Rice Anthers Research Articles

3D Imaging and In Situ Hybridization for Uncovering the Functions of MicroRNA in Rice Anther.

Small RNAs specifically expressed in reproductive tissues are key regulators of germline development in eukaryotes. Rice microRNA2118 (miR2118), which is enriched during reproduction in grasses, is a trigger to produce phased small interfering RNAs (phasiRNAs). These phasiRNAs demonstrate the temporal regulation with premeiotic phasiRNAs and meiotic phasiRNAs in anther development. Furthermore, the site-specific regulation via miR2118 and phasiRNAs is of importance in soma and germ development in anthers. Accordingly, histological imaging methods are essential tools for understanding spatiotemporal regulation during reproduction and elucidating the reproductive roles of miRNAs and phasiRNAs. We successfully developed a method to visualize the three-dimensional (3D) structure of entire rice anthers, which can also be used for distinguishing the internal structure of the anthers in other plants. Here, we describe the detailed methods of in situ hybridization for miR2118 localization and the visualization of the 3D structure of entire anthers of rice.

Reproductive cells and peripheral parietal cells collaboratively participate in meiotic fate acquisition in rice anthers.

In flowering plants, the transition from mitosis to meiosis is the precondition for gametogenesis, which is the most crucial event during sexual reproduction. Here, we report an intriguing mechanism whereby germ cells and surrounding somatic cells cooperatively involve in the meiotic switch during anther development in rice (Oryza sativa). In double mutants with loss function of both leptotene chromosome establishment- and somatic cell layer differentiation-associated genes, chromosome morphology in the reproductive cells remains the same as that in somatic cells, and sporogenous cells fail to differentiate into pollen mother cells. OsSPOROCYTELESS and MICROSPORELESS1, two pivotal genes involved in meiosis entry, are prominently downregulated in anthers of plants with mutations in both MULTIPLE SPOROCYTE1 and LEPTOTENE 1. In addition, the transcription of redox-related genes is also affected. Therefore, germ cells and the surrounding somatic cells collaboratively participate in meiosis initiation in rice.

Two rice MYB transcription factors maintain male fertility in response to photoperiod by modulating sugar partitioning.

Photoperiod-dependent male fertility is a critical enabler of modern hybrid breeding. A MYB transcription factor, CSA, is a key regulator of sugar partitioning in rice anthers, disruption of which causes photoperiod-sensitive male sterility. However, little is known about the molecular mechanisms governing plant fertility in response to photoperiod. Here, we have obtained another rice photoperiod-sensitive male sterile mutant, csa2, which exhibits semi-sterility under long-day (LD) conditions, with normal fertility under short-day (SD) conditions. CSA2 specifically expressed in anthers, and here is shown to be indispensable for sugar partitioning to anthers under LD conditions. The CSA2 protein can restore the fertility of csa mutants under SD conditions when expressed in a CSA-specific pattern, indicating that the two proteins share common downstream regulatory targets. Transcriptomic analyses also reveal discrete regulatory targets in anthers. Furthermore, the regulatory role of CSA2 in sugar transport was influenced by the photoperiod conditions during floral initiation, not simply during anther development. Collectively, we propose that rice evolved at least two MYB proteins, CSA2 and CSA, that regulate sugar transport in anthers under LD and SD conditions, respectively. This finding provides insight into the molecular mechanisms that regulate male fertility in response to photoperiod.

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1 (OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator (GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of OsGPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. OsGPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that AtGPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of OsGPT1 in the tapetum degeneration and pollen development. The divergent functions of OsGPT1 and AtGPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.

Effect of extensin-like OsPEX1 on pollen fertility in rice.

LRXs (leucine-rich repeat extensins) are chimeric cell wall proteins containing an N-terminal leucine-rich repeat (LRR) and a C-terminal extensin domain. Increasing evidences suggest that LRXs family genes play important roles in pollen germination and pollen tube growth in Arabidopsis thaliana. However, the functions of rice (Oryza sativa L.) LRX genes in pollen development remain poorly understood. Bioinformatics analysis showed that the rice LRX gene family consist of eight members, namely OsPEX3, OsLRX3 and OsLRX5 located on chromosome 1, OsLRX1, OsLRX3, OsLRX2,OsPEX1 and OsPEX2 located on chromosome 2, 5, 6, 11 and 12, respectively. The OsPEX1 gene is preferentially expressed in rice anther, suggesting that it may be involved in the regulation of pollen development. Next, we further investigated the role of the OsPEX1 gene in rice by knockdown of its expression using an RNAi approach. The OsPEX1 RNAi transgenic lines showed a significant decrease in seed setting rate (10%~30%) due to pollen sterility. Further quantitative RT-PCR analysis indicated that the OsPEX1 gene was significantly down-regulated in the RNAi transgenic lines. The results indicate that the OsPEX1plays an important role in the regulation of rice pollen development. Further studies on this gene could provide insights on the molecular and genetic mechanisms in this developmental process.

