Impaginato 3 Adv. Hort. Sci., 2020 34(1): 3­10 DOI: 10.13128/ahsc­8251 Development of interspecific hybrids between Habenaria radiata and Habenaria rhodocheila complex P. Sinumporn (*), T. Narumi­Kawasaki, S. Fukai Faculty of Agriculture, Kagawa University, Miki‐cho, Kagawa 761‐0795, Japan. Key words: apomixis, cross combination, PCR­RFLP, reciprocal crossing, seed germination. Abstract: Reciprocal crosses between Habenaria radiata and H. rhodocheila complex were investigated to develop new hybrids. The fruit­setting frequency and seed germination in the cross combination of H. radiata × H. rhodocheila complex were higher than those of H. rhodocheila complex × H. radiata. The hybridity of the obtained progenies was confirmed through PCR­RFLP analysis of the rRNA gene. Cross combinations producing true hybrids, apomicts, or both were observed, indicating that both H. radiata and H. rhodocheila com­ plex were facultative apomixis. The obtained hybrids, H. radiata × H. rhodocheila (orange flower), showed the intermediate plant form and flower shape of the parents, and both petals and lip were pale yellow. 1. Introduction Habenaria is a large genus in the family Orchidaceae, consisting of more than 800 species distributed in tropical and subtropical areas such as Southern America, Southern and Central Africa, and East Asia (Pridgeon, 1992; Kurzweil, 2009; Pedron et al., 2012; Batista et al., 2013; Jin et al., 2014). Habenaria species show diverse plant forms, flower shapes, and petal colors. There are many Habenaria species having high ornamental value, but only a few species are commercialized. In this study, we focused on two Habenaria species: H. radiata and H. rhodocheila. Habenaria radiata is a species native to Japan in the wet­ lands of Honshu, Shikoku, and Kyushu Islands. This species is low­temper­ ature tolerant. The form of the flowers is unique and beautiful, and the white petals look like a white egret bird. This species has been used as ornamental pot plants (Kim et al., 2007, 2010; Mitoma and Kanno, 2018), but it can be used as cut flowers (Sinumporn et al., 2015). Habenaria rhodocheila is found in Southeast Asia, Laos, Myanmar, southeast China, Thailand, Malaysia, and the Philippines. The flowers of H. rhodocheila have a large lip and four lobes, with side lobes and oblique mid lobes. The lips show a wide range of color such as orange, pink, red, and yellow. Formerly, the pink­flowered genotype was accepted under the name H. (*) Corresponding author: punpaka.sinumporn@gmail.com Citation: SINUMPORN P., NARUMI­KAWASAKI T., FUKAI S., 2020 ­ Development of interspecific hybrids between Habenaria radiata and Habenaria rho‐ docheila complex ­ Adv. Hort. Sci., 34(1): 3­10 Copyright: © 2020 Sinumporn P., Narumi­Kawasaki T., Fukai S. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 20 October 2018 Accepted for publication 6 March 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(1): 3­10 4 erichmichelii, and the yellow­flowered genotype was H. xanthocheila. The morphological characteristics are also different in each genotype, beside petal color (Kurzweil, 2009; Batista et al., 2013). H. xan‐ thocheila is distinguished from H. rhodocheila in its tuber shape, i.e. H. xanthocheila has a crown­shaped tuber but H. rhodocheila has a round tuber (Cullen et al., 2011). Because these genotypes are very closely related, they are integrated into one species, called H. rhodocheila complex. In this report, we adopted the name H. rhodocheila complex and distinguished the genotypes only by the color of the petals. Producing hybrids between the two completely dif­ ferent Habenaria species, H. radiata and H. rhodocheila complex, could result in new hybrids having vigor, low­temperature tolerance, and beauti­ ful flower shape with colorful petals. Recently, many orchid species including Habenaria are at risk of extinction. The numbers of both H. radiata and H. rhodocheila complex are decreasing in their natural habitats, which are being destroyed through urbanization, agricultural