the effect of 3 different rooting media, namely MSr [19], N6I medium [33] modified version of N6 medium [35], and ½N6I + Ca rooting medium (Supplementary Table S3). The ½N6I + Ca medium was a modified N6I medium containing half the amount of N6 macro-, microelements and vitamins supplemented with 220.4 mg/L CaCl2.
The well-rooted green plantlets were transplanted into plastic plates (66 plantlets/plate) which contained a 1:1 peat and sandy soil mix. During the 5–7-day-long acclimatization period, the anther culture-derived plantlets were covered by a transparent plastic cover to keep humidity high. The number of acclimatized plantlets per treatment was counted three weeks after transplantation, and the percentage of acclimatization was calculated based on these data (acclimatized plantlets/transplanted plantlets*100). The acclimatized green plantlets were planted in the nursery in fall.

2.4. Ploidy-Level Analyses of Anther Culture-Derived Green Plantlets

The ploidy level of 111 acclimatized plantlets (52 and 59 plantlets from the ‘SU Ellen×GKH-88-19’ and the ‘GKH-88-19×GKH-39-18’ F1 cross-combinations, respectively) was determined by flow cytometric analysis (CytoFLEX Flow Cytometer, Beckman Coulter International S. A., Nyon, Switzerland). Leaf samples (20 mg/plantlets) were collected from the acclimatized plantlets, and seed-grown plants were used as a control. The samples were cut into 0.5 cm pieces and put into 2 mL Eppendorf tubes that contained 1 mL Galbraith buffer and two stainless steel beads. The collected leaf samples were homogenized using TissueLyser II (Qiagen, Hilden, Germany) at 30 Hz for 30 s to isolate the nuclei from the young leaves of plantlets [36]. After the isolation of nuclei, the suspensions of samples were filtrated using 20 μm sieves. RNA content was reduced in the samples by adding 6 μL RNase solution (1 mg/mL) added to each sample (150 μL) at room temperature for 60 min. Propidium iodide (PI) solution (1 mg/mL, 40 μL/sample) was used to paint the DNA content of samples for 30 min at room temperature. The DNA content of the samples was determined by flow cytometer, and the different ploidy levels (haploid, diploid and tetraploid) of the samples were identified by the histograms.
The fertility of the 111 plants tested by flow cytometer was checked after harvest, and three groups (sterile, fully fertile and partially fertile plants) were distinguished among the tested plantlets. These data were compared with the results of flow cytometric analyses.

2.5. Statistical Analyses

Each treatment was repeated at least eight times. The parameters of in vitro androgenesis (number of ELSs; total regenerated (green and albino) plantlets; percentage of acclimatization; and number of spontaneous DH plants) were recorded and analyzed by statistical methods. The number of ELSs was analyzed by one-way ANOVA, while the number of regenerated, green, albino, rooted and acclimatized plantlets and the percentage of acclimatization was analyzed by two-way ANOVA. The assumptions of ANOVA, such as normality and homogeneity, were checked by the Shapiro–Wilk and Levene tests. The means of genotypes and treatments were separated using the LSD values. The coefficient of the variation was also calculated to determine the variability of each parameter. The statistical analyses were performed using Microsoft Excel 2019 statistical software (Microsoft Ltd., Redmond, WA, USA).

3. Results

3.1. Androgenesis Induction in Anther Culture of Barley F1 Combinations

The spikes of donor F1 cross-combinations containing uninucleated microspores were selected for implementation of in vitro anther culture experiments (Figure 1a). Androgenesis was induced in the anther culture for each genotype and treatment. The dividing microspores were monitored under a microscope, and multicellular structures were observed after two weeks of androgenesis induction (Figure 1b).
Large numbers of ELSs were visible on the fourth week of anther culture (Figure 1c). The produced ELSs regenerated (green and albino) plantlets (Figure 1d); the green plantlets were rooted in glass tubes (Figure 1e). The acclimatized green plantlets (Figure 1f) adapted well to the field conditions (Figure 1g).

3.2. Production of Anther Culture-Derived ELSs for Plant Regeneration Experiments

Four high-responding F1 cross-combinations (‘SU Ellen×GKH-88-19’, ‘GKH-88-19×GKH-39-18’, ‘Leopard×GK Judy’, ‘GK Stramm×Leopard’) were selected for carrying out the plant regeneration experiments. The statistical analyses revealed significant differences among the number of ELSs of F1 cross-combinations, but a large number of ELSs were produced from each of them (Table 1). The number of ELSs varied between 221.71 and 435.50 ELSs/100anthers depending on genotype, with an average of 324.8 ELSs/100 anthers.

3.3. Plant Regeneration of Anther Culture-Derived ELSs

Two plant regeneration media (FHGR and K4NB) were compared to test their effects on the efficiency of plant regeneration. Based on the statistical analyses (Table 2), the genotype significantly influenced the number of regenerated (p ≤ 0.001), green (p ≤ 0.001) and albino plantlets (p ≤ 0.001). The regeneration media also significantly influenced the measured parameters, while the treatment interactions were not significant based on the data of experiments.
Green and albino plantlets were regenerated in each treatment. The number of regenerated plantlets ranged from 26.86 to 164.83 regenerated plantlets/100 anthers depending on genotype and media (Table 3). The mean of values was 105.38 plantlets/100 anthers on K4NB medium while the average of regenerated plantlets was 77.09 plantlets/100 anthers using FHGR medium.
The albinism was also influenced by the regeneration media and genotype. The most albinos were regenerated from ELSs of anther culture of ‘GKH-88-19×GKH-39-18’ F1 cross-combination (50.33 albinos/100 anthers) using K4NB regeneration media, while the lowest number of regenerated albinos (7.00 albinos/100 anthers) was observed in anther culture of ‘GK Stramm×Leopard’ F1 cross-combination on FHGR regeneration medium. The means of regenerated albinos were 21.32 albinos/100 anthers and 30.85 albinos/100 anthers on FHGR and K4NB regeneration medium, respectively. The production of green plantlets significantly exceeded the number of albinos in each genotype.
The number of regenerated green plantlets varied between 12.57 green plantlets/100 anthers and 129.67 green plantlets/100 anthers (Table 3). On average, 55.77 green plantlets/100 anthers and 74.53 green plantlets/100 anthers were regenerated using FHGR and K4NB regeneration media, respectively. The quantity of regenerated green plantlets was more on the K4NB medium than on the FHGR medium in the tested F1 cross-combinations, these differences were significant in the case of ‘SU Ellen×GKH-88-19’, ‘GKH-88-19×GKH-39-18’ and ‘GK Stramm×Leopard’ F1 cross-combinations (Table 3).
The data of plant regeneration experiments (regenerated (green and albino) plantlets) were higher in 6-row barley cross-combinations compared to 2-row barley cross-combinations.

3.4. Rooting of Anther Culture-Derived Green Plantlets

The effect of different rooting medium was tested on the rooting and acclimatization of the regenerated green plantlets. Statistical analyses revealed that the medium and genotype significantly influenced the measured parameters (Table 4). The regeneration and rooting media had a strong effect on the rooting (p ≤ 0.001) and acclimatization (p ≤ 0.001) of green plantlets (Table 4).
Remarkable differences were observed among the four different plant regeneration methods regarding the rooting of green plantlets (Table 5).
On average, the highest number of rooted plantlets was achieved with MSr rooting medium using plantlets regenerated on K4NB plant regeneration medium (139 rooted plantlets on average), while this value was lowest using FHGR and MSr media (54.2 rooted plantlets on average of F1 cross-combinations).
The rooted plantlets acclimatized to the greenhouse conditions, and the number of acclimatized plantlets was counted 3 weeks after transplantation (Table 5). The number of acclimatized plants was highest in that treatment when K4NB plant regeneration medium and MSr rooting medium were combined (63.8 acclimatized plantlets on average), and similar values were achieved with K4NB plant regeneration medium and ½N6I + Ca rooting medium (60.2 acclimatized plantlets on average). The percentage of acclimatization was also highest in the case of these plant regeneration methods (Table 5). The application of K4NB plant regeneration medium and MSr rooting medium was proved an efficient plant regeneration way which was also tested with the other four F1 cross-combinations.

