Microspore Developmental Stage Research Articles

Overexpression of sesame O-acetylserine(thiol)lyase induces male sterility by delaying tapetum programmed cell death

Male sterile lines are key resources for hybrid seed production and for ensuring high varietal purity. However, the genes and mechanisms underlying sesame male sterility remain largely unknown. Hence, this study identified an O-acetylserine(thiol)lyase gene SiOASTL1 and functionally characterized its roles in inducing defective anther development. Spatiotemporal expression analysis revealed that SiOASTL1 is significantly (2.7 fold) up-regulated in sterile sesame anthers at the microspore stage compared with fertile ones. Sequence and phylogenetic analyses showed that SiOASTL1 is homologous to Arabidopsis OAS-TL plastid isoforms. We thus overexpressed SiOASTL1 in Arabidopsis to unravel its regulatory roles. Cytological observation revealed that SiOASTL1 overexpression transformed transgenic plants into male sterile lines arising at the microspore development stage. SiOASTL1 overexpression decreased cysteine biosynthesis and down-regulated the expression of the sporopollenin synthesis-related genes, including AtTKPR1, AtTKPR2, AtPKSA, and AtPKSB in transgenic Arabidopsis. Consequently, the tapetum programmed cell death (PCD) was delayed, resulting in the formation of defective pollen grains with irregular walls and empty cytoplasm. Our findings prove that the induction of SiOASTL1 expression disrupts pollen development and contributes to sesame male sterility. Moreover, these results suggest that genetic manipulation of SiOASTL1 expression may facilitate the development of new hybrid varieties in sesame and other crops.

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.

Identification and functional characterization of Si4CLL1 controlling the male sterility in the sesame genic male sterile mutant, ms1812

AbstractSesame (Sesamum indicum L.), 2n = 2x = 26, is an important oilseed crop with high heterosis potential. In this study, we explored the histological and genetic characters and causal gene of a male sterile sesame mutant, ms1812 (ms3). The microspore abortion of ms1812 that led to male sterility (MS) occurred at the tetrad stage of microspore development. No normal pollens were formed and observed in anther chambers of ms1812 at the microspore mature stage. With the aid of high‐efficient association mapping and the sesame var. Yuzhi 11 reference genome, we identified the single nucleotide polymorphism 10505640 on SiChr.3 segregating with the MS and the mutated gene, Si4cll1, controlling the recessive MS in sesame. Si4CLL1 encodes a 4‐coumarate‐CoA ligase‐like protein. The 1292th nucleotide (T1292) of Si4CLL1 mutated into A1292, resulting in the amino acid change from Ile239 to Lys239. Si4CLL1 is mainly expressed in young developing buds. Compared with the wild type, the transcript abundance of Si4cll1 in the developing buds of ms1812 reduced significantly, which might affect the phenylpropanoid biosynthesis pathway and finally, the lignin formation of microspore cell wall. These findings provide key new information for deciphering the molecular mechanism underlying MS and hybrid variety breeding assisted by MS in sesame.

Androgenesis in tomato (Solanum lycopersicum L.)-Effect of genotypes, microspore development stage, pre-treatments and media composition on induction of haploids

