Haploid Plants Research Articles

Effects of hormone concentrations on anther cultures and the acquisition of regenerated plants of five awnless triticale genotypes

BackgroundThe rapid production of doubled haploids by anther culture technology is an important breeding method for awnless triticale. The aim of this study was to explore the effects of triticale genotype and the types and ratios of exogenous hormones in the medium on the efficiency of triticale anther culture.ResultsAnthers of five triticale genotypes were cultured on four different callus induction media and the calli were induced to differentiate into green plants by culture on three different differentiation media. The triticale genotype T8004 showed the best performance in anther culture, with a callus induction rate of 28.64%, a green plantlet differentiation frequency of 33.33%, and a green plantlet production rate of 2.78%. The highest callus induction rates were obtained by culturing anthers on C3 medium (the main components were potassium nitrate, glutamine, inositol, etc.), and the highest green plantlet differentiation frequency was obtained by culturing calli on D2 differentiation medium (the main components were potassium nitrate, ammonium nitrate, calcium chloride dihydrate, etc.). Flow cytometry analyses showed that 15 of the 20 DH0 generation plants that grew normally in the field were doubled haploids. The average chromosome doubling success rate was 55.6%. Analyses of agronomic traits showed that the 11 DH1 doubled haploid plants reached the standard for awnless triticale, so they are candidate materials for breeding new awnless triticale varieties.ConclusionThe anther culture technology of triticale was optimized in this paper, which made it possible to rapidly breed homozygous varieties of awnless triticale.

Development of haploid winter wheat by the method of distant hybridization with maize

In order to improve the breeding process and expand the genetic diversity of winter wheat, haploid lines can serve as new initial material. One of the methods for developing haploids in wheat plants is distant hybridization when crossing with maize plants and subsequent selective elimination of chromosomes. Postgamic incompatibility of parent plants can be overcome by growing embryos in vitro. The purpose of the current study was to develop haploid winter common wheat plants using the method of distant hybridization when crossing with maize and selective elimination of chromosomes. The current paper has presented the hybridization results of winter wheat varieties and hybrids ([(‘Odesskaya 200’ x ‘Agrofak 100’) x ‘Razdolie’], ‘1608/21’, ‘1638/19’, ‘Stanichnaya’, ‘221/20’, ‘Volnitsa’, ‘Knyaginya Olga’, ‘Bezostaya 100’, ‘Alekseich’) and haploproducer lines of maize (SM7, Kr 935/86, Kr 651) using the embryoculture method in vitro. As a result, there was established that the mean setting rate of hybrid grains was 64 %. The highest percentage of grains with embryos was in the combinations ‘Stanichnaya x SM7’ (30 %), ‘Bezostaya 100 x SM7’ (33 %) and ‘Volnitsa x SM7’ (36 %). The maximum number of haploid sprouts was formed in the hybrid combinations ‘Stanichnaya x SM7’ (8 pcs.), ‘Bezostaya 100 x SM7’ and ‘Volnitsa x SM7’ (7 and 7 pcs.). There were developed 37 haploid plants from 13 cross combinations. The survival rate of plants after putting into soil was 62 %. It is being planned to evaluate the developed breeding material.

Obtaining Haploid Plants Via Anther Culture in Some Eggplant Varieties

In plant breeding studies, anther culture is used to breed hybrid varieties, to transform tetraplaid plants that can be obtained through interspecific somatic hybridization into fertile dihaploid plants, and in recent years, to obtain diploid in protoplast fusion of haploid embryogenic calli obtained from anther culture. The frequency of obtaining haploid plants in anther culture depends on many factors such as the variety, the season in which it was taken and the use of suitable media for regeneration. In this study, it was aimed to determine the appropriate regeneration environment in the anther culture of Aydın Siyahı and Kemer eggplant varieties, which are widely grown in our country. In the anther culture study, C (callus), R (regeneration) and V3 media recommended by Dumas de Valux et al. (1982) were used as nutrient media. However, as C (callus) medium, Dumas de Vaulx et al. (1982) 3 different doses of 2,4 D, Kinetin and sucrose of the C medium reported were tested. The highest plant yield (36.4%) of the Aydin Siyahı variety was obtained from the modified Dumas de Vaulx et al. (1982), from 1 mg/l 2.4-D +1 mg/l Kinetin + 120 g/l sucrose medium, the highest plant yield (33.8%) of the Kemer variety was reported by Dumas de Vaulx et al. (1982), obtained from 5 mg/l 2.4-D + 5 mg/l Kinetin + 120 g/l sucrose medium. Of the 79 plants obtained from anther culture and whose ploidy level was examined, 60 were determined as haploid, 13 as diploid, 4 as triploid, 1 as tetraploid and 1 as mixoploid.