use, ecological mismanagement of habitat, changes in cli­ mate conditions, and overcollection by people (Stewart and Kane, 2006; Mitsukuri et al., 2009; Tanaka et al., 2015). Supply of new interspecific hybrids with increased ornamental value is expected to reduce the illegal collection of the species in their habitats. There is little research on intraspecific cross breeding using these two Habenaria species. Only a successful of intraspecific cross between wild­type and petaloid­sepal genotypes in H. radiata was done (Kim et al., 2010; Mitoma et al., 2019). In this study, we carried out reciprocal crossing between H. radiata and H. rhodocheila complex, and evaluated the obtained progenies. This report is the first on suc­ cessful interspecific crossing of H. radiata and H. rhodocheila complex. 2. Materials and Methods Plant materials Tubers of H. radiata ‘Aoba’ (HRA) (Fig. 1A) were planted in April, every year, in 12 cm plastic pots (5 tubers per pot) with sphagnum moss and tubers of H. rhodocheila complex (orange, pink, and yellow petal genotypes, RCO, RCP, and RCY, respectively) (Fig. 1B, 1C, 1D) were planted in 12 cm plastic pots filed with a medium consisting of Growing Mix (Metro Mix 350; Sun Gro Horticulture, MA USA): Kanuma (volcanic porous soil): Vermiculite, 1:2:1. Then, H. radiata were placed in a greenhouse in natural temperature with solar radiation. H. rhodocheila complex were placed in a growth chamber controlled at a constant tem­ perature of 20°C with solar radiation. Interspecific cross and in vitro germination A preliminary crossing experiment, HRA × RCO, was carried out in 2015. HRA and two H. rhodocheila complexes, RCO and RCY, were then cross­pollinated reciprocally in 2017. Five plants of each genotype were used in those cross combinations. Twenty plants of HRA and RCP were also cross­pollinated rec­ iprocally in 2017. A total of six cross combinations were made (Table 1). The pollinia of mother plants were removed in advance to prevent self­pollination and the aimed pollinia of other plants were placed on the stigma. Reciprocal crosses were also conducted. Hand­pollinated flowers were labelled individually, and the capsules were harvested before dehiscence about 2 months after pollination. The capsules were surface sterilized with 70% (v/v) ethanol for 30 s and 0.1% (v/v) sodium hypochlorite solution for 15 min Fig. 1 ­ Plant morphology of Habenaria species used in this study. (A) H. radiata ‘Aoba’, (B) H. rhodocheila complex (orange), (C) H. rhodocheila complex (pink), and (D) H. rhodocheila complex (yellow). Sinumporn et al. ‐ Development of interspecific hybrids of Habenaria spp. 5 and then rinsed three times with sterilized water. Seeds were removed from the capsules and mixed in a petri dish, and then a batch of seeds picked up with tweezers was placed on a seed germination medium in 5 cm petri dishes. The medium was MM (Malmgren, 1996) supplemented with 20 g/l sucrose and 0.7% agar, and adjusted to pH 5.75 prior to auto­ claving at 0.103 MPa pressure and 121°C for 20 min. The cultures were kept at 20°C under dark condi­ tions. Extract numbers of seeds placed on the medi­ um was unknown, the germination was evaluated in four stages: well (+++), fair (++), poor (+) and no­ger­ mination (­). Six months after sowing, protocorms were transplanted to 6 × 6 cm plastic culture vessels with MM medium. The protocorm cultures were kept under light inflorescence lamps (FL40S. BRN; Toshiba Lighting & Technology Co. Ltd.) for 16 h with a light intensity of 31.5 µmol m­2 s­1at 24°C. The protocorms were subcultured every month. After 12 months, the developed plantlets were acclimatized and planted in 12 cm plastic pots using the same growing medium as for the mother plants. The obtained progenies were grown in a growth chamber controlled at 20°C. PCR‐RFLP The total DNA of both parents and progenies was extracted from 0.1 g of leaf tissue according to a modified ABBAS DNA extraction method (Abbas et al., 2013). PCR was performed in a 50 µl reaction mix­ ture containing 