Doubled haploid (DH) technology remarkably accelerates the crop breeding by obtaining homozygous lines in a single generation. The present study was targeted in generating haploid plants through androgenesis. Anthers from immature flower buds of six tomato genotypes viz., LE-1230, LE-1236, LE-1256 TLCV 2, PKM 1 and TNAU tomato hybrid CO 3 were used for induction of haploids. A preliminary study based on callus induction frequency (CIF), more than 5% was helpful in short listing flower bud size, pre-treatments and growth regulator combinations. Subsequently, anthers from two different sized flower buds (4 and 6 mm length), dissected either from fresh or pre-treated flower buds (2 and 5 days in dark at 4 °C or gamma irradiated) were inoculated in MS medium fortified with different growth regulators for callus induction. Among the genotypes, TLCV 2 had recorded the maximum CIF (38.80%) from anthers of 4 mm long flower buds followed by TNAU tomato hybrid CO 3 (34.70%). Throughout the study, anthers from 4 mm long flower buds responded the best for callus induction. Among the pre-treatments, anthers from gamma irradiated flower buds recorded the highest CIF (31.90%) when compared to others. Cold shock (4 °C) in dark to flower buds for 2 days had improved the CIF of anthers when compared to fresh in LE 1230, LE 1238, TLCV2 and TNAU tomato hybrid CO 3, but when the cold shock was increased to 5 days, invariably there was a reduction in CIF in all the six genotypes. TA 8 (MS + 2iP (0.5 mg L-1) + NAA (0.5 mg L-1)) medium was found to be the best for maximum CIF in LE 1230 and PKM1, TA1 (MS + 2iP (1.0 mg L-1) + IAA (2.0 mg L-1)) in LE 1238, LE 1256 and TNAU tomato hybrid CO 3 and TA7 (MS + 2iP (0.5 mg L-1) + Kinetin (1.5 mg L-1) + NAA (1.0 mg L-1)) for TLCV 2 genotypes. The callus induced was sub cultured at monthly intervals in the same medium for proliferation and later transferred to regeneration medium. A good number of shoots got regenerated only from anther calli of TNAU hybrid CO 3 that was sub cultured in MS medium fortified with Zeatin (0.5 mg L-1). The clumps of shoots induced were separated and inoculated in MS medium supplemented with GA3 (0.5 mg L-1) for shoot elongation. After 4-6 weeks, the elongated shoots were transferred to half strength MS medium enhanced with IBA (1 mg L-1). Profuse rooting from the base of the shoot was noticed in 4-5 weeks. The stomatal count with leaves from the diploid plants and in vitro plants observed were 3-4 and 1 respectively indicating the haploidy nature of in vitro plants.

Effects of culture conditions on isolated microspore culture of melon (Cucumis melo L.)

Isolated microspore culture is a useful tool to produce pure, fully homozygous parental lines in a short time. This study evaluated factors including the microspore developmental stage, cell culture density, and heat treatment influencing callus formation from melon microspores (Cucumis melo L.). The results showed that the obtained number of calli induced was highest (11.00±1.02) when cultivating flower buds with sizes 7.0-7.9 mm that contained microspores at middle-to-late uninucleate stages. The optimal microspore density for culture is4×104 cells. The cultured medium was NLN, containing 130 g/l of sucrose at pH 5.8. Heat treatment at 40ºC for 48 hours was best suited for callus induction of all flower bud sizes. The survival rate of microspores after 7 days of culture was lower than before inoculation and was only 75.6%. The development of the microspores and the arising of calli and embryos have been observed and evaluated for morphological cell characteristics. However, in this study, no mature embryo formation or seedling regeneration was observed.

Determination of the Microspore Development Concerning Floral Morphology for Improving Drought Tolerant Citron Watermelon (Citrullus lanatus var. citroides) Rootstocks Via Androgenesis.

The negative effects of abiotic stress factors (especially drought and salinity) that arise due to global climate change are increasing in today’s agricultural production. To eliminate the negative effects of these stress factors, some cultural practices have been implemented such as the breeding of tolerant cultivars and rootstock, exogenous applications of PGPR, AMF, and chemical substances, etc. Contrary to exogenous applications, providing tolerance with breeding methods has an important role in efficient and sustainable agriculture. Androgenesis is a phenomenon for the induction and development of microspore-derived pure lines, as initial material for F1 cultivar breeding, in different vegetable crops. Success in microspore embryogenesis is considerably related to the culture of microspores at an appropriate developmental stage. There is no standard protocol for the anther culture in citron watermelon (Citrullus lanatus var. citroides). Thus, a relation between floral morphology (bud length and width, bud index, sepal position, petal color), and microspore development (microspore diameter, uninucleated and binucleated phases) was investigated in citron. The male flower buds were collected at different sizes and microspore development was observed in both light and fluorescence microscopy. A strong positive relationship was detected between flower bud morphology and the specific stages of microspore development. To the author’s knowledge, this is the first report to indicate that the microspore developmental stage can be estimated by flower bud morphology, and applied in the anther/microspore culture of citron watermelon.

Stages of Microspore Development in Eggplant (Solanum melongena L.)

Background: Microspores are small haploid spores that develop into the male gametophyte. Microsporocytes undergo meiotic division to form microspores. Microspores can be found in seedless and seed plants. The microspores in each flowering plant are different. This study aims to observe microspores on eggplant flowers. Method: Microspore observations were carried out on different flower bud sizes until the flower buds bloomed. Result: The results showed microspores in eggplant had different stages of development for each flower bud size. The stages of microspore development observed were Early uninucleate (Young microspore), Mid-uninucleate, Late uninucleate (Vacuolate microspore), early binucleate (Young bicellular pollen), mid-binucleate, and mature pollen. Conclusion: In eggplant microspore culture, anther length is a strong parameter to predict the stage of microspore development contained there in.

