In Vitro Cellular & Developmental Biology - Plant最新文献

{"title":"Low-temperature storage in dark condition improved the in vitro regeneration of Plumbago zeylanica synthetic seeds: a medicinally valuable species","authors":"Ravishankar Chauhan, Priya Banjare, Subir Kumar Parey, Afreen Anjum, Afaque Quraishi","doi":"10.1007/s11627-024-10416-1","DOIUrl":"https://doi.org/10.1007/s11627-024-10416-1","url":null,"abstract":"<p>Medicinal applications of <i>Plumbago</i> <i>zeylanica</i> and its metabolites on various diseases and low viability and inconsistent germination of its seeds are the reasons behind the loss of its genetic diversity. Hence, an efficient protocol for the short-term storage of <i>P.</i> <i>zeylanica</i> synthetic seeds, which is an overexploited medicinally valuable plant, was developed. Initially, <i>in vitro</i> culture was performed from nodal explants to develop synthetic seeds from its proliferated shoots. Murashige and Skoog (MS) medium augmented with 0.5 mg L⁻¹ 6-benzylaminopurine (BAP) resulted in the best morphogenetic response. Thereafter, the developed synseeds were stored for 2 wk at a temperature of 10 or 25°C in different conditions and further evaluated for regeneration. Higher re-growth rate (80%) and the identical morphogenetic response were recorded for the <i>P.</i> <i>zeylanica</i> synthetic seeