Effects of different ectomycorrhizal fungi on somatic embryogenesis of <Emphasis Type="Italic">Abies cephalonica</Emphasis> Loud

The effects of ectomycorrhizal (ECM) fungi, including Laccaria bicolor (Maire) Orton, Laccaria laccata (Scop., Fr.) Berk. and Br., along with two strains of Pisolithus tinctorius (Pers.) Coker and Couch, on the proliferation and subsequent maturation of two embryogenic cell lines of Abies cephalonica Loud., designated lines 6 and 8, were investigated. In the presence of these ECM fungi, the proliferation of both embryogenic cell lines was inhibited. L. bicolor and P. tinctorius strain 2 resulted in the highest inhibition rates. On the other hand, cultivation of embryogenic cultures along with ECM fungi, termed a dual culture, increased radial growth of both P. tinctorius strains; whereas, L. bicolor and L. laccata did not grow as well in the presence of embryogenic cell masses. The dual culture during the proliferation period of embryogenic cells, however, enhanced the subsequent embryo formation and maturation of A. cephalonica; i.e. the capability of embryogenic cell lines to form somatic embryos as well as increasing the mean number of somatic embryos per 1 g fresh weight of embryogenic cell mass. However, levels of responses were highly dependent on the interaction between the specific embryogenic cell line and fungal strain.     

Ectomycorrhizal community structure of different genotypes of Scots pine under forest nursery conditions

In this paper, we report the effect of Scots pine genotypes on ectomycorrhizal (ECM) community and growth, survival, and foliar nutrient composition of 2-year-old seedlings grown in forest bare-root nursery conditions in Lithuania. The Scots pine seeds originated from five stands from Latvia (P1), Lithuania (P2 and P3), Belarus (P4), and Poland (P5). Based on molecular identification, seven ECM fungal taxa were identified: Suillus luteus and Suillus variegatus (within the Suilloid type), Wilcoxina mikolae, Tuber sp., Thelephora terrestris, Cenococcum geophilum, and Russuloid type. The fungal species richness varied between five and seven morphotypes, depending on seed origin. The average species richness and relative abundance of most ECM morphotypes differed significantly depending on pine origin. The most essential finding of our study is the shift in dominance from an ascomycetous fungus like W. mikolae in P2 and P4 seedlings to basidiomycetous Suilloid species like S. luteus and S. variegatus in P1 and P5 seedlings. Significant differences between Scots pine origin were also found in seedling height, root dry weight, survival, and concentration of C, K, Ca, and Mg in the needles. The Spearman rank correlation coefficient revealed that survival and nutritional status of pine seedlings were positively correlated with abundance of Suilloid mycorrhizas and negatively linked with W. mikolae abundance. However, stepwise multiple regression analysis showed that only survival and magnesium content in pine needles were significantly correlated with abundance of ECM fungi, and Suilloid mycorrhizas were a main significant predictor. Our results may have implications for understanding the physiological and genetic relationship between the host tree and fungi and should be considered in management decisions in forestry and ECM fungus inoculation programs.     

Mycorrhiza formation is not needed for early growth induction and growth-related changes in polyamines in Scots pine seedlings in vitro

Ectomycorrhizal (ECM) fungi have been shown to improve growth of the host plant before the formation of physical ECM structures, i.e. during the so-called pre-mycorrhizal phase. In the present study, changes in growth and the concentrations of individual polyamines (PAs) were followed during the mycorrhiza formation in Scots pine (Pinus sylvestris) seedlings in the presence of two ECM fungi, Pisolithus tinctorius and Paxillus involutus. The two fungus stains were chosen because they differed in infection characteristics as well as in PA and auxin production. The results were compared to our earlier study with two Suillus variegatus strains forming ECMs with Scots pine seedlings in vitro. Paxillus was not able to form ECMs whereas Pisolithus formed ECM association with Scots pine seedlings within two weeks. However, Paxillus enhanced the growth of the seedlings more than Pisolithus. Paxillus also increased putrescine (Put) concentrations of the seedlings in the pre-mycorrhizal phase much more than Pisolithus. A similar trend was observed in the free spermidine (Spd) in stems, whereas in the needles Paxillus decreased free Spd concentration. Pisolithus caused a threefold greater increase in root free Spd than Paxillus. Effects of Paxillus on the growth and PA fluctuation, excluding root free Spd, of the host plant resembled that observed in our previous in vitro study on S. variegatus–Scots pine interaction. Therefore, changes in specific PA concentrations in the pre-mycorrhizal phase seem to be related to growth induction by the ECM fungus rather than to mycorrhiza formation. Moreover, we suggest that growth induction in host plants is not necessarily followed by ECM formation.     