Identification of the most sensitive stage of cotton microspore development to water deficit and analysis of carbohydrate metabolism related to pollen viability

Previous studies have shown that drought occurring at anther development period will have a certain effect on pollen viability, however, it is not clear which developmental stage of cotton microspore is most sensitive to drought and the mechanism of the effect of drought during this period on pollen viability is not explored. To that end, a polyethylene glycol simulated drought experiment (Experiment 1), where drought stress was imposed at different stages of microspore development, was conducted, using cv. Yuzaomian 9110, in order to identify the most sensitive developmental stage to water deficit based on changes in microspore structure and pollen germinability. The results indicated that the microspore mother cell meiosis (MCM) stage was the most sensitive to water deficit. According to that, a second study (Experiment 2) was conducted, using the same cultivar, and the effects of drought stress during MCM-stage on pollen’s carbohydrate and energy metabolism were monitored during and after the stress and their influences on pollen viability were revealed. As per our results, MCM-stage drought down-regulated the sucrose transporter gene GhSUT9 expression which inhibited sucrose import to the anthers; however, increases in sucrose phosphate synthase activity with concurrent decreases in sucrose synthase (SuSy) and invertase (INV) activities resulted in high sucrose content in the water-stressed anthers. In spite of the decreased sucrose decomposition, anther hexose contents were higher in the water-stressed anthers than well-watered anthers, probably due to a decrease in hexose oxidation through the tricarboxylic acid cycle as it was later confirmed by the reduced anther adenosine triphosphate (ATP) content under drought. Contrastingly to sucrose and hexose, anther starch content was greatly reduced by drought, due to decreases in the activities of starch synthesizing enzymes, ADP-glucose pyrophosphorylase (AGPase) and soluble starch synthase (SSSase). After the relief of stress, anther sucrose content decreased to levels lower than well-watered anthers, due to unrecovered GhSUT9 expression and increased alkaline INV activity which enhanced sucrose decomposition. Despite the increased fructose content, glucose content decreased to levels lower than well-watered anthers, without however, resulting in higher energy supply as it was indicated by the lower pollen ATP content after the relief of stress. Anther starch remained lower than well-watered anthers, even after the stress was relieved, as starch biosynthesizing enzymes, AGPase and SSSase, remained suppressed. All these findings suggest that drought stress occurring at the MCM-stage has the most detrimental effect to cotton’s pollen viability due to alterations in carbohydrate metabolism that result in inadequate energy supply.

Anther development in Brachiaria brizantha (syn. Urochloa brizantha) and perspective for microspore in vitro culture.

Brachiaria, a genus from the Poaceae family, is largely cultivated as forage in Brazil. Among the most cultivated varieties of Brachiaria spp., B. brizantha cv. Marandu (syn. Urochloa brizantha) is of great agronomical importance due to the large areas cultivated with this species. This cultivar is apomictic and tetraploid. Sexual diploid genotype is available for this species. The difference in levels of ploidy among sexual and apomictic plants contributes to hindering Brachiaria breeding programs. The induction of haploids and double haploids is of great interest for the generation of new genotypes with potential use in intraspecific crosses. A key factor for the success of this technique is identifying adequate microspore developmental stages for efficient embryogenesis induction. Knowledge of the morphological changes during microsporogenesis and microgametogenesis and sporophytic tissues composing the anther is critical for identifying the stages in which microspores present a higher potential for embryogenic callus and somatic embryo through in vitro culture. In this work, morphological markers were associated with anther and pollen grain developmental stages, through histological analysis. Anther development was divided into 11 stages using morphological and cytological characteristics, from anther with archesporial cells to anther dehiscence. The morphological characteristics of each stage are presented. In addition, the response of stage 8 anthers to in vitro culture indicates microspores initiating somatic embryogenic pathway.