Induction and genetic verification of homologous double haploid plants obtained through the anther culture of Citrus cultivars

In this study, an anther culture system was developed for two Citrus varieties known for their genetic value: blood orange (Moro) and mandarin (Lee). Anthers were inoculated on N6 solid medium containing thidiazuron (TDZ, 0.44 mg/L), 6-benzylaminopurine (0.8 mg/L), zeatin (0.43 mg/L), kinetin (0.44 mg/L), 1-naphthaleneacetic acid (0.2 mg/L), 2,4-Dichlorophenoxyacetic acid (0.2 mg/L), and malt extract (500 mg/L). The inoculated anthers were treated with N6 liquid medium containing spermidine (200 µM) and gibberellic acid (GA3, 1 mg/L), and cultured for 6 weeks. Thereafter, the swollen anthers were transferred to a Murashige and Skoog (MS) basal medium enriched with malt extract (500 mg/L), sucrose (50 g/L), TDZ (0.5 mg/L), GA3 (1 mg/L), and gelrite (0.2%), which induced callus and somatic embryos. These somatic embryos, from both varieties, were then transferred to a germination medium (MS basal medium containing sorbitol [0.05 M], galactose [0.05 M], malt extract [500 mg/L], GA3 [0.5 mg/L], and gelrite [2 g/L]) to develop into normal plants. However, Lee exhibited significantly slower shoot and root growth compared to Moro. Genetic analysis using barley microsatellite-derived cleaved amplified polymorphic sequence markers indicated that Lee likely originated from haploid plants, whereas Moro retained heterozygosity similar to the parent. Ploidy analysis confirmed Lee as a diploid, identical to the control. Internal transcribed spacer region analysis confirmed that Lee was an anther-cultured haploid-derived plant, estimated to be a homozygous diploid carrying recessive genes. These findings highlight potential applications in marker development for haploid-derived plants focused on recessive trait-associated phenotypes and genotypes.

Production of Microspore-Derived Plants by Anther Culture of Cyclamen coum

Cyclamen is one of the most important ornamental crops sold worldwide as a potted flower for winter production. Cyclamen species take up a wide swathe of habitats across Türkiye. Ten wild Cyclamen species grow naturally in Türkiye and some of them are endemic. This study aimed to produce haploid plants of C. coum using anther culture. The microspore developmental stage was evaluated by staining anther with acetocarmine (%2), and then the stage was correlated with bud size. It was determined that the buds between 7.64 and 8.23 mm had the appropriate bud size for the late uninuclear stage. Anthers were cultured in B5 medium containing different levels of 1-Naphthaleneacetic acid (NAA) (0.1, 1, 2 mgL-1), 2,4-Dichlorophenoxyacetic acid (2,4-D) (0.1, 1, 2 mgL-1), and kinetin (0, 1 mgL-1), 90 gL-1 sucrose and 3 gL-1 gelrite for haploid embryo production. Anthers were kept at 4°C for 4 days after culture. The explants were incubated at 24°C in a completely dark condition until the embryo was formed, then embryos were transferred to hormone-free media in 16:8 hours (light (75 µmolm-2s-1): dark) photoperiod. The experiment was carried out for two years. In the first year, 12 different media were examined in view of regeneration and the experiments were continued with selected 7 media in the second year. The highest callus regeneration rates were %5.71 and 14.5% and the highest embryo induction rates varied between 8.57% and 4.0% in the first and second year respectively. Embryo/callus formation was observed in 7 of a total of 12 different media tested for haploid plant production, and the best media were kinetin (1 mgL-1) + NAA (1, 2 mgL-1) and kinetin (1 mgL-1) +2,4-D (2 mgL-1). Our findings indicated that cold pre-treated anther explants collected at appropriate flower bud size resulted in embryo production. Additionally, B5 medium supplemented with NAA and kinetin ensured successful embryo regeneration from anther explants in wild C. coum.

Crucial Factors Influencing the Efficiency of Androgenesis in Oat (Avena sativa L.) Through Anther and Microspore Cultures

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.).

Doubled Haploid Technology: Generation of Doubled Haploid Maize Lines Using Haploid Inducers.

Doubled haploid (DH) technology allows for the development of completely homozygous lines from heterozygous plants in only two generations. This approach has been widely adopted in maize breeding programs, as it expedites the generation of inbred lines compared to traditional methods. The DH approach is based on the use of maize genotypes that have the ability to induce haploid seeds when used as the pollen parent. The most common method for producing maize haploid plants for the generation of DH lines is in vivo maternal haploid induction. The process involves pollination with a haploid inducer maize line to generate haploid seeds. Then, haploids are screened for and identified (typically via the expression of a particular marker gene), germinated, treated with an exogenous doubling agent to induce genome duplication, and transplanted to the field. Following successful self-pollination, seeds harvested from the ear represent fully homozygous lines. The seed set at this stage, however, is often low, necessitating one or two additional rounds of self-pollination to increase the number of fully homozygous inbred lines. Here, we describe a protocol for the generation of maize DH lines using maternal haploid-inducing maize lines. We outline the steps for setting up the donor material, performing induction crosses, selecting haploids based on two different marker alleles, treating seedlings with colchicine to double the genome, transplanting the treated seedlings to the field, and self-pollinating the treated plants.

Development of Haploid Plants by Shed-Microspore Culture in Platycodon grandiflorum (Jacq.) A. DC.

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.

An efficient and easy-to-use protocol for induction of haploids in cucumber through parthenogenic embryo development

BackgroundCucumber (Cucumis sativus L.) is a model crop to study cell biology, including the development of haploids and doubled haploids in vegetable crops. In plant breeding, haploid and doubled haploids are valuable tools for developing pure homozygous inbred lines and accelerating genetic progress by reducing the time required for breeding cycles. Besides, the haploids are also valuable in genomic studies. We are reporting the induction of haploids in cucumber involving gynoecious and parthenocarpic genotypes for the first time. This study aimed to assess the efficient induction of haploids through pollination with gamma-irradiated pollen in cucumber. The effect of gamma irradiation dose on pollen viability and germination, fruit setting percentage, seed development, and haploid embryo development in cucumber hybrid genotypes were studied in detail. The goal was to utilize this information to produce haploid plants for genomics and transformation works in this model vegetable crop.ResultsPollination was done on six cucumber genotypes using varying doses of gamma rays (100, 200, 300, 400, and 500 Gy). Genotypes, doses of irradiation, and embryo developmental stage influenced the successful generation of in-vitro haploid plants. The optimal timeframe for embryo rescue was found to be 25 to 30 days after pollination. Haploid embryos were effectively induced using irradiated pollen at 400 to 500 Gy doses. Parthenogenetic plantlets were analyzed, and their ploidy level was confirmed through stomatal physiology, cytology (mitosis), and flow cytometry methods.ConclusionThrough parthenogenic embryo development, it is possible to induce a large number of haploids in cucumber. This technique’s power lies in its ability to streamline the breeding process, enhance genetic gain, and produce superior cultivars that contribute to sustainable agriculture and food security.

Stress-Induced Autophagy Is Essential for Microspore Cell Fate Transition to the Initial Cell of Androgenesis.