Effects of Pisolithus tinctorius and Cenococcum geophilum inoculation on pine in copper-contaminated soil to enhance phytoremediation

We used Pisolithus tinctorius and Cenococcum geophilum to determine the copper (Cu) resistance of ectomycorrhizal (ECM) fungi and their potential for improving phytoremediation of Cu-contaminated soil by Chinese red pine (Pinus tabulaeformis). The results showed that nutrient accumulation in C. geophilum mycelium was significantly lower under higher Cu concentrations in the soil, which was not observed in P. tinctorius. Meanwhile, P. tinctorius exhibited greater Cu tolerance than C. geophilum. Inoculation with ECM fungi significantly improved the growth of pine shoots planted in polluted soil in pot experiments (p < 0.01). The total accumulated Cu in pine seedlings planted in Cu-contaminated soil increased by 72.8% and 113.3% when inoculated with P. tinctorius and C. geophilum, respectively, indicating that ECM fungi may help their host to phytoextract heavy metals. Furthermore, the majority of the total absorbed metals remained in the roots, confirming the ability of ECM fungi to promote heavy metal phytostabilization. There were no differences between the effects of the two fungi in helping the host stabilize and absorb Cu, even though they have different Cu tolerances. Inoculation with ECM fungi can benefit plant establishment in polluted environments and assist plants with phytoremediating heavy-metal-contaminated soils.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

Changes in the activity of enzymes involved with primary nitrogen metabolism due to ectomycorrhizal symbiosis on jack pine seedlings

Jack pine (Pinus banksiana Lamb.) seedlings were inoculated with either one of the ectomycorrhizal (ECM) fungi, Laccaria bicolor (Maire) Orton or Pisolithus tinctorius (Pers.) Coker and Couch, and grown for 16 weeks in a growth chamber along with non-ECM controls. Five enzymes involved with the assimilation of nitrogen or the synthesis of amino acids were measured in the 3 jack pine root systems as well as in the pure fungal cultures. Pisolithus tinctorius in pure culture had no detectable activity of nitrate reductase (NR. EC 1.6.6.1), glutamate dehydrogenase (GDH. EC 1.4.1.2), glutamate decarboxylase (GDCO. EC 4.1.1.15) or glutamate oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13) but did have some glutamine synthetase (GS, EC 6.3.1.2) activity. Laccaria bicolor in pure culture had no NR activity, small levels of GDCO activity, and high GS, GDH and GOGAT activity. The high levels of enzymatic activity present in L. bicolor indicate that it may play a greater role in the nitrogen metabolism of its host plant than P. tinctorius. ECM infection clearly altered the enzymatic activity in jack pine roots but the nature of these changes depended on the fungal associate. Non-ECM root systems had higher specific activities than ECM root systems for NR, GS, GDH and GDCO but GOGAT activites were the same for both the ECM and non-ECM roots. Root systems infected with L. bicolor had significantly greater NR and GDCO activity than those infected with P. tinctorius. Differences in the GS activity of the two fungi in pure culture corresponded to the GS activity of jack pine roots in symbiotic association with these fungi. While the free amino acid profiles in roots were significantly affected by ECM infection, the profile of free amino acids exported to the stem was the same for all treatments. High asparagine and low glutamine in roots infected with P. tinctorius indicates that asparagine synthetase (EC x.x.x.x) activity should be higher within this symbiotic association than in the L. bicolor association or in the non-mycorrhizal roots.     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.     