Triticale haploidy <i>in vitro</i> (literature review)

Triticale (× Triticosecale Wittmack) is a hybridized grain crop developed from wheat and rye crossings. Today, triticale is a multipurpose commercial grain crop with great potential as a human food and animal feed. The sown area of the new grain crop in the world reached 4 million hectares in 2018, grain production was about 14 million tons. The current climate change, the rapid evolution of pathogens, as well as the requirements of the modern market dictate the necessity for accelerated development of varieties while reducing the cost of their development. The production of double haploids makes it possible to reduce the time required for the development of homozygous lines by an average of 5–7 years. For the mass production of haploid triticale plants in vitro, there are used two methods, namely anther/microspore culture and distant hybridization followed by selective chromosome elimination of the pollinator. The most critical factors for the success of developing haploids in anther culture are a genotype, growing conditions of donor plants, a microspore development stage, stress effects on heads or anthers, and a nutrient media. Among the unresolved problems of the method are a genotypic dependence, a high incidence of albinism and a presence of aneuploids in the androgenic plant progeny. The rye genome is more often involved in chromosomal transformations than the wheat genome. Most aneuploids are nullisomics, most often on the 2R and 5R chromosomes. Nullisomic plants for 2R and 5R chromosomes have fewer number of spikelets per head and fewer number of kernels per head. In order to develop haploids by the method of selective chromosome elimination during distant hybridization, there have been successfully used such grain crops whose pollen is insensitive to Kr-genes, as maize (Zea mays L.) and wild cereal grass ‘Imperata Cylindrical’ (Imperata cylindrical L.). The advantages of the method are less genotypic dependence, absence of albino plants, genetic stability of regenerants, and reduced costs for developing haploid plants. The length of flowering period of ‘Imperata Cylindrical’ and the absence of the need to combine the timing of flowering period of the parents ensure the economy and efficiency of using this species when developing haploid triticale plants. The purpose of the current review was to characterize the methods of mass development of haploid triticale plants, to describe their advantages and disadvantages when being used in the breeding process.

Androgenic ability and plant regeneration potential in some tomato varieties

Aiming to evaluate the in vitro regeneration potential, five varieties of tomatoes (Solanum lycopersicum) were studied for their response in anther culture. Anther explants at an early stage of microspore development were inoculated onto three culture media. The first differentiation processes were recorded during the first three weeks of culture, in darkness. The statistical analysis of the data recorded during the anther culture showed significant differences between genotypes regarding their specific response to culture conditions and the significant influence of the initiation medium composition in triggering the differentiation processes. Under the tested conditions were induced: the embryogenic potential in three genotypes (ʻȘtefănești 22ʼ, ʻCostate 21ʼ and ʻChihlimbarʼ) and the indirect organogenesis in two genotypes (ʻArgeș 20ʼ and ʻCostate 21ʼ). Morphological characteristics of anther-derived plants from ʻArgeș 20ʼ variety, grown in greenhouse conditions (growth rate, features of leaf, flower, and fruit), as well as analyses with nine SSR markers (banding patterns, the coefficient of genetic similarity, and the polymorphism information content) in DNA samples from each regenerant and the donor variety, provided clear evidence of the occurrence of spontaneous genetic variation during in vitro anther culture, and of the existence of somaclonal variation in regenerated plants. The amplified products obtained with SSR primers revealed a total number of scorable bands of 160 and a mean percentage of polymorphic bands of 21.09%. Two out of the nine tested primers, SSR63 and SLM6-7 proved to be efficient in detecting genetic differences not only among regenerants but also between them and the donor genotype.

Eggplant Anther Culture: Association Between Bud/Anther Size and Microspore Developmental Stage in Different Eggplant Genotypes

Anther culture is a valuable method for obtaining haploid and doubled haploid (DH) plants from microspores and there are several factors influencing the induction of androgenesis such as genotype and microspore development stage. The aim of the present work was, therefore, to identify the bud stages, with maximum amount of young microspores (YM) and mid microspores (MM), thought to be most responsive to embryogenesis induction in anther cultured, and to investigate the influence of genotype on embryogenesis. In this work, first of all anthers and buds containing the highest percentage of YM and MM in four different F1 eggplant genotypes were identified. Results revealed that a certain bud/ anther size group in each genotype might correspond to different microspore / pollen development stage in different genotypes. After determining the best anther length in order to increase the presence of YM and MM corresponding anthers were collected for four genotypes and cultured. Embryo and regenerated plantlet production were taken into consideration to evaluate the response to anther culture for each genotype. Embryos were obtained in all 4 genotypes with variable percentages, ranging from 3,57% to 40.67 %. As a conclusion, related bud and anther length determined for each genotype could be used as fast and reliable criteria to determine the most responsive microspore/pollen developmental stage, which has maximum amount of YM and MM, to increase the efficiency in eggplant anther culture.