70 ng of total DNA, 0.2mM each of rRNA gene specific primers (5ʹ­ACA CAC CGC CCG TCGCTC CTA­3ʹ and 5ʹ­ACT CGA TGG TTC ACG GGA TTC TG­3ʹ), 2.5 mM dNTPs, 20mM of 10× PCR Ex Taq buffer, and 5 U/µl of Ex Taq polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan) according to Haruki et al. (1997). PCR was conducted under the following ther­ mocycling conditions: 1 cycle of 96°C, 10 s; 25 cycles of 96°C, 10 s, 55°C, 30 s, 72°C, 60 s; and 1 cycle of 72°C, 10 min. The amplified products of both the parents and the progenies were digested with selected restriction endonucleases (Alu Ι, Hha Ι, Rsa Ι, and Sty Ι; Nippon Gene Co. Ltd., Toyama, Japan) at 37°C for 1 h. The digested products were separated by electrophoresis in 1.8% agarose gels (Invitrogen, Carlsbad, California, USA) containing 0.1 µl/ml ethidium bromide solution and photographed. 3. Results Pod set and seed germination in the reciprocal cross‐ ings Six reciprocal cross combinations were made, and the pod set frequencies varied depending on both cross combinations and the ovule parents. The HRA × RCO cross combination resulted in 8 pod sets from 13 flowers (61.5%), and the opposite cross of RCO × HRA resulted in 2 pod sets from 2 flowers (100%). HRA × RCP had 16 pod sets from 28 flowers (57.1%), and the opposite cross RCP × HRA had 12 from 41 flowers (29.3%). HRA × RCY had only 1 pod set from 11 flow­ ers (9.1%), and the opposite cross RCY × HRA had 1 from 3 flowers (33.3%) (Table 1). All pods were harvested before dehiscence, and the seeds were cultured on MM medium without plant growth regulators. The seeds from the cross combination of HRA × RCO germinated well, but the seeds from RCO × HRA did not germinate. The seeds of HRA × RCP also had rather high germination, but the seeds from RCP × HRA did not germinate. Both cross combinations of HRA × RCY and RCY × HRA had poor seed germination (Table 1). The sown seeds from HRA × RCO (Fig. 2A) swelled within 50 days of culture (Fig. 2B), developed proto­ corms around 90 days of culture (Fig. 2B), and the protocorms produced rhizoids (Fig. 2C). After being Table 1 ­ Reciprocal crossing between Habenaria radiata and Habenaria rhodocheila complex HRA = H. radiate; RCO = H. rhodocheila (orange petal); RCP = H. rhodocheila (pink petal); RCY = H. rhodocheila (yellow petal). *++++ = Germination well, ++ = Germination fair, + = Germination poor, ­ = No germination. Cross combination Number of flowers pollinated Number of pod sets (%) Seed germination Number of plantlets tested PCR­RFLP Number of true hybrids (%) HRA × RCO 13 8 (61.5) ++++ 36 36 (100) RCO × HRA 2 2 (100) ­ ­ ­ HRA × RCP 28 16 (57.1) +++ 21 0 (0) RCP × HRA 41 12 (29.3) ­ ­ ­ HRA × RCY 11 1 (9.1) + 18 1 (5.5) RCY × HRA 3 1 (33.3) + 8 0 (0) Adv. Hort. Sci., 2020 34(1): 3­10 6 subjected to light, the protocorms turned green (Fig. 2D), produced the first leaf within 120 days of culture (Fig. 2E), and then developed plantlets around 150 days of culture (Fig. 2F). ed both apomixis and true hybrids (Fig. 3C). The prog­ enies of the reciprocal cross RCY × HRA (YXW) showed the same band pattern as the female parent (RCY), suggesting that they were apomicts. Fig. 2 ­ Seed development of Habenaria radiate × Habenaria rhodocheila on MM medium. (A) Sown seeds (day 0), (B) Protocorm deve­ lopment (50 days), (C) Enlarged embryo rupture testa and developed protocorm (3 months), (D) Green protocorm with protome­ ristem (3 months), (E) Emergence of first leaf (4 month), (F) Plantlets (5 months). Confirmation of hybridity through PCR‐RFLP analysis To confirm the hybridity of the obtained progenies, PCR­RFLP analysis targeting the ribosomal RNA gene was used according to Haruki et al. (1997). The early growth stage of two progenies of HRA × RCO, named WXO1 and WXO2, were used. Expected single PCR product was amplified in all the tested plants. Three restriction enzymes (Alu ӏ, Hha ӏ and Rsa ӏ) that showed polymorphism in the digested PCR products between both parents were applied. The band pattern of both WXO1 and WXO2 was intermediate