Studies on Ectomycorrhizae of Pine II. Growth Inhibition of Mycorrhizal Fungi by Volatile Organic Constituents of Pinus silvestris (Scots Pine) Roots

The effects of several monoterpenes and the sesquiterpene longifolene on the vegetative growth of two mycorrhizal fungi, Boletus variegatus and Rhizopogon roseolus, were studied in petri plate experiments on an agar medium. The test substances are natural constituents of Pinus silvestris (Scots pine) roots. Vapors from 20 μl of all the test substances, upon a 5 day exposure inhibited the growth of both the mycorrhizal fungi 55% to 86%. The two fungi, however, differed in their tolerance to individual compounds. It is suggested that the terpenes and the sesquiterpenes play an important role in the development of the symbiotic condition in ectomycorrhizae by restricting the growth of the mycorrhizal fungi within the host root.     

Transgene expression in regenerating cotyledons and embryogenic cultures of Scots pine

Regenerating cotyledons and embryogenic cultures of Scots pine (Pinus sylvestris L.) were used as targets for gene delivery via particle bombardment. Both target tissues differed in their response to selective agents, regulative sequences of transferred genes, and conditions optimal for particle bombardment. Of the gene constructs tested, the pB1410 including CaMV 35S promoter, AMV-translation enhancer and gusA:npt reporter and selectable marker gene fusion was the most suitable for cotyledon transformation, resulting on average in 3.7 ( 0.1 SE) -glucuronidase (GUS) expression units per cotyledon. In embryogenic cell masses the pB1221.1 gene construct including the gusA reporter gene driven by the 35S promoter gave the highest transient expression, 63 (plusmn; 15 SE) GUS expression units of g^-1 fresh weight of embryogenic cell mass. Kanamycin and geneticin concentrations suitable for selection of cotyledons were 10-15 mg dm^-3 and 0.5-1.0 mg dm^-3, respectively. Kanamycin (10 mg dm^-3) and phosphinotricin (1 mg dm^-3) as selectors caused a significant decline in growth, but geneticin did not significantly affect the growth of the embryogenic cultures during the 8 week cultivation period. The production of transgenic plantlets seems to be more dependent on the regeneration and multiplication efficiency of organogenesis and somatic embryogenesis of Scots pine than on gene delivery into regenerating tissues.     

The threatened plant intermediate wintergreen (<Emphasis Type="Italic">Pyrola media</Emphasis>) associates with a wide range of biotrophic fungi in native Scottish pine woods

The plant intermediate wintergreen (Pyrola media, Ericaceae) is in need of conservation action in Scotland. Although widespread, it is locally distributed in dwarf shrub heath and more commonly in Scots pine (Pinus sylvestris) woodlands. A recent study on the mycorrhizal status of Pyrola suggested that they associate with a restricted range of ectomycorrhizal (ECM) fungi. Here, we examined the hypothesis that specialisation by P. media for fungi usually associated with Scots pine is a factor in promoting its occurrence in this habitat. The fungal community associated with the roots of P. media growing in a Scots pine forest was determined by morphotyping, polymerase chain reaction, cloning and sequencing. Molecular identification found 49 taxa representing ecto- and ericoid mycorrhizal fungi, dark septate endophytes, saprotrophs, and fungi of unknown trophic status. The majority of the taxa (67.4%) were Basidiomycota, with 24.4% known to be ECM fungi specific to Pinus sp. or conifers. However, a wide range of other mycorrhizal fungi with varying degrees of host specificity were also found, including taxa usually associated with deciduous hosts. In conclusion, the broad range of mycorrhizal fungi recovered from the roots of P. media suggests that specialization is not a major factor in determining its distribution.     

