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

KEY MESSAGE: In 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.

Analysis of histone H3K4me3 modifications in bovine placenta derived from different calf-production methods.

In ruminants, the overgrowth of offspring produced by in vitro fertilization (IVF) is a common problem. Abnormal epigenetic modifications caused by environmental factors during the early embryonic period are suspected as an aetiology of overgrowth. In this study, we investigated the genome-wide histone H3K4me3 profiles of bovine placentae that play a pivotal role in foetal development and compared their characteristics between artificial insemination (AI)- and IVF-derived samples. Cotyledons were harvested from the placentae obtained at parturition of 5 AI- and 13 IVF-derived calves, and chromatin immunoprecipitation sequencing was performed for H3K4me3. We confirmed no significant maternal tissue contamination in the samples we used. The revealed H3K4me3 profiles reflected the general characteristics of the H3K4me3 modification, which is abundantly distributed in the promoter region of active genes. By extracting common modifications from multiple samples, the genes involved in placenta-specific biological processes could be enriched. Comparison with the H3K4me3 modifications of blastocyst samples was also effective for enriching the placenta-specific features. Principal component analysis suggested the presence of differential H3K4me3 modifications in AI- and IVF-derived samples. The genes contributing to the difference were related to the developmental biological processes. Imprinted genes such as BEGAIN, ZNF215 and DLX5 were among the extracted genes. Principal component and discriminant analyses using only male samples categorized the samples into three groups based on foetal weight and calf-production methods. To our knowledge, this is the first study to profile the genome-wide histone modifications of bovine foetal placentae and reveal their differential characteristics between different calf-production methods.

Evaluating histone modification analysis of individual preimplantation embryos.

BACKGROUND: We previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT). RESULTS: WOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing. CONCLUSIONS: These results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

Automated estimation of stomatal number and aperture in haskap (Lonicera caerulea L.).

MAIN CONCLUSION: This study developed the reliable Mask R-CNN model to detect stomata in Lonicera caerulea. The obtained data could be utilized for evaluating some characters such as stomatal number and aperture area. The native distribution of haskap (Lonicera caerulea L.), a small-shrub species, extends through Northern Eurasia, Japan, and North America. Stomatal observation is important for plant research to evaluate the physiological status and to investigate the effect of ploidy levels on phenotypes. However, manual annotation of stomata using microscope software or ImageJ is time consuming. Therefore, an efficient method to phenotype stomata is needed. In this study, we used the Mask Regional Convolutional Neural Network (Mask R-CNN), a deep learning model, to analyze the stomata of haskap efficiently and accurately. We analyzed haskap plants (dwarf and giant phenotypes) with the same ploidy but different phenotypes, including leaf area, stomatal aperture area, stomatal density, and total number of stomata. The R-square value of the estimated stomatal aperture area was 0.92 and 0.93 for the dwarf and giant plants, respectively. The R-square value of the estimated stomatal number was 0.99 and 0.98 for the two phenotypes. The results showed that the measurements obtained using the models were as accurate as the manual measurements. Statistical analysis revealed that the stomatal density of the dwarf plants was higher than that of the giant plants, but the maximum stomatal aperture area, average stomatal aperture area, total number of stomata, and average leaf area were lower than those of the giant plants. A high-precision, rapid, and large-scale detection method was developed by training the Mask R-CNN model. This model can help save time and increase the volume of data.

Deep learning-based high-throughput detection of in vitro germination to assess pollen viability from microscopic images.

In vitro pollen germination is considered the most efficient method to assess pollen viability. The pollen germination frequency and pollen tube length, which are key indicators of pollen viability, should be accurately measured during in vitro culture. In this study, a Mask R-CNN model trained using microscopic images of tree peony (Paeonia suffruticosa) pollen has been proposed to rapidly detect the pollen germination rate and pollen tube length. To reduce the workload during image acquisition, images of synthesized crossed pollen tubes were added to the training dataset, significantly improving the model accuracy in recognizing crossed pollen tubes. At an Intersection over Union threshold of 50%, a mean average precision of 0.949 was achieved. The performance of the model was verified using 120 testing images. The R2 value of the linear regression model using detected pollen germination frequency against the ground truth was 0.909 and that using average pollen tube length was 0.958. Further, the model was successfully applied to two other plant species, indicating a good generalizability and potential to be applied widely.