The isolated microspores can be reprogrammed towards embryogenesis via stress treatment during in vitro culture, and produce (doubled) haploid plants as a breeding source of new genetic variability. However, the mechanism underlying the cell fate transition from gametogenesis to embryogenesis remains largely unknown. Here, we report that autophagy plays a key role in cell fate transition for microspore embryogenesis (referred to as androgenesis) in Nicotiana tabacum. Immunofluorescence and transmission electronic microscopy detection unveiled that autophagy was triggered in microspores following exposure to inductive stress, and a transient wave of the numerous autophagy-related genes (ATGs) expression occurred before the initiation of microspore embryogenesis. Suppression or promotion of the original autophagy levels could inhibit microspore embryogenesis, indicating that stress-induced autophagic homeostasis is essential for cell fate transition. Furthermore, quantitative proteomics analysis revealed that autophagy might be involved in lignin biosynthesis and chromatin decondensation for promoting reprogramming for androgenesis initiation. Altogether, we reveal an essential role of autophagy in the microspore cell fate transition and androgenesis initiation, providing novel insight for understanding this critical developmental process.

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

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.

Current Status of Haploidization in Cool-Season Grain Legume Crop Species

Doubled haploid technology is, so far, the fastest route to induce a true homozygous state in plants. True homozygous plants are particularly important for breeders, as they can facilitate hybrid breeding and are useful in fixing traits in a breeding line. Fabaceae species are of great importance in food and feed production; however, they are far behind other families with respect to the development of effective haploidization protocols. Here, we present the most recent status of research on haploidization protocols in cool-season grain legume crops, including dry peas, chickpeas, faba beans, lentils, lupines, and grass peas. The first four species are primarily for human consumption; the latter are utilized as forage. All the mentioned species have been subject to haploidization trials; however, repeatable protocols, including the regeneration of confirmed haploid or doubled haploid plants, have not been elaborated. Research in field pea, chickpea, grass pea, and lupine is promising, with the reported regeneration of microspore-derived embryos in all four species. Repeatable plant regeneration has been reported only in field peas and chickpeas. The most recent achievements on haploidization through male and female gametophytes in faba bean are also presented. The key factors for the effective stimulation of haploid cell development in cool-season legumes are reviewed, providing a useful basis for future efforts toward haploidization in this group.

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

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

Androgenesis and gynogenesis in tomato (<I>Solanum lycopersicum</I> L.) <I>in vitro</I>

Tomato (Solanum lycopersicum L.) is one of the most consumed vegetable crops worldwide. Tomato fruits are rich in vitamins, minerals, and pigments, including lycopene. The high demand and the need to enhance tomato production call for new improved cultivars and F1 hybrids.Biotechnological methods reduce the time for source material development and the labor intensity of breeding efforts. Obtaining doubled haploid plants makes it possible to fix and analyze new gene combinations faster than with conventional breeding techniques, and produce homozygous genotypes. Tomato is highly unsusceptible to haploid induction, which has been continuously studied for more than 40 years and is still of special interest. The main methods for producing haploids are based on androgenesis and gynogenesis. Androgenesis is the production of haploids from the cells of the male gametophyte, and gynogenesis from the cells of the female gametophyte.The objective was to review the research on the induction of tomato haploids based on androgenesis and gynogenesis. No standardized, efficient or reproducible protocols are currently available to produce doubled haploids of tomato. It is necessary to determine the incubation conditions, physicochemical environments, dependence of the genotype in vitro, physiological state of the donor plant, and development of the anther, which affect the reproducibility of protocols to achieve haploid induction. Anther culture for obtaining haploid tomato plants has not yielded successful results, and the studies on microspore culture were too few, so it is difficult to understand the effectiveness of this technique. The method of gynogenesis is poorly investigated, but the culture of unfertilized ovules can become a successful way to obtain tomato haploids, with more research on this subject.

Vascular system of rice leaves depending on ploidy level

The current paper has provided data on the study of the vascular system of flag leaves of rice plants grown in a greenhouse in the period 2022–2023. The objects of the study were 24 rice samples developed at the FSBSI “ARC “Donskoy” using the method of androgenesis and cell culture. The purpose of the work was to determine the degree of development of the vascular system of flag leaves of regenerated rice plants with different ploidy levels (1n, 2n, 4n). When studying the vascular system of plant leaves (the number and area of bundles), it was found that haploid plants had smaller vascular bundles compared to diand tetraploid samples. The average beam diameter had dimensions of 59.5, 69.3 and 75.3 µm, the area of one beam was 2815.6, 3827.2 and 4540.5 µm², respectively. In the leaves of rice samples, a larger number of small and large vascular bundles have been formed with increasing ploidy level. Their number was 36–44 in haploids, 40–52in diploids, 52–60 in tetraploids. The average number of bundles was 40.3, 46.6 and 55.2, respectively. The venation pattern was individual for each sample. Between single large bundles there are from one to seven small veins, most often 4–6 pieces. Thus, samples with different ploidy levels differ in the anatomical structure of the leaves, which ultimately affects their morphology and productivity.

Doubled haploidy methodology for three forage grasses [crested wheatgrass (Agropyron cristatum (L.) Gaertn.), hybrid bromegrass (Bromus riparius x B. inermis), and meadow bromegrass (Bromus riparius Rehm.)

Doubled haploidy (DH) methodology is used in many plant species to accelerate crop improvement and cultivar development; however not all species are amenable to the tissue culture technique. Experiments were undertaken to develop DH protocols for three perennial grasses [crested wheatgrass (Agropyron cristatum (L.) Gaertn.), hybrid bromegrass (Bromus riparius x B. inermis), and meadow bromegrass (Bromus riparius Rehm.)]. The initial experiment screened these forage grass species to established wheat (Triticum aestivum L.) microspore culture protocols. Following the initial screen, several factors influencing microspore embryogenesis were evaluated. These included genotype, donor plant conditions, developmental stage of the microspore, pretreatments, media composition, and culture conditions. For regeneration of the embryos to plants, media composition and culture conditions were assessed. Microspore-derived embryos/calli as well as green haploid/doubled haploid plants were regenerated from all three forage grasses. Differences were observed between species and genotypes within species in terms of embryogenic response. Modifications to the initial wheat DH protocol included the donor plant conditions, developmental stage of the microspore to late uninucleate to early binucleate and media composition. Regenerated plants were grown in the greenhouse.

Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam

Key messageIn Cyrtanthus mackenii, development of embryo and endosperm were differentially affected by fertilization of male gametes with DNA damage and mutations.Pollen irradiation with ionizing radiations has been applied in plant breeding and genetic research, and haploid plant induction has mainly been performed by male inactivation with high-dose irradiation. However, the fertilization process of irradiated male gametes and the early development of embryo and endosperm have not received much attention. Heavy-ion beams, a type of radiation, have been widely applied as effective mutagens for plants and show a high mutation rate even at low-dose irradiation. In this study, we analyzed the effects of male gametes of Cyrtanthus mackenii irradiated with a carbon-ion beam at low doses on fertilization. In immature seeds derived from the pollination of irradiated pollen grains, two types of embryo sacs were observed: embryo sac with a normally developed embryo and endosperm and embryo sac with an egg cell or an undivided zygote and an endosperm. Abnormalities in chromosome segregation, such as chromosomal bridges, were observed only in the endosperm nuclei, irrespective of the presence or absence of embryogenesis. Therefore, in Cyrtanthus, embryogenesis is strongly affected by DNA damage or mutations in male gametes. Moreover, various DNA contents were detected in the embryo and endosperm nuclei, and endoreduplication may have occurred in the endosperm nuclei. As carbon-ion irradiation causes chromosomal rearrangements even at low doses, pollen irradiation can be an interesting tool for studying double fertilization and mutation heritability.