3.5. Application of Anther Culture with Eight F1 Cross-combinations

The anther culture was tested with eight F1 cross-combinations to check the efficiency of the method using the above-mentioned plant regeneration protocol (K4NB plant regeneration medium and MSr rooting medium). The androgenesis was induced in each combination (Table 6) and the number of green plantlets (66.93 on average) overproduced the number of regenerated albinos (24.44 on average). The in vitro green plantlet production varied between 24.95 and 129.67 green plantlets/100 anthers depending on genotype. Furthermore, more than two thousand in vitro green plantlets were transplanted into the greenhouse. The percentage of acclimatization was 61.83%, which ranged from 26.4% to 82.9% depending on genotype. Altogether, 1403 acclimatized plantlets were produced for their own barley breeding programs.

3.6. Determination of Ploidy Level of Anther Culture-Derived Plantlets

The ploidy level of 111 acclimatized anther culture-derived plantlets of two F1 cross-combinations was determined by flow cytometric analyses (Figure 2). Based on the results of measurements, 25 haploid, 77 spontaneous DH and 9 tetraploid plantlets were identified among the anther culture-derived plantlets (Table 7). The tetraploid plants showed a more robust phenotype (thicker stem and wider leaves) compared to the diploids, which flowered and matured later.
The fertility of these plantlets was checked after harvest. The haploid plants were sterile, while the spontaneous DH plants produced full fertile spikes (Figure 3). The spikes of tetraploid plants contained a few seeds, and partial fertility of tetraploid lines was also observed in the DH1 generation.