Evaluation of pollen tube growth ability in Petunia species having different style lengths.

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.

Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag).

Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the conventional method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive and negative peaks and lower resolution for broad modifications. Despite these limitations, we consider NTU-CAT a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

Genetic Structure of Native Blue Honeysuckle Populations in the Western and Eastern Eurasian Ranges.

Blue honeysuckle (Lonicera caerulea L.) is a promising berry crop producing edible early-ripening berries with a valuable chemical composition. We evaluated the genetic diversity of native L. caerulea populations from the western (Baltic states) and eastern (the Russian Far East and Japan) edges of the Eurasian range using inter-simple sequence repeat (ISSR) and chloroplast DNA (psbA-trnH and trnL-trnF) markers. The genetic relationships of populations and genotypes were analyzed using principal coordinate and cluster analyses (neighbor joining and Bayesian clustering). Sampling was carried out in two disjunct areas of this circumpolar species and the analyses showed clustering of individuals and populations according to geographic origin. The analysis of genetic structure based on ISSR markers showed that the studied populations of L. caerulea were highly differentiated. However, sequence analysis of two chloroplast DNA (cpDNA) regions revealed no phylogeographic structure among the populations. We also found that the eastern populations of blue honeysuckle had significantly greater genetic diversity parameters than the populations from the Baltic region. This finding correlates with the endangered status of blue honeysuckle in the Baltic states.

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate.

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species.

In F1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer-protector model.

Comparison of anthocyanin distribution in berries of Haskap (Lonicera caerulea subsp. edulis (Turcz. ex. Herder) Hultén), Miyama-uguisukagura (Lonicera gracilipes Miq.), and their interspecific hybrid using imaging mass spectrometry.

Haskap (Lonicera caerulea subsp. edulis), a shrub with violet-blue fruits, is distributed mainly in Hokkaido, Japan. Miyama-uguisukagura (Lonicera gracilipes), a species related to Haskap, produces red fruits. Interspecific hybridization of Miyama-uguisukagura and Haskap was performed to introduce novel characteristics in the resulting hybrids. The shape and color of the interspecific hybrid fruits differed from those of the parent fruits. A comparison of anthocyanin distribution among these three fruit types by imaging mass spectrometry (IMS) revealed the presence of five different anthocyanins. The average cyanidin 3,5-diglucoside and peonidin 3,5-diglucoside intensities in the interspecific hybrid fruit were higher than those of the parent fruits, whereas the average pelargonidin 3-glucoside, cyanidin 3-glucoside, and peonidin 3-glucoside intensities were the highest in Haskap. All anthocyanins were mainly accumulated in the inner and outer skins of Haskap and interspecific hybrid fruits, and in the skin of Miyama-uguisukagura fruits. The order of signal intensities of all anthocyanins among the three fruits was unchanged in different regions. Additionally, a comparison of IMS and LC/MS data from our previous study confirmed the possibility of comparing multiple fruits in the same plate by IMS. Thus, we elucidated anthocyanin distribution patterns of the interspecific hybrid and parent fruits by IMS.

Age-related changes in DNA methylation levels at CpG sites in bull spermatozoa and in vitro fertilization-derived blastocyst-stage embryos revealed by combined bisulfite restriction analysis.