Effect of ectomycorrhizal fungi on nitrogen mineralisation and the growth of Scots pine seedlings in natural peat

The aim of this study was to investigate the potential of different isolates of ectomycorrhizal (EM) fungi to enhance the growth of Pinus sylvestris seedlings in five natural peat substrates with different nitrogen concentrations, and the effect of the Scots pine seedlings and fungal inoculum on the formation of dissolved inorganic and organic nitrogen in peat. Utilization of different organic nitrogen compounds by microbial community in the peat was also investigated using Biolog MT MicroPlates. Inoculation of the seedlings with EM fungi enhanced seedling growth. Piloderma croceum increased root growth especially, whereas Lactarius rufus increased needle growth and Suillus variegatus I, II and III improved both root and needle growth. All the EM fungi also significantly affected stem growth. Nitrogen concentration of the peat did not affect seedling growth as much as the EM fungi. At the lowest peat N concentration (1.17%) NH ₄ ⁺ mineralisation was lower and DON (dissolved organic nitrogen) accumulation higher than at higher peat N concentrations. The EM fungal isolates had different effects on NH ₄ ⁺ and DON accumulation/degradation in peat. The EM fungal isolates significantly increased NH ₄ ⁺ formation in the peat, whereas L. rufus and P. croceum had an opposite effect on DON accumulation. S. variegatus I significantly decreased the DON concentrations during peat incubation. The N concentration of the peat slightly affected the utilization of amino acids and polyamines by the microbial community, whereas inoculation with S. variegatus I, II or III had no effect.     

Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death

Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.     

Embryogenic cell lines of <Emphasis Type="Italic">Juniperus communis</Emphasis>; easy establishment and embryo maturation,limited germination

Several coniferous species belonging to the Pinaceae family can be propagated via somatic embryogenesis, while species belonging to the Cupressaceae family cannot. The aim of this study was to identify possibilities and limitations with somatic embryogenesis in Cupressaceae. Juniperus communis was chosen as model species. We show that a high initiation frequency of embryogenic cell lines can be established from intact megagametophytes at the time when intensive cleavage polyembryogeny takes place. The embryogenic cell lines proliferate fast on medium lacking plant growth regulators. Early somatic embryos develop after transfer to medium with decreased content of nitrogen and calcium. The early embryos mature after exposure to abscisic acid. Mature cotyledonary embryos germinate after partial desiccation. A high proportion, over 40%, of the germinating embryos retain the embryogenic potential in the basal part, resulting in development of new embryogenic tissue.     

Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Clonal plant production from self- and cross-pollinated seed families of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.     

Mobilisation of potassium and phosphorus from iron ore by ectomycorrhizal fungi

Mutualistic roles of ectomycorrhizal (ECM) fungi have been linked to their ability to produce organic acids that aid in the dissolution of insoluble minerals in the rhizosphere. This ability of ECM fungi was utilised to investigate their potential participation in the mobilisation of nutrients such as phosphorus (P) and potassium (K) from a typical insoluble ore—iron ore. In vitro pure cultures of four different ECM fungi; Pisolithus tinctorius, Paxillus involutus, Phialocephala fortini, and Suillus tomentosus were screened for their ability to mobilise P and K from two types of non-exportable Sishen iron ore. When present in iron ore, these elements are deleterious and reduce the commercial values of the ore. Experiment was set up with different treatments that included two ore types (KGT and SK) and five particle sizes of each ore type. Results indicated the potential of the four fungi to mobilise P and K from the two iron ore types though at different levels. Ore type, particle size, organic acid production and attachment of the fungi to the iron ore were all found to play important roles in the mobilisation of nutrients from these ores.     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest

Pinus halepensis Mill. is a common forest species in the Mediterranean area and it is important for environmental conservation. This study established a method of regenerating Pinus halepensis Mill. through somatic embryogenesis. The effect of culture medium (mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was analysed during the first steps of embryogenesis process. This study showed a marked effect of the culture medium tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture medium and the proliferation stage was significantly affected by the combination of plant growth regulators. At the end of the maturation phase the presence of activated charcoal was also evaluated. Finally, maturation of embryogenic tissue was affected by the nitrogen source in the culture medium and these results were different for high and low mature embryo producing cell lines. To the best of our knowledge, this is the first report on Aleppo pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.