Gümüş Nitrat ve Ön Soğuk Uygulamalarının Çilek Anter Kültüründe Kallus Oluşumuna Etkileri

Strawberry (Fragaria x ananassa Duch.) which is one of the widely grown berry species in the world has economic and commercial importance. In commercial strawberry varieties, in order to increase yield and quality, it is necessary to obtain starting materials that are resistant/tolerant to biotic and abiotic stress factors. Biotechnological methods have an important place in strawberry breeding studies due to the long and costly process of classical breeding methods, the genetic expansion of seed production, high ploidy level and strong heterozygosity. Haploid plant production is an efficient breeding method that has been successfully applied to most plant species. However, due to the lack of sufficient haploid studies on strawberry and the fact that a specific protocol for this species has not yet been developed the necessary progress has not been made in this regard. In this study, the effectiveness of some factors determine the success in anther culture which has a significant place in obtaining haploid strawberries was investigated. For this reason, first, different sodium hypochlorite doses (NaOCl; 1%, 2%, 3%) and application durations (10, 15, 20 min) were used to determine the appropriate method for sterilisation, then cold pre-treatments (24, 36, 48, 72 hours at +4 °C) and different silver nitrate doses (AgNO3; 10, 20, 30, 40 mg l-1) were employed for callus induction in Festival strawberry variety. At the conclusion of the study it was observed that the lowest contamination rate (1%) was obtained by soaking in 1% sodium hypochlorite solution for 10 minutes. Cold pre-treatment of flower buds at +4 °C for 36 hours produced the highest callus induction rate (96%). The evaluation of the effect of AgNO3 application at different doses on the callus induction rate revealed that the highest callus induction (82%) was obtained from 20 mg l-1 AgNO3 dosage. This study showed that anther culture practices in strawberry can be improved by using cold pre-treatment, appropriate sterilization method and silver nitrate addition to the medium.

Anther-derived microspore embryogenesis in pepper hybrids orobelle and Bomby

BackgroundTraditional breeding methods have long been employed worldwide for the evaluation and development of pepper cultivars. However, these methods necessitate multiple generations of screening, line development, evaluation, recognition, and crossing to obtain highly homozygous lines. In contrast, in vitro anther-derived microspore culture represents a rapid method to generate homozygous lines within a single generation. In the present study, we have optimized a protocol for microspore embryogenesis from anther cultures of pepper hybrids Orobelle and Bomby.ResultsWe achieved early and successful embryo formation from both genotypes by subjecting the buds to a cold pretreatment at 4 °C for 4 days. Our optimized culture medium, comprised of MS medium supplemented with 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 2.6 g/L gelrite, 30 g/L sucrose, and 15 mg/L silver nitrate, exhibited the highest efficiency in embryo formation (1.85% and 1.46%) for Orobelle and Bomby, respectively. Furthermore, successful plant regeneration from the anther derived microspore embryos was accomplished using half-strength MS medium fortified with 2% sucrose and 0.1 mg/L 6-benzylaminopurine (BA), solidified with 2.6 g/L gelrite. The ploidy status of the microspore-derived plantlets was analyzed using flow cytometry technique. Notably, the haploid plants exhibited distinct characteristics such as reduced plant height, leaf length, leaf width, and shorter internode length when compared to their diploid counterparts derived from seeds.ConclusionOur findings highlight the potential of anther culture and microspore embryogenesis as an advanced method for accelerating pepper breeding programs, enabling the rapid production of superior homozygous lines.

Creation of rice doubled haploids with low amylose content using in vitro anther culture.

In vitro androgenesis is a unique model for producing homozygous doubled haploid plants. The use of haploid biotechnology accelerates to obtain of doubled haploid plants, which is very important in rice breeding. The purpose of this work is to improve the production of doubled haploids in rice anther culture in vitro and selection of doubled haploid plants with valuable traits. The study the influence of nutrient media on the production of calli and plant regeneration processes in anther culture of 35 rice genotypes was revealed a significant influence of nutrient media on callus production. It was shown that the addition to culture medium phytohormones ratio with high level of cytokinin (5.0 mg/L BAP) and a low level of auxin (0.5 mg/L NAA), supplemented with amino acid composition promotes high production of green regenerated plants (68.75%) compared to albino plants (31.25%). As a result, doubled haploid lines of the glutinous variety Violetta were selected, which characterized by a low amylose content variation (from 1.86 to 2.80%). These doubled haploids are superior to the original variety in some yield traits and represent valuable breeding material.