between both parents (HRA and RCO), indicating that both WXO1 and WXO2 were true hybrids. Thirty­six HRA × RCO progenies in the in vitro stage were chosen ran­ domly and their hybridity was tested in the same manner. The results showed that all the tested proge­ nies were true hybrids (Table1) (Fig. 3A). In contrast, an early growth­stage progeny of HRA × RCP, named WXP1, showed a band pattern the same as the female parent (HRA) when the PCR products were digested with Alu Ι, Hha Ι, Rsa Ι, and Sty Ι, suggesting that the plants were apomicts. Twenty­one HRA × RCP proge­ nies in the in vitro stage were tested in the same man­ ner. The results showed all the tested progenies were apomicts (Fig.3B). One rapidly grown apomict was designed WXY1 and the one that was judged to be a true hybrid was designed WXY2. Moreover, the results of HRA × RCY (WXY) showed that the progenies includ­ Fig. 3 ­ PCR­RFLP profile of parents and progenies. A: HRA × RCO. 1. HRA, 2. WXO1, 3.WXO2, 4. RCO. B: HRA × RCP. 1. HRA, 2. WXP, 3. RCP. C: HRA × RCY. 1. HRA, 2. WXY1, 3.WXY2, 4.YXW, 5. RCY. Morphological characteristics of the obtained progenies The obtained progenies WXO1 and WXO2 (con­ firmed as hybrids through PCR­RFLP analysis), WXY1 (assumed to be an apomict), and the parent RCO were grown in a growth chamber controlled at 20°C. HRA grown in a greenhouse without heating was used for morphological comparison. Sinumporn et al. ‐ Development of interspecific hybrids of Habenaria spp. 7 6A). RCO had a flower consisting of two grey­brown orange petals and an orange lip and three sepals. The dorsal sepal was egg­shaped and the slanted lateral sepals sometimes rolled­in. The lip had three main lobes, with the two side lobes elliptical and the mid­ dle lip had two ovate­oblong lobes (Fig. 6B). The petals of both WXO1 and WXO2 were pale yellowish, which was an intermediate characteristic of the par­ ents. The sepals of WXO1 and WXO2 were green, with the dorsal sepal being egg­shaped, the same as the female parent (HRA), and the lateral sepals were slanted and rolled­in, the same as the male parent (RCO). In addition, two petals were attached, forming an egg­shaped hood with the dorsal sepal. The lip had three main lobes, with the two lateral lobes being slightly fringed and the center lobe had two slightly ovate­oblong lobes, the same as the male parent (RCO) (Fig. 6D, 6E).The apomixtic progeny WXY1 produced flowers resembling H. radiata (Fig. 6F). One progeny derived from the cross combination of HRA × RCY (WXY2), confirmed as a true hybrid through PCR­RFLP analysis, showed an intermediate plant morphology between the parents (data not shown) but had not flowered because only one year had passed after acclimatization. Plants in the genus Habenaria produce a storage organ having species­specific morphology. HRA pro­ duced stolons during the growing season, and new tubers formed at the top of the stolon. The HRA tuber was oval with a smooth surface (Fig. 7A). RCO produced a long oval tuber at the bottom end of the stem. The RCO tuber was bigger than that of HRA. The tuber had a rough surface and was densely cov­ ered with hair (Fig. 7C). Both WXO1 and WXO2 had Both WXO1 and WXO2 grew vigorously, and the first flowering was observed in WXO1 one year after transfer to ex vitro and in WXO2 two years after transfer to ex vitro. Both WXO1 and WXO2 showed an intermediate plant form (Fig. 4A, 4B, respectively) and leaf morphology of theirs parents. HRA had nar­ row light green leaves (Fig. 5A). RCO had wide lance­ olate leaves with an undulate leaf margin, and the leaves were green or greyish green, sometimes with red­brown spots (Fig. 5D). WXO1 had lanceolate light green leaves, and WXO2 had lanceolate light green leaves with an undulate leaf margin (Fig. 5B, 5C, respectively). The inflorescence morphology of WXO1 and WXO2 was intermediate between the par­ ents. WXO1 had an inflorescence with two flowers, and WXO2 had an inflorescence with eight flowers. Habenaria radiata produced two to four flowers (average 2.8 