Transcriptome Analysis Reveals Photoperiod-Associated Genes Expressed in Rice Anthers.

Environmental conditions, such as photoperiod and temperature, can affect male fertility in plants. While this feature is heavily exploited in rice to generate male-sterile lines for hybrid breeding, the underlying molecular mechanisms remain largely unknown. In this study, we use a transcriptomics approach to identify key genes and regulatory networks affecting pollen maturation in rice anthers in response to different day lengths. A total of 11,726 differentially expressed genes (DEGs) were revealed, of which 177 were differentially expressed at six time points over a 24-h period. GO enrichment analysis revealed that genes at all time points were enriched in transport, carbohydrate, and lipid metabolic processes, and signaling pathways, particularly phytohormone signaling. In addition, co-expression network analysis revealed four modules strongly correlated with photoperiod. Within these four modules, 496 hub genes were identified with a high degree of connectivity to other photoperiod-sensitive DEGs, including two previously reported photoperiod- and temperature-sensitive genes affecting male fertility, Carbon Starved Anther and UDP-glucose pyrophosphorylase, respectively. This work provides a new understanding on photoperiod-sensitive pollen development in rice, and our gene expression data will provide a new, comprehensive resource to identify new environmentally sensitive genes regulating male fertility for use in crop improvement.

Comparative Analysis of Callus Induction and Plant Regeneration Rates Using One-step and Two-step Cultures for Rice Anther Cultivation

Comparative Analysis of Callus Induction and Plant Regeneration Rates Using One-step and Two-step Cultures for Rice Anther Cultivation

The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

OsMFS1/OsHOP2 Complex Participates in Rice Male and Female Development

Meiosis plays an essential role in the production of gametes and genetic diversity of posterities. The normal double-strand break (DSB) repair is vital to homologous recombination (HR) and occurrence of DNA fragment exchange, but the underlying molecular mechanism remain elusive. Here, we characterized a completely sterile Osmfs1 (male and female sterility 1) mutant which has its pollen and embryo sacs both aborted at the reproductive stage due to severe chromosome defection. Map-based cloning revealed that the OsMFS1 encodes a meiotic coiled-coil protein, and it is responsible for DSB repairing that acts as an important cofactor to stimulate the single strand invasion. Expression pattern analyses showed the OsMFS1 was preferentially expressed in meiosis stage. Subcellular localization analysis of OsMFS1 revealed its association with the nucleus exclusively. In addition, a yeast two-hybrid (Y2H) and pull-down assay showed that OsMFS1 could physically interact with OsHOP2 protein to form a stable complex to ensure faithful homologous recombination. Taken together, our results indicated that OsMFS1 is indispensable to the normal development of anther and embryo sacs in rice.

Effect of sucrose on calus induction and green plantlet regeneration in anther culture of Indica x Indica rice

Anther culture is an important biotechnological tool. By rice anther culture homozygous pure lines in the form of doubled haploid (DH) plants can be produced within a year as compared to the long inbreeding method, which might take 8-10 years. Application of anther culture technique in breeding and genetics research is limited due to the very low regeneration frequency of anthers of rice in general, and indica cultivars in particular. Therefore, the successful use of the technique depends on the adequate production of DH plants for selection and field evaluation. Effect of various concentrations of sucrose in callus induction media was investigated on callus induction and regeneration of green plantlets from anther culture of several F1s derived from indica x indica crosses. Cold pretreated anthers of five genotype of anther donor plants (F1s) at 5 °C for 8 days were cultured on N6 callus induction medium containing sucrose at a concentration of 6.0%, 6.5%, and 7.0%. Results revealed that all genotypes showed similar response to callus induction, but a significantly different responses to plant regeneration depended on sucrose level in the callus induction media. Two genotypes, i.e. IR78788 / Inpara 5 and Dendang/Inpari 30 only regenerated albino plantlets. In this experiment, 6.5% sucrose compared to two other sucrose treatments was suitable for inducing high frequency callus induction and high green plant regeneration in all genotypes. The high concentration of sucrose (7.0%) in the culture medium not only resulted in a decrease in the number and percentage of callus formation but also decreased the regeneration of green plantlets.

OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways.

Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

Evolution of PHAS loci in the young spike of Allohexaploid wheat

BackgroundPhasiRNAs (phased secondary siRNAs) play important regulatory roles in the development processes and biotic or abiotic stresses in plants. Some of phasiRNAs involve in the reproductive development in grasses, which include two categories, 21-nt (nucleotide) and 24-nt phasiRNAs. They are triggered by miR2118 and miR2275 respectively, in premeiotic and meiotic anthers of rice, maize and other grass species. Wheat (Triticum aestivum) with three closely related subgenomes (subA, subB and subD), is a model of allopolyploid in plants. Knowledge about the role of phasiRNAs in the inflorescence development of wheat is absent until now, and the evolution of PHAS loci in polyploid plants is also unavailable.ResultsUsing 261 small RNA expression datasets from various tissues, a batch of PHAS (phasiRNA precursors) loci were identified in the young spike of wheat, most of which were regulated by miR2118 and miR2275 in their target site regions. Dissection of PHAS and their trigger miRNAs among the diploid (AA and DD), tetraploid (AABB) and hexaploid (AABBDD) genomes of Triticum indicated that distribution of PHAS loci were dominant randomly in local chromosomes, while miR2118 was dominant only in the subB genome. The diversity of PHAS loci in the three subgenomes of wheat and their progenitor genomes (AA, DD and AABB) suggested that they originated or diverged at least before the occurrence of the tetraploid AABB genome. The positive correlation between the PHAS loci or the trigger miRNAs and the ploidy of genome indicated the expansion of genome was the major drive force for the increase of PHAS loci and their trigger miRNAs in Triticum. In addition, the expression profiles of the PHAS transcripts suggested they responded to abiotic stresses such as cold stress in wheat.ConclusionsAltogether, non-coding phasiRNAs are conserved transcriptional regulators that display quick plasticity in Triticum genome. They may be involved in reproductive development and abiotic stress in wheat. It could be referred to molecular research on male reproductive development in Triticum.

Global identification and analysis revealed differentially expressed lncRNAs associated with meiosis and low fertility in autotetraploid rice

BackgroundAutotetraploid rice is a useful germplasm for polyploid rice breeding. Our previous research showed that non-coding RNAs might be associated with low fertility in autotetraploid rice. However, little information is available on long non-coding RNAs (lncRNAs) involved in the low fertility of autotetraploid rice. In the present study, RNA-seq was employed to detect the differentially expressed meiosis-related lncRNAs in autotetraploid rice, and gene overexpression and knock out experiments were used to validate the potential function of candidate lncRNA.ResultsA total of 444 differentially expressed lncRNAs (DEL) were detected during anther and ovary meiosis in autotetraploid rice. Of these, 328 DEL were associated with the transposable elements, which displayed low expression levels during meiosis in autotetraploid rice. We used rapid amplification of cDNA ends (RACE) assay to validate 10 DEL and found that the lncRNAs were not assembly artifacts, and six of them were conserved in tetraploid rice. Moreover, 237 and 20 lncRNAs were associated with pollen mother cell (PMC) and embryo sac mother cell (EMC) meiosis in autotetraploid rice, respectively. The differential expressions of some meiosis-related targets and its DEL regulator, including MEL1 regulated by TCONS_00068868, LOC_Os12g41350 (meiotic asynaptic mutant 1) by TCONS_00057811 in PMC, and LOC_Os12g39420 by TCONS_00144592 in EMC, were confirmed by qRT-PCR. TCONS_00057811, TCONS_00055980 and TCONS_00130461 showed anther specific expression patterns and were found to be highly expressed during meiosis. CRISPR/Cas9 editing of lncRNA57811 displayed similar morphology compared to wild type. The overexpression of lncRNA57811 resulted in low pollen fertility (29.70%) and seed setting (33%) in rice.ConclusionThe differential expression levels of lncRNAs, associated with transposable elements and meiosis-regulated targets, might be endogenous noncoding regulators of pollen/embryo sac development that cause low fertility in autotetraploid rice. The results enhance our understanding about rice lncRNAs, and facilitate functional research in autotetraploid rice.

Dynamic Transcriptome Analysis of Anther Response to Heat Stress during Anthesis in Thermotolerant Rice (Oryza sativa L.).

High temperature at anthesis is one of the most serious stress factors for rice (Oryza sativa L.) production, causing irreversible yield losses and reduces grain quality. Illustration of thermotolerance mechanism is of great importance to accelerate rice breeding aimed at thermotolerance improvement. Here, we identified a new thermotolerant germplasm, SDWG005. Microscopical analysis found that stable anther structure of SDWG005 under stress may contribute to its thermotolerance. Dynamic transcriptomic analysis totally identified 3559 differentially expressed genes (DEGs) in SDWG005 anthers at anthesis under heat treatments, including 477, 869, 2335, and 2210 for 1, 2, 6, and 12 h, respectively; however, only 131 were regulated across all four-time-points. The DEGs were divided into nine clusters according to their expressions in these heat treatments. Further analysis indicated that some main gene categories involved in heat-response of SDWG005 anthers, such as transcription factors, nucleic acid and protein metabolisms related genes, etc. Comparison with previous studies indicates that a core gene-set may exist for thermotolerance mechanism. Expression and polymorphic analysis of agmatine-coumarin-acyltransferase gene OsACT in different accessions suggested that it may involve in SDWG005 thermotolerance. This study improves our understanding of thermotolerance mechanisms in rice anthers during anthesis, and also lays foundation for breeding thermotolerant varieties via molecular breeding.

Monitoring rice anther proteome expression patterns during pollen development

Rice (Oryza sativa L.) pollen grains are highly sensitive to extreme temperatures. Studies on the proteomic analysis of pollen at different developmental stages are limited. In this study, to identify proteins expressed during pollen development, we performed shotgun proteomic analysis of rice anthers harvested at 8, 3, and 0 days before heading (DBH). A total of 4509 non-redundant proteins were identified in rice anthers at each of the three time points with three replications per time point. Among these, 2968 proteins were reproducible, of which 1919 were specifically expressed at a given stage of anther development. Clustering and gene ontology (GO) enrichment analyses categorized the specifically expressed proteins into four groups. Group I comprised proteins involved in cell differentiation, reproduction, and stress response; Group II contained proteins related to cell growth and carbohydrate metabolic process; Group III included proteins related to starch accumulation and lipid metabolic process; Group IV comprised proteins related to structure morphogenesis and cellular components. Proteins in Group I steadily increased during anther development, whereas those in Group I steadily decreased. Group III proteins showed the highest expression at 3 DBH, whereas Group IV proteins exhibited the lowest expression at 3 DBH.

Development of rice pre-breeding resources with blast resistance

Within the framework of this study, the first Russian-Chinese joint program for development of rice varieties with long-term blast resistance was launched on the basis of rice germplasm exchange and the subsequent comprehensive study of the obtained breeding samples, hybridization of Russian and Chinese varieties with specified traits. The genetic diversity of the Chinese rice samples obtained by exchange was studied by biological and morphological traits of plants, taxonomic belonging to a botanical variety, elements of the yield structure, and resistance to lodging. 31 hybrid combinations (F1) from the crossing of Russian and Chinese varieties were used to obtain the BC1 generation. The technologies of cultivation of isolated rice anthers in vitro have been optimized in relation to Chinese genotypes in order to accelerate the genetic stabilization of breeding material obtained from crossing. Phenotyping of Russian and Chinese breeding samples was carried out on the basis of resistance to the local population of the blast pathogen (Magnaporthe grisea (T.T. Hebert) M.E. Barr)), on natural and artificial infectious backgrounds. DNA identification of genes for blast resistance was carried out for the same breeding samples. Based on the experimental data obtained, promising samples were selected - prototypes of new rice varieties.

Structure and expression analysis of TaGW5 in common wheat

OsGW5 (OsGSE) regulates cell proliferation and is involved in regulating grain size and thousand-grain weight in rice. Our knowledge about its wheat ortholog TaGW5 is limited. In the present study, we characterized the structure and expression of TaGW5 at molecular level in wheat and predicted the cis-elements and transcription factor binding sites (TFBS) of its promoter. The GW5 orthologs in barley (HvGW5), rice (OsGW5), Triticum turgidum L. (TtGW5) and Brachypodium distachyon (BdGW5) were also identified for comparative analyses. TaGW5 was mapped onto the short arms of group 1 chromosomes (1AS, 1BS, and 1DS). Multiple alignments indicated GW5 possesses three exons and two introns in all the analyzed species except for rice and the exon-intron junction composed of exon 2 and intron 2 was highly conserved. GW5 has a conserved domain (DUF 4005) and two neighboring IQ domains and was mainly expressed in wheat young spikes, in barley immature inflorescences and in rice anthers. Drought, heat and biotic-stress treatments had no significant effects on HvGW5 and TaGW5 expression. Significant correlation between the expression patterns of predicted transcription factor ABF2 and OsGW5 was also detected. Taken together, these results broaden our understanding of GW5 in wheat, barley and rice and will be helpful for further manipulating GW5 and uncovering its roles in plants