Age-associated methylation changes in genomic DNA have been recently reported in spermatozoa, and these changes can contribute to decline in fertility. In a previous study, we analyzed the genome-wide DNA methylation profiles of bull spermatozoa using a human DNA methylation microarray and identified one CpG site (CpG-1) that potentially reflects age-related methylation changes. In the present study, cryopreserved semen samples from a Japanese Black bull were collected at five different ages, which were referred to as JD1-5: 14, 19, 28, 54, and 162 months, respectively, and were used for genome-wide DNA methylation analysis and in vitro fertilization (IVF). Distinct age-related changes in methylation profiles were observed, and 77 CpG sites were found to be differently methylated between young and adult samples (JD1-2 vs. JD4-5). Using combined bisulfite restriction analysis (COBRA), nine CpG sites (including CpG-1) were confirmed to exhibit significant differences in their age-dependent methylation levels. Eight CpG sites showed an age-dependent increase in their methylation levels, whereas only one site showed age-dependent hypomethylation; in particular, these changes in methylation levels occurred rapidly at a young age. COBRA revealed low methylation levels in some CpG regions in the majority of the IVF blastocyst-stage embryos derived from spermatozoa at JD2-5. Interestingly, bulls with different ages did not show differences in their methylation levels. In conclusion, our findings indicated that methylation levels at nine CpG sites in spermatozoa changed with increasing age and that some CpG regions were demethylated after fertilization. Further studies are required to determine whether age-dependent different methylation levels in bull spermatozoa can affect fertility.

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa.

This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1-3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0-2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.

Resistance of Sclerotinia homoeocarpa Field Isolates to Succinate Dehydrogenase Inhibitor Fungicides.

Sclerotinia homoeocarpa isolates were collected from golf courses in Japan and the United States (2016-2017). Japan isolates were collected during a monitoring study and the U.S. isolates were collected due to field failure. Five succinate dehydrogenase inhibitor (SDHI) active ingredients (boscalid, fluopyram, fluxapyroxad, isofetamid, and penthiopyrad) were examined using in vitro sensitivity assays to determine cross-resistance. Sequence analysis revealed a point mutation leading to an amino acid substitution (H267Y) and a silent mutation (CTT to CTC) at codon 181 in the SdhB subunit gene. Isolates with the B-H267Y (n = 10) mutation were resistant to boscalid and penthiopyrad and had increased sensitivity to fluopyram. SdhB silent mutation 181C>T isolates (n = 2) were resistant to boscalid, isofetamid, and penthiopyrad. Sequence analysis revealed 3 mutations leading to an amino acid substitution (G91R, n = 5; G150R, n = 1; G159W, n = 1) in the SdhC subunit gene. Isolates harboring the SdhC (G91R or G150R) mutations were resistant to boscalid, fluxapyroxad, isofetamid, and penthiopyrad. A genetic transformation system was used to generate mutants from a SDHI sensitive isolate to confirm the B-H267Y and C-G91R mutations were direct determinants of SDHI resistance and associated with in vitro sensitivity assay results.

Evaluation of female gamete fertility through histological observation by the clearing procedure in Lilium cultivars.

Lilies (Lilium spp.) are one of the most important floricultural crops. As most lily cultivars have originated from interspecific hybridization, they usually have complex genome composition and occasionally fail to develop normal gametes. Further improvement of lily cultivars by sexual crossing requires evaluation of gamete development and subsequent male and female fertility. Although male fertility is easily evaluated through microscopic observation after staining or by pollen culture for germination, evaluation of female fertility is difficult, because gametes develop inside an ovule within an ovary. Lilium species have the Fritillaria type of embryo sac, which, at maturity, consists of a haploid egg apparatus, including one haploid egg cell and two haploid synergids, two polar nuclei (one haploid nucleus and one triploid nucleus) and three triploid antipodal cells. Compared to the Polygonum type of embryo sac, composition of the embryo in the Fritillaria type of embryo sac is complex. We developed an efficient microscopic observation technique for ovules using the clearing procedure, which allowed us to categorize abnormal patterns of female gametes and to elucidate the frequency of abnormal female gamete development. The relationship among normal embryo sac, pollen stainability and seed formation in lily cultivars is discussed.

Expression levels of brown/beige adipocyte-related genes in fat depots of vitamin A-restricted fattening cattle1.