flowers) per inflorescence. RCO had eight to ten flowers (average 9.4 flowers) per inflo­ rescence (Fig. 1B). The inflorescence morphology of WXY1 was similar to HRA, although it could not be accurately determined due to the poor growth of WXY1. HRA had a flower consisting of two pure white petals and a lip with three green ovate sepals. The lip had three main lobes; the two lateral lobes were highly fringed and the center lobe was simple (Fig. Fig. 6 ­ Floral morphology of the parents and hybrids. (A) HRA, (B) RCO, (C) RCY, (D) WXO1, (E) WXO2, (F) WXY1. Fig. 5 ­ Leaves of the parents and hybrids. (A) HRA, (B) WXO1, (C) WXO2, (D) RCO. Fig. 4 ­ Plant morphology of hybrids. (A) WXO1, (B) WXO2, (C) WXY1. 8 Adv. Hort. Sci., 2020 34(1): 3­10 tubers with intermediate morphological characteris­ tics of the parents (Fig. 7B) (Table 2). 4. Discussion and Conclusions Interspecific hybridization is a powerful breeding method that can produce new traits in ornamental plants including orchids. This study aimed to produce new Habenaria hybrids by using two different eco­ type species (H. radiata and H. rhodocheila complex). The two species chosen in this study are different in not only plant morphology but also flowering physiol­ ogy. Because the flowering time of H. radiata is dur­ ing June to August under natural conditions, H. rhodocheila complexes were grown in a growth chamber controlled at 20°C to match the flowering time. The fruit settings in H. radiata × H. rhodocheila complex were higher than in the opposite crosses. The germination of the obtained seeds was also dif­ ferent depending on the cross combination (Table 1). The seeds obtained from H. radiata × H. rhodocheila complex showed higher seed germination than those in the opposite crosses. Unilateral cross incompatibil­ ity is often observed in interspecific crossings includ­ ing orchids (Johansen, 1990; Borba et al., 1999). The hybridity of the obtained progenies was investigated by using RFLP analysis. The results showed that all the tested progenies of HRA × RCO were true hybrids, whereas all the tested progenies of HRA × RCP and RCY × HRA were apomicts. Both true hybrids and apomicts were found in the progenies of HRA × RCY. The results of RFLP analysis were consistent with the observed plant morphological characteris­ tics of the obtained progenies. The production of apomixis, including oblique and facultative, is known in some genera including Habenaria in the family Orchidaceae (Batygina et al., 2003). Zhang and Gao (2018) reported obligate apomixis in H. malintana. The present study showed that both true hybrids and apomicts appeared in H. radiata and H. rhodocheila. Successful crossing of different flower types of H. radiata has been reported (Kim et al. , 2010). Adthalungrong et al. (2015) reported successful reci­ procal crossing between RCP and RCY. These findings indicate that both H. radiata and H. rhodocheila do not have obligate apomixis. We consider that the fac­ ultative apomixis observed in this study is induced by interspecific crossing between distantly related species. In many plants, pollination is necessary for induction of apomixis (den Nijs and van Dijk, 1993). When HRA was used as a female parent, the frequen­ cy of apomict production varied depending on the pollen parents. The results suggest that the growth of the pollen tube in the ovary differed depending on the H. rhodocheila complex genotype. In HRA × RCY, only one pod was harvested, and both an apomict and true hybrid were obtained from the pod. This finding suggests that different embryogenesis, sexual and asexual, occurred at the same time. Further detailed morphological and genetic investigation of embryo development in interspecific hybridization is required to explain these phenomena. If the occur­ rence of apomixis is unpredictable in a practical breeding program, efficient selection of true hybrids is essential. The present study selected true hybrids at the early developmental stage of progenies through PCR­RFLP analysis. Selecting plants in