Variability of Rice Haploids Obtained From In Vitro Anther Culture

Morphological variability of haploid plants and doubled haploids of rice obtained from one callus line in in vitro anther culture has been studied. The study was performed on rice (Oryza sativa L. subsp. japonica Kato) plants of the Kaskad varietу. Regenerants of the same callus line derived from one rice anther (four lines in total) were divided into 2–3 groups of 20–30 plants (depending on the sample size) in the order of their differentiation on a callus and transplantation on the rooting medium. Two callus lines (15.1 and 18.1) generated both haploids and doubled haploids (in equal proportion), while two other callus lines (5.1 and 7.2) formed numerous haploids. In the case of the 5.1 and 7.2 lines, regenerants with a larger ordinal number were characterized by decreased size indices of derived plants (plant height, number of flowers on the main panicle, and the number of panicles). No statistically significant difference has been revealed between the haploids and doubled haploids originating from the 15.1 and 18.1 lines. To induce the doubling of a chromosome number in haploid regenerants by colchicine or other antitubulin compounds, it is recommended to use the earliest plants developing on a callus. Using the Hotelling’s T2 criterion calculated for the whole complex of biometric indices, a statistically significant (p = 0.001) difference was revealed between haploids of four callus lines and doubled haploids of two callus lines. Haploids of different lines differed by 3–4 traits, while doubled haploids differed by three of five traits (panicle length, productive tilling capacity, and plant height). Rice varieties interesting for breeders may be improved using in vitro anther cultures.

Structural Organization and Functional Activity of the Orthologous TaGLW7 Genes in Bread Wheat (Triticum aestivum L.)

It is reported that GLW7 encoding the transcription factor OsSPL13, positively regulates grain size and shape in rice. We have limited knowledge about its orthologs in wheat. Here, based on the rice OsGLW7 we isolated and identified the TaGLW7 gene in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, analyzed its expression patterns. The orthologs in barley (HvGLW7), Brachypodium (BdGLW7), wild emmer (TtGLW7), Aegilops tauschii (AtGLW7) were also used for comparative analysis. As predicated, TaGLW7, HvGLW7, TtGLW7, and AtGLW7 were mapped onto group 2 chromosomes in the respective species. Multiple alignments indicated GLW7 possesses two exons and one intron in the analyzed species. GLW7 contains a conserved domain SBP and two neighboring low complexity regions. GLW7 was highly expressed in spike organs including wheat young spikes, barley inflorescence, and rice anthers. Additionally, biotic stress significantly down-regulated GLW7 in wheat and barley. Significant correlations between the expression patterns of predicted transcription factor ABF2 and TaGLW7 were detected. In conclusion, the conserved structure and expression of GLW7 among the investigated species and the predicted transcription factors significantly related to GLW7 are helpful for further manipulating GLW7 and uncovering its roles in plants.

Variability of rice haploids obtained in anther culture in vitro

Morphological variability of haploid plants and doubled haploids of rice obtained on one callus line in anther culture in vitro was studied. The work was carried out on rice plants Oryza sativa L. subspecies japonica Kato varietу Cascade. Regenerant plants of one callus line obtained from one rice anther (four in total) were divided into two or three groups of 20-30 plants, depending on the sample size in order of their differentiation on callus and transplantation on the rooting medium. Two callus lines (15.1 and 18.1) formed half of the haploids, half of the doubled haploids, and two other callus lines (5.1 and 7.2) numerous haploids. On callus lines with numerous haploids (5.1 and 7.2), as the regenant number increases, the size of plants decreases (plant height, number of flowers on the main panicle, number of panicles). On the lines 15.1 and 18.1 between groups of haploids and between groups of doubled haploids statistically significant differences absent. In breeding purposes for the induced doubling of the number of chromosomes in haploid regenerants with antitubulin substances such as colchicine, it is advisable to use plants that form on callus among the first. Between haploids of four callus lines and doubled haploids of two callus lines, statistically significant differences (at p=0.001) were revealed using the Hotelling's T2-criterion, calculated for the whole complex of biometric features. Haploids of different lines differed in three or four of them, doubled haploids on three of the five signs (length of panicle, productive bushiness and plant height). Varieties of interest to breeders may be improved by anther culture in vitro.