4. Discussion

The methods of DH plant production have proven to be an effective tool in the breeding of new barley varieties in many breeding programs [7,8,37,38,39,40]. Furthermore, the methods of in vitro androgenesis (anther- and isolated microspore culture) work most effectively in barley among cereals. The method of isolated microspore culture may be more efficient than anther culture for the production of barley DH plants [41], but the implementation of anther culture is technically simpler [27]. Although the methods of in vitro androgenesis are usable effectively in barley, some relevant research groups reported genotype dependency and albinism in barley, which can limit the wide range production of DH plants in some genotypes [33,42,43,44]. Thus, the continuous improvement of these methods has remained the focus of research and breeding programs.
Induction of in vitro androgenesis, ELS production, green plant regeneration, acclimatization of plantlets and genome duplication are the most critical steps of an efficient in vitro anther culture method. In this study, different plant regeneration and rooting media were compared to improve the efficiency of DH plant production for barley breeding programs. The anther culture-derived ELSs of four F1 cross-combinations were used to compare the plant regeneration efficiency of FHGR [19] and K4NB [33] medium. FHGR medium is a frequently used medium in anther- and isolated microspore culture of barley [6,19,45]. The K4NB medium was originally improved for plant regeneration of barley after the genetic transformation of haploid tissues [34], and it was modified [33] to produce DH plants for different research and breeding programs [13,25,29,33,46]. The macro elements of the two media are the same, but the quantity of inorganic N components is significantly higher in the K4NB regeneration medium. A notable difference is that the K4NB medium contains 100 times more CuSO4·5H2O, than the other medium. Optimization of Cu2+ ion concentrations in the induction medium can increase the number of green plants in anther culture of barley [47]. The vitamin compositions of the FHGR medium are poorer and less. In case of growth regulators added in media, the FHGR medium contained 1 mg/L BAP and 0.5 mg/L IAA, while K4NB contains only 0.225 mg/L BAP. The carbon source is maltose in both of them. The number of regenerated plantlets on the K4NB medium overproduced the efficiency of the other regeneration medium in each tested F1 cross-combination. On average, the regenerated plantlets were 105.38 and 77.09 plantlets/100 anthers on K4NB and FHGR regeneration media, respectively. The more complex composition of K4NB medium was more favourable for plant regeneration (both albino and green) of anther culture-derived ELSs in barley.
Albinism can mitigate the large-scale production of DH lines in some barley genotypes; a higher proportion of albino plantlets were regenerated from some 6-row genotypes [6,48,49]. Furthermore, the frequency of albinos is much higher in spring-type barley than in winter-type barley [33,50]. The number of albinos was mitigated using both of the two plant regeneration media. The percentage of albinos was less than 30% based on the data of experiments. The regenerated plantlets were dominantly green both in anther cultures of 2-row and 6-row F1 cross-combinations. The number of green plantlets on the K4NB medium surpassed the data of the FHGR medium in each tested cross-combination. On average, the regenerated green plantlets were 74.53 and 55.77 on K4NB and FHGR regeneration media, respectively. The genotype influenced the efficiency of anther culture, the quantity of green plantlets was higher in 6-row F1 cross-combinations than in 2-row F1 cross-combinations in harmony with the observations of Davies [51].
The well-shooted green plantlets were used to compare the efficiency of three rooting media (MSr, N6I and ½N6I + Ca). The MS-based regeneration medium [6,19,45] and N6I plant regeneration medium [25,29,33,52] are frequently applied for rooting the anther- or microspore culture-derived barley plantlets. Some significant differences can be realized between the rooting media. The macro elements of the two media are the same but in different quantities. The MSR rooting medium contains more microelement components (MnSO4·4H2O, H3BO3, ZnSO4·7H2O, KI, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O) than the N6I medium (MnSO4·H2O, H3BO3, ZnSO4·7H2O, KI). The MSR medium has fewer vitamin components (only thiamine-HCl and myo-inositol). N6I medium contained 2 mg/L IAA as a growth regulator, while MSr was 2 mg/L NAA. Casein hydrolysate was also added to the MSr rooting medium. The carbon source was sucrose in both of them, and in this case there was no difference. Significant differences were observed in the effect of rooting medium regarding the quantity of well-rooted and acclimatized plantlets and the percentage of acclimatization. The highest values were observed using the K4NB regeneration medium and MSr rooting medium. Furthermore, the effect of these media was validated with eight F1 cross-combinations including wide genetic background (11 different parents in cross-combinations). The percentage of acclimatization was 61.83% on average. Altogether, nearly a thousand DH lines were used in the local barley breeding program.
The regenerated plantlets were dominantly spontaneous DH plants. The ratio of spontaneous chromosome doubling was high (69.37%) according to flow cytometric analyses in harmony with the earlier published data [6,23,27,51]. The percentage of the haploids was 22.52%, while the remaining part (8.11%) of the tested plantlets were tetraploid. Devaux and Kasha [23] also observed 8% polyploid plants among the anther culture-derived plantlets. Generally, tetraploid plants are eliminated from breeding programs due to poor performance [51]. The tetraploid plant produces a higher biomass than diploid or haploid ones [27], but their incomplete fertility hinders the direct application of these tetraploid lines in practical breeding programs.

5. Conclusions

An efficient anther culture method has been described for barley breeding. The application of K4NB regeneration medium and MSr rooting medium enhanced the green plantlet production and acclimatization of green plantlets to the in vivo conditions. The produced DH plants have been integrated into own barley breeding program. This method can open new perspectives to produce tetraploid barley plants for research programs in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14091440/s1, Table S1: Compositions of FHGI induction medium [19]; Table S2: Compositions of plant regeneration media; Table S3: Compositions of rooting media.

Author Contributions

Conceptualization, C.L. and J.P.; methodology, F.M.; resources, R.M.; data curation, F.M.; writing—original draft preparation, C.L.; writing—review and editing, R.M. and J.P.; supervision, J.P. All authors have read and agreed to the published version of the manuscript.

Funding

The experiments were supported by the Ministry for Innovation and Technology and the National Research, Development and Innovation Office (FK_21-FK138042, TKP2020-NKA-21 and K_21-K138416).

Data Availability Statement

Data is contained within the article and supplementary materials.

Acknowledgments

The authors greatly appreciate the hard work of Szilvia Palaticki, Krisztina Beregszászi-Kéri and Márta Pataki. We thank Árpád Székely for his help in the evaluation of statistical analyses.