Effect of sowing dates of donor plants and concentration of 2,4-D on the purity of productive anther formation of common barley (Hordeum vulgare L.) in anther culture in vitro.

Creating dihaploid lines of agricultural plants is a labour-intensive but essential step in variety production in modern plant breeding. This stage allows significantly accelerate the process of creating new varieties of common barley and other crops. Barley digaploids are produced mainly by anther culture and microspore culture. The authors preferred anther culture in vitro. In the present study, the influence of climatic factors in the cultivation of donor plants on the yield of productive anthers at different sowing dates was established. The authors also identified the more stable culti- vars with a high anther production regardless of sowing date (Signal, Laureate and Eifel). Varieties showed the highest number of embryo-like structures formation at the first and third sowing dates (Zu Suren, Zu Zaza); and sorts with a high rate of productive anther formation at the second sowing date (Acha, Exploer) were identified. Different concentrations of 2,4-D in N6 medium on the frequency of embryogenesis and yield of productive anthers were studied. As a result of this study, the authors found that different concentrations of 2,4-D (1 mg/l and two mg/l) had no significant effect on the for- mation frequency of productive anthers in all the varieties studied. When the embryogenesis capacity of the cultivars was reviewed, all the samples were found to be positive in anther culture. However, the array Zu Suren had a significantly lower effective anthers yield than the samples Signal and Acha. As a result of correlation analysis, the authors found a close relationship between the length of the ear tube of donor plants and the frequency of formation of productive anthers (r = -0.69). A close relationship with the development of optimal microspore phase for the induction of androgenesis in anthers extracted from the ear tube with an average length of 6 cm was determined. This information can significantly speed up the selection of donor plants, but it is recommended to confirm the stage of microspore development microscopically for each new cultivar used.

Comparative transcriptome analysis reveals key insights into male sterility in Salvia miltiorrhiza Bunge

BackgroundLarge-scale heterosis breeding depends upon stable, inherited male sterility lines. We accidentally discovered a male sterility line (SW-S) in the F1progeny of a Salvia miltiorrhiza Bunge from Shandong, China (purple flowers) crossed with a S. miltiorrhiza f. alba from Sichuan, China (white flowers). We sought to provide insights into the pollen development for male sterility in S. miltiorrhiza.MethodsThe phenotypic and cytological features of the SW-S and fertile control SW-F were observed using scanning electron microscopy and paraffin sections to identify the key stage of male sterility. Transcriptome profiles were recorded for anthers at the tetrad stage of SW-S and SW-F using Illumina RNA-Seq.ResultsThe paraffin sections showed that sterility mainly occurred at the tetrad stage of microspore development, during which the tapetum cells in the anther compartment completely fell off and gradually degraded in the sterile line. There was little-to-no callose deposited around the microspore cells. The tetrad microspore was shriveled and had abnormal morphology. Therefore, anthers at the tetrad stage of SW-S and fertile control SW-F were selected for comparative transcriptome analysis. In total, 266,722,270 clean reads were obtained from SW-S and SW-F, which contained 36,534 genes. There were 2,571 differentially expressed genes (DEGs) in SW-S and SW-F, of which 63.5% were downregulated. Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes were enriched in 56 functional groups (GO terms); of these, all DEGs involved in microgametogenesis and developmental maturation were downregulated in SW-S. These results were confirmed by quantitative RT-PCR. The two GO terms contained 18 DEGs, among which eight DEGs (namely: GPAT3, RHF1A, phosphatidylinositol, PFAS, MYB96, MYB78, Cals5, and LAT52) were related to gamete development. There were 10 DEGs related to development and maturation, among which three genes were directly related to pollen development (namely: ACT3, RPK2, and DRP1C). Therefore, we believe that these genes are directly or indirectly involved in the pollen abortion of SW-S. Our study provides insight into key genes related to sterility traits in S. miltiorrhiza, and the results can be further exploited in functional and mechanism studies.

Direct embryogenesis from anther culture of hot chilli Capsicum annuum L.

In Viet Nam, local varieties of chili have a distinctive aroma and pungency. However, the generation of pure lines from pollen culture in local hot chilli has been very limited reported. Therefore, this study was to relate flower bud size with microspore developmental stages and the culture media have concentration changed of growth regulator effects on the in vitro androgenesis. Flower buds were randomly collected and visually divided into three stage based on both petal and sepal size. Then anther was cultured on MS basal medium with different concentration of hormones NAA changed 0.1 - 0.7 mg/L and kinetin changed 1.0 - 3.0 mg/L, BA changed 0.5 - 1.5 mg/L. The results showed that bud flower have anthers are light violet in color, 2.5 mm long, consisted of anthers with 80 % uni-nucleate and 20 % bi-nucleate microspores were selected. In induction culture media, it was observed that MS medium with 2.0 mg/L Kinetin and 0.5 mg/L NAA gave higher embryo frequency. MS medium with 1 mg/L BA is the best medium for embryo germination and inducting shoots. And ½ MS medium for shoot elongation and rooting.

Качество пыльцы сосны обыкновенной (Pinus sylvestris L.) в зоне действия выбросов АО «Карабашмедь»

The study results of Common pine mature pollen state in the smoke emissions from the plant JSC «Karabashmed» in connection with the level of technogenic impact are presented. It is shown that the state of the vegetation cover is in accordance with the level of technogenic pollution, determined by the accumulation degree of air pollutants in the snow cover. It was established that the soil and vegetation were degraded in the flare zone of the plant. There are no pine stands at a distance of less than 3.5 km from the source of smoke emissions. Symptoms of chronic and acute damage to the assimilation apparatus and disturbance of the generative sphere of pine at a distance of 8 km to the southeast were revealed. A significant similarity in the vital state of pine stands at a distance of 13 km (zone of low aerosol pollution) and 19 km (background conditions) from the source was established. At the same time, the frequency of small and degenerated pollen was many times higher in the pool of mature pine pollen in the stand under conditions of a low level of pollution than under background conditions. A high frequency of pollen grains with anomalies of air pockets was detected in the stand under background conditions. The results of the study indicate a high susceptability of the male generative system to technogenic pollution (accumulated soil, aerosol, gas) and the possibility of its bioindication in the absence of symptoms of damage to the assimilation apparatus. It was shown that disturbances in the development of male gametophyte in the zones of technogenic pollution are already realized at the early stages of microspore development and are revealed by the high frequency of small underdeveloped pollen. It was found that most of the pine trees growing on the soil of fine-stone-sandy composition correspond to the categories of weakened and dying ones, which makes it possible to recognize the conditions of the man-made land as extremely unfavorable for the growth and development of pine. The mature pine pollen of these stands is characterized by the peculiar features that distinguish it from the pollen of other stands. The conclusion is made about the negative effect of smoke emissions from the JSC «Karabashmed» plant on the condition of pine plantations and the male generative system of pine, even at a considerable distance from the source of emissions.

An integral insight into pollen wall development: involvement of physical processes in exine ontogeny in Calycanthus floridus L., with an experimental approach.

We aimed to understand the underlying mechanisms of development in the sporopollenin-containing part of the pollen wall, the exine, one of the most complex cell walls in plants. Our hypothesis is that distinct physical processes, phase separation and micellar self-assembly, underpinexine development by taking the molecular building blocks, determined and synthesised by the genome, through several phase transitions. To test this hypothesis, we traced each stage of microspore development in Calycanthus floridus with transmission electron microscopy and then generated invitro experimental simulations corresponding to every developmental stage. The sequence of structures observed within the periplasmic space around developing microspores starts with spherical units, which are rearranged into columns to then form rod-like units (the young columellae) and, finally, white line centred endexine lamellae. Phase separation precedes each developmental stage. The set of experimental simulations, obtained as self-assembled micellar mesophases formed at the interface between lipid and water compartments, was the same: spherical micelles; columns of spherical micelles; cylindrical micelles; and laminate micelles, separated by gaps, resembling white-lined lamellae. Thus, patterns simulating structures observed at the main stages of exine development in C.floridus were obtained from invitro experiments, and hence purely physicochemical processes can construct exine-like patterns. This highlights the important part played by physical processes that are not under direct genomic control and share influence on the emerging ultrastructure with the genome during exine development. These findings suggest that a new approach to ontogenetic studies, including a consideration of physical factors, is required for a better understanding of developmental processes.

Effect of Chemical Composition of Nutritive Medium and Explant Size Over Androgenetic Response in Microspore Culture of Brassica oleracea L.