Brown/beige adipocytes dissipate energy as heat. We previously showed that brown/beige adipocytes are present in white adipose tissue (WAT) of fattening cattle. The present study examined the effect of vitamin A restriction on mRNA expression of brown/beige adipocyte-related genes. In Japan, fattening cattle are conventionally fed a vitamin A-restricted diet to improve beef marbling. Twelve Japanese Black steers aged 10 mo were fed control feed (n = 6) or vitamin A-restricted feed (n = 6) for 20 mo. Subcutaneous WAT (scWAT) and mesenteric WAT (mesWAT) were collected, and mRNA expression levels of molecules related to the function of brown/beige adipocytes (Ucp1, Cidea, Dio2, Cox7a, and Cox8b) as well as transcriptional regulators related to brown/beige adipogenesis (Zfp516, Nfia, Prdm16, and Pgc-1α) were evaluated. The vitamin A restriction significantly increased or tended to increase expression levels of Cidea and Pgc-1α in scWAT, and Cidea, Dio2, and Nfia in mesWAT. Previous studies revealed that the bone morphogenetic protein (Bmp) pathway was responsible for commitment of mesenchymal stem cells to brown/beige adipocyte-lineage cells. The vitamin A restriction increased expression of Bmp7 and some Bmp receptors in WAT. The interrelationship between gene expression levels indicated that expression levels of Nfia, Prdm16, and Pgc-1α were closely related to those of genes related to the function of brown/beige adipocytes in scWAT. Also, expression levels of Nfia, Prdm16, and Pgc-1α were highly correlated with those of Alk3 in scWAT. In summary, the present results suggest that the vitamin A restriction increases the number or activity of brown/beige adipocytes through regulatory expression of transcriptional regulators to induce brown/beige adipogenesis, especially in scWAT of fattening cattle, which may be governed by the Bmp pathway.

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa.

Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the ß-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field.

Branch regeneration induced by sever damage in the brown alga Dictyota dichotoma (dictyotales, phaeophyceae).

Tissue wounds are mainly caused by herbivory, which is a serious threat for macro-algae, and brown algae are known to regenerate branches or buds in response to wounding. In the present paper, we describe a branch regeneration system, induced by sever damage, in the brown alga Dictyota dichotoma. Segmentations of juvenile thalli induced branch regenerations unless explants possessed apical cells. Apical excisions in distinct positions elucidated that disruption of an apical cell or disconnection of tissue with an apical cell triggered the branch regeneration. Furthermore, spatial positions of regenerated branches seemed to be regulated by the apical region, which was assumed to generate inhibitory effects for lateral branch regeneration. Mechanical incision, which disrupted tissue continuity with the apical region, induced branch regeneration preferentially below the incision. Although we were unable to identify the candidate inhibitory substance, our results suggested that the apical region may have an inhibitory effect on lateral branch regeneration. Additionally, observations of branch regeneration showed that all epidermal cells in D. dichotoma possess the ability to differentiate into apical cells, directly. This may be the first report of algal transdifferentiation during the wound-stress response.

Suppression of B function strongly supports the modified ABCE model in Tricyrtis sp. (Liliaceae).

B class MADS-box genes play important roles in petal and stamen development. Some monocotyledonous species, including liliaceous ones, produce flowers with petaloid tepals in whorls 1 and 2. A modified ABCE model has been proposed to explain the molecular mechanism of development of two-layered petaloid tepals. However, direct evidence for this modified ABCE model has not been reported to date. To clarify the molecular mechanism determining the organ identity of two-layered petaloid tepals, we used chimeric repressor gene-silencing technology (CRES-T) to examine the suppression of B function in the liliaceous ornamental Tricyrtis sp. Transgenic plants with suppressed B class genes produced sepaloid tepals in whorls 1 and 2 instead of the petaloid tepals as expected. In addition, the stamens of transgenic plants converted into pistil-like organs with ovule- and stigma-like structures. This report is the first to describe the successful suppression of B function in monocotyledonous species with two-layered petaloid tepals, and the results strongly support the modified ABCE model.