Conflicts of Interest

Authors Csaba Lantos, Ferenc Markó, Róbert Mihály and János Pauk were employed by the company Cereal Research Non-Profit Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. The anther culture of barley: (a) uninucleated microspores, (b) development of microspore-derived multicellular structures and (c) ELSs in anther culture of barley. (d) Regeneration of plantlets from anther culture-derived ELSs. (e) Rooting of anther culture-derived green plantlets in glass tubes. (f) Acclimatized plantlets in the greenhouse. (g) Anther culture-derived barley plants in field conditions. Bars = 10 µm for (a), 100 µm for (b), 10 mm for (c–e). nc = nucleus.
Figure 1. The anther culture of barley: (a) uninucleated microspores, (b) development of microspore-derived multicellular structures and (c) ELSs in anther culture of barley. (d) Regeneration of plantlets from anther culture-derived ELSs. (e) Rooting of anther culture-derived green plantlets in glass tubes. (f) Acclimatized plantlets in the greenhouse. (g) Anther culture-derived barley plants in field conditions. Bars = 10 µm for (a), 100 µm for (b), 10 mm for (c–e). nc = nucleus.
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Figure 2. Histograms of flow cytometric analyses of anther culture-derived barley plantlets: figures show the relative DNA content of samples: (a) control, (b) spontaneous diploid, (c) haploid and (d) tetraploid plantlets.
Figure 2. Histograms of flow cytometric analyses of anther culture-derived barley plantlets: figures show the relative DNA content of samples: (a) control, (b) spontaneous diploid, (c) haploid and (d) tetraploid plantlets.
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Figure 3. Fertility of anther culture-derived haploid (a), diploid (b) and tetraploid (c) plantlets: (a) sterile, (b) fertile, (c), and partially fertile spikes (red arrow shows a fertile seed in spike).
Figure 3. Fertility of anther culture-derived haploid (a), diploid (b) and tetraploid (c) plantlets: (a) sterile, (b) fertile, (c), and partially fertile spikes (red arrow shows a fertile seed in spike).
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Table 1. ELS production in anther culture of the four tested barley F1 cross-combinations.
Table 1. ELS production in anther culture of the four tested barley F1 cross-combinations.
Genotype‘SU Ellen×GKH-88-19’‘GKH-88-19×GKH-39-18’‘Leopard×GK Judy’‘GK Stramm×Leopard’Mean of Genotypes
Mean of ELSs/100 anthers317.00 b435.50 a221.71c325.00 b324.80
Note: Data were analyzed by one-way ANOVA. Data with the same letters are not significantly different (p ≤ 0.05) using LSD value.
Table 2. Statistical analyses (Two-way ANOVA) of the effect of regeneration media and genotype on the number of regenerated (green and albino) plantlets in anther culture of barley.
Table 2. Statistical analyses (Two-way ANOVA) of the effect of regeneration media and genotype on the number of regenerated (green and albino) plantlets in anther culture of barley.
dfMS of Green Plantlets/100 AnthersMS of Albinos/100 AnthersMS of Total Plantlets/100 Anthers
Regeneration Media15632.145 *1453.062 **12,806.690 **
Genotype331,738.660 ***3007.715 ***49,421.820 ***
Interaction3117.447 ns435.030 ns1003.139 ns
Error56930.846169.3341555.050
df: Degrees of Freedom; MS = Mean Square; *** sign. at p < 0.001; ** sign. at p < 0.01; * sign. at p < 0.05; ns non-sign.
Table 3. Effect of regeneration media and genotype on the number of regenerated plantlets (green and albino). Data with same lower-case letters (a, b) are not significantly different (p ≤ 0.05) within the same genotype based on LSD value. Data with same capital letters (A–D) are statistically identical (p ≤ 0.05) within the same treatments based on LSD value.
Table 3. Effect of regeneration media and genotype on the number of regenerated plantlets (green and albino). Data with same lower-case letters (a, b) are not significantly different (p ≤ 0.05) within the same genotype based on LSD value. Data with same capital letters (A–D) are statistically identical (p ≤ 0.05) within the same treatments based on LSD value.
GenotypeNumber of Regenerated Plantlets/100 Anthers
Plant Regeneration Media
FHGRK4NBMean of Media (CV)
‘SU Ellen×GKH-88-19’ (6-row)143.5 b A164.83 a A154.17 (9.78)
‘GKH-88-19×GKH-39-18’ (6-row)99.5 b B138.67 a A119.08 (23.26)
‘Leopard×GK Judy’ (2-row)26.86 a C36.19 a C31.52 (20.94)
‘GK Stramm×Leopard’ (2-row)38.5 b C81.83 a B60.17 (50.93)
Mean of Genotypes77.09105.3891.24 (21.93)
Number of Albinos/100 Anthers
Plant Regeneration Media
FHGRK4NBMean of Media (CV)
‘SU Ellen×GKH-88-19’ (6-row)30.00 a A35.17 a B32.58 (11.21)
‘GKH-88-19×GKH-39-18’ (6-row)34.00 b A50.33 a A42.17 (27.39)
‘Leopard×GK Judy’ (2-row)14.29 a B11.24 a D12.76 (16.89)
‘GK Stramm×Leopard’ (2-row)7.00 b B26.67 a C16.83 (82.61)
Mean of Genotypes21.3230.8526.09 (25.83)
Number of Green Plantlets/100 Anthers
Plant Regeneration Media
FHGRK4NBMean of Media (CV)
‘SU Ellen×GKH-88-19’ (6-row)113.50 b A129.67 a A121.58 (9.4)
‘GKH-88-19×GKH-39-18’ (6-row)65.50 b B88.33 a B76.92 (20.99)
‘Leopard×GK Judy’ (2-row)12.57 a C24.95 a D18.76 (46.66)
‘GK Stramm×Leopard’ (2-row)31.50 b C55.17 a C43.33 (38.62)
Mean of Genotypes55.7774.5365.15 (20.36)
Table 4. Statistical analyses (Two-way ANOVA without repetition) of the effect of regeneration, rooting media and genotype on the number of rooted and acclimatized plantlets in anther culture of barley F1 cross-combinations.
Table 4. Statistical analyses (Two-way ANOVA without repetition) of the effect of regeneration, rooting media and genotype on the number of rooted and acclimatized plantlets in anther culture of barley F1 cross-combinations.
dfMS of Rooted PlantletsMS of Acclimatized Plantlets
Genotype38597.083 *90.816 **
Media344,734.420 ***187.982 ***
Error91341.25010.583
df: Degrees of Freedom; MS = Mean Square; *** significance at p < 0.001; ** significance at p < 0.01; * significance at p < 0.05.
Table 5. The effect of regeneration, rooting media and genotype on the number of rooted and acclimatized plantlets in anther culture of barley. Data with the same lower-case letters (a–c) are not significantly different (p ≤ 0.05) within the same genotype using LSD value. Data with the same capital letters (A–C) are statistically identical (p ≤ 0.05) within the same treatments using LSD value.
Table 5. The effect of regeneration, rooting media and genotype on the number of rooted and acclimatized plantlets in anther culture of barley. Data with the same lower-case letters (a–c) are not significantly different (p ≤ 0.05) within the same genotype using LSD value. Data with the same capital letters (A–C) are statistically identical (p ≤ 0.05) within the same treatments using LSD value.
GenotypeNumber of Rooted Plantlets
Plant Reg. (FHGR or K4NB) and Rooting Media (MSr, N6I or ½N6I + Ca)