The dimension of the bud is a key factor for the orientation of microspore culture and the success of obtaining double haploid plants as it is a strong correlation between bud size and the developmental stages of microspores, and it is specific for each plant species and genotype. Our study was focused to determine the correlation between morphological characteristics, namely floral bud size and specific microspore developmental stages in order to determine the proper size, suitable for a successful protocol of obtaining double haploid plants in Brassica oleracea var. italica. Thus, we tested four bud sizes ranging from 2.0 to 4.0 mm measured from the base to the tip of the bud. After the statistical analysis of the results it can be emphasized that the best results were obtained in the case of using as a source of microspores the flower buds with the size between 3.1-3.5 mm. At this dimension, the share of microspores in the uninucleate stage, predominantly in the late uninucleate stage, is 90%, thus ensuring a homogeneous population of microspores in the optimum stage of development. Their evolution is predominantly embryogenic, the percentage of microspores following the gametophytic pathway is reduced, by only 9.12%.

Expression pattern analysis of key genes related to anther development in a mutant of male-sterile Betula platyphylla Suk.

Male-sterile mutants are ideal materials for studying the gene regulatory pathway of anther development. Here, the cytological morphology of mutant and wild-type anthers during the whole development stage was analyzed. At the microspore developmental stage, the tapetum cells of two types of mutants (mutant male inflorescences (MM), normal-like male inflorescences (NLM) showed additional vacuoles and a reduced cytoplasm, while the wild-type tapetum cells began to degenerate. Before pollination, compared with formed mature pollen grains in wild-type male inflorescences, collapsed microspores were discovered in mutants. Based on the transcriptome data of wild-type male inflorescence (NM) and mutant MM, ten key differentially expressed genes (ABORTED MICROSPORES (AMS), MYB38, MALE STERILITY 1 (MS1), MYB32, ODORANT1 (ODO1), QUARTET3 (QRT3) and CYTOCHROME P450 77A3 (CYP77A3), ATP-Binding Cassette Transporter G26 (ABCG26), FIL1, LIPID-TRANSFER PROTEIN 14 (LTP14)) related to anther development were selected for further study. Furthermore, MYB32, MYB38, AMS, MS1, and ABCG26 were highly expressed at the tetrad stage in MM and NLM, along with ODO1, QRT3, and CYP77A3, which were significantly upregulated at the tetrad stage in NLM, FIL1 and LTP14 were upregulated at the tetrad stage in MM. These events suggested that these genes may accelerate the abortion process in mutant birch anther. Tissue-specific expression analysis of ten genes indicated diverse spatiotemporal expression patterns. QRT3 and CYP77A3 were expressed in all five tissues, including leaves, stems, roots, female inflorescences, and male inflorescences, while the other eight genes had high transcription levels during male development, indicating that these genes may play different roles in these developmental processes. These results facilitate further investigation of the functional and molecular mechanisms underlying anther and microspore development in Betula platyphylla.

Factors affecting DH plants in vitro production from microspores of European radish.

Over the recent years the market demand for scaling up the production of European radish (Raphanus sativus L.) varieties and hybrids for open and protected production, varying in ripeness group, root shape and color, has drastically increased. Therefore, the expansion of genetic diversity and acceleration of the selection process are important. Doubled haploid technology considerably curtails the time required for creation of homozygous constant parental cell lines when in vitro microspore culture is used as the most promising method. For the first time, we were able to realize the full production cycle of DH plants of European radish by in vitro microspore culture up to inclusion of the produced material into the selection process. We have selected: preferable flower bud size, heat shock parameters, induction and regeneration media. It was revealed that linear length on the flower buds with the best possible stage of microspore development is genotype-specific: the flower bud length 2.8–3.3 mm is optimal for accessions of Rhodes and 3.7–4.2 mm is optimal for accessions of Teplichny Gribovsky. Heat shock at 32 °C for 48 hours is the most suitable for most genotypes. For the first time Murashige and Skoog based culture medium has been used for embryogenesis induction, and a major dependence of embryogenesis induction on the genotype × medium interaction was found. At regeneration and tiller stage it is advisable to add 1 mg/mL of benzylaminopurine and 0.1 mg/L of gibberellic acid to the medium, and rotting of micro-sprouts is performed with the use of hormone-free medium. Analysis of the produced regenerant plants by chromosome count and cell nucleus flow cytometry showed that 69 % of plants have a diploid chromosome set, 9 % have a haploid chromosome set, and 22 % have mixoploids and aneuploids chromosome sets. The seed progeny from doubled haploids and mixoploids were obtained by self-pollination, where all R1 plants had a doubled set of chromosomes. This study launches the development of an efficient method of radish doubled haploid production to be used in the selection process.