the in vitro stage will be useful in a practical breeding pro­ gram. Fig. 7 ­ Tubers of the parents and hybrids. (A) HRA, (B) WXO2, (C) RC. Name Plant form (cm) Leaves (cm) Number of leaves Flower (cm) Stigma (cm) Number of flowers Inflorescences (cm) Spur Tuber (cm) Storage organ characteristics Height Spread Length Width Length Width (cm) Length width H. radiata (n=5) 13.8±2.4 15.1±0.4 6.9±0.7 0.5±0.1 5.0±3.0 3.2±0.1 2.2±0.1 0.3±0.1 2.8±0.4 18.8±2.0 3.3±0.8 1.5±0.1 1.1±0.2 Round­oval with many long stolons H. rhodocheila (orange) (n=5) 22.3±3.1 23.4±0.6 9.5±1.9 2.2±0.4 6.4±0.2 3.1±0.0 2.3±0.1 0.4±0.1 9.4±1.2 25.8±2.4 3.5±0.5 3.6±1.5 1.5±0.2 Long oval WXO1 (n=2) 10.8±2.8 9.3±1.0 3.2±0.7 0.8±0.1 4.0±1.0 2.4±0.2 1.6±0.2 0.4±0.1 1.5±0.5 12.9±3.4 3.1±0.1 1.4±0.1 1.0±0.1 Round­oval WXO2 (n=2) 16.0±2.5 20.4±1.6 7.4±0.1 2.4±0.1 6.0±1.0 2.7±0.1 1.8±0.1 0.3±0.0 8.5±1.5 22.0±1.8 3.3±0.0 1.7±0.2 0.9±0.2 Round­long oval with many short stolons Table 2 ­ Morphology characteristics of two Habenaria species and interspecific hybrids Sinumporn et al. ‐ Development of interspecific hybrids of Habenaria spp. 9 We obtained new interspecific Habenaria hybrids, WXO1, WXO2, and WXY2. Both WXO1 and WXO2 showed intermediate morphological characteristics of the parents (Table 2). It is well­known in Orchidaceae that interspecific hybrids exhibit an intermediate mor­ phological characteristic of parent. When the interme­ diate characteristics is observed in detail, the charac­ teristics of either the female parent or the male par­ ent often appear strongly in each organ as shown in this study. In case of Ascocentrum ampullaceum var. auranticum × Vanda coerulea, the progenies showed flowers having pink petal color and spur which come from female parent and orange mottles on the petals come from male parent (Kishor et al., 2006). Interspecific hybrids between Vanilla planifilia and V. aphylla showed two types, light green plants without leaf resembling male parent and green plants with leaves resembling female parent (Divakaran et al., 2006). The flowers of WXO1 and WXO2 were pale yel­ low. The major flower pigment of H. rhodocheila com­ plex is considered to be carotenoids (Sinumporn et al., 2015). In flowers that contain carotenoids as a main flower pigment, the flowers are orange when the amount of carotenoid is high and yellow when it is low (Kishimoto et al., 2007). It is assumed that the carotenoid content of the WXO1 and WXO2 petals is very low because one parent HRA has white petals, resulting in pale yellow petals in the hybrids. The inter­ specific hybrid of HRA × RCY named WXY2 showed intermediate morphological characteristics in leaves between parents (data not shown). Because that the hybrid was not large enough to give flowering, the flo­ ral characteristics was not determine yet. Interspecific hybrids often have flower color intermediate between the parents as reported in the reciprocal crossing progenies of RCP and RCY, which produced pale orange and pink flowers (Adthalungrong et al., 2015). The hybrids obtained in this study are new, but the ornamental value is not high enough. Further breeding steps such as backcrossing and self­pollination are required for flower pigment accumulation and flower form improvement. Furthermore, the temperature response of these hybrids is not yet clear because the resulting hybrids were grown under constant temper­ ature conditions. It is necessary to clarify the charac­ teristics of cultivation to evaluate the hybrids. We obtained new interspecific hybrids between H. radiata and H. rhodocheila complex. The interspecific hybridization produced both true hybrids and apomicts. The apomicts were distinguished at the early developmental stage of the progenies through PCR­RFLP analysis. The obtained hybrids showed an interesting flower shape and color, which were inter­ mediate between the parents. Further breeding processes, especially back crossing, is required to improve the ornamental value of the hybrids. 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