FHGR, MSrK4NB, N6IK4NB, ½N6I + Ca K4NB, MSrMean of Media (CV)
‘SU Ellen×GKH-88-19’ (6-row)130 b A285 a A309 a A341 a A266.25 (49.09)
‘GKH-88-19×GKH-39-18’ (6-row)102 b A214 a B35 c B75 bc B172.75 (46.32)
‘Leopard×GK Judy’ (2-row)11 a B35 a C29 a B53 a B32.00 (58.52)
‘GK Stramm×Leopard’ (2-row)27 b B75 ab C66 ab B110 a B69.50 (55.78)
Mean of Genotypes54.2122.2119.0139.0108.6
Number of Acclimatized Plantlets
Plant Reg. (FHGR or K4NB) and Rooting Media (MSr, N6I or ½N6I + Ca)
FHGR, MSrK4NB, N6IK4NB, ½N6I + CaK4NB, MSrMean of Media
‘SU Ellen×GKH-88-19’ (6-row)11 c A72 b A186 a A183 a A113.00 (71.84)
‘GKH-88-19×GKH-39-18’ (6-row)1 b A60 ab AB85 a B70 a B54.00 (66.73)
‘Leopard×GK Judy’ (2-row)1 a A3 a B11 a C14 a B7.25 (78.16)
‘GK Stramm×Leopard’ (2-row)0 a A7 a B16 a C48 a B17.75 (101.14)
Mean of Genotypes2.828.860.263.8038.90
Percentage of Acclimatization (%)
Plant Reg. (FHGR or K4NB) and Rooting Media (MSr, N6I or ½N6I + Ca)
FHGR, MSrK4NB, N6IK4NB, ½N6I + CaK4NB, MSrMean of Media
‘SU Ellen×GKH-88-19’ (6-row)8.50 c A25.30 b A60.20 a A53.70 a A36.9 (65.3)
‘GKH-88-19×GKH-39-18’ (6-row)1.00 c A28.00 b A45.20 a B37.40 ab B27.9 (67.3)
‘Leopard×GK Judy’ (2-row)9.10 b A8.60 b B37.90 a BC26.40 a B20.5 (67.64)
‘GK Stramm×Leopard’ (2-row)0.00 c A9.30 c B24.20 b C43.60 a AB19.3 (86.66)
Mean of Genotypes3.9114.64 34.1233.0321.42 (59.44)
Table 6. The acclimatization of anther culture-derived green plantlets of eight barley F1 cross-combinations.
Table 6. The acclimatization of anther culture-derived green plantlets of eight barley F1 cross-combinations.
GenotypeNumber of Green plantlets/100 AnthersNumber of Albinos/100 AnthersTotal Transplanted PlantletsTotal Acclimatized PlantletsPercentage of Acclimatization
‘GK Árpád×Quadriga’89.3815.3843235882.9%
‘GKH-36-20×GKH-88-19’56.3313.7539129074.2%
‘SU Ellen×GKH-88-19’129.6735.1734118353.7%
‘GKH-88-19×GKH-39-18’88.3350.331877037.4%
‘GW3801×GKH-36-20’28.1317.3819514674.9%
‘Leopard×GK Judy’24.9511.24531426.4%
‘Monroe×Leopard’63.5025.6356029452.5%
‘GK Stramm×Leopard’55.1726.671104843.6%
Summa (Mean)(66.93)(24.44)2269 (283.625)1403 (175.375)61.83%
Table 7. Ploidy levels of barley plantlets derived from anther culture according to flow cytometric analyses.
Table 7. Ploidy levels of barley plantlets derived from anther culture according to flow cytometric analyses.
GenotypeHaploid
(n)		DH
(2n)		Tetraploid
(4n)		Number of Tested Acclimatized Plantlets
‘SU Ellen×GKH-88-19’12 (23.08%)36 (69.23%)4 (7.69%)52
‘GKH-88-19×GKH-39-18’13 (22.03%)41 (69.49%)5 (8.47%)59
Total Number (Percentage) of Plantlets with Different Ploidy Levels25 (22.52%)77 (69.37%)9 (8.11%)111 (100%)
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Lantos, C.; Markó, F.; Mihály, R.; Pauk, J. Comparative Analyses of Green Plantlet Regeneration in Barley (Hordeum vulgare L.) Anther Culture. Agriculture 2024, 14, 1440. https://doi.org/10.3390/agriculture14091440

AMA Style

Lantos C, Markó F, Mihály R, Pauk J. Comparative Analyses of Green Plantlet Regeneration in Barley (Hordeum vulgare L.) Anther Culture. Agriculture. 2024; 14(9):1440. https://doi.org/10.3390/agriculture14091440

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Lantos, Csaba, Ferenc Markó, Róbert Mihály, and János Pauk. 2024. "Comparative Analyses of Green Plantlet Regeneration in Barley (Hordeum vulgare L.) Anther Culture" Agriculture 14, no. 9: 1440. https://doi.org/10.3390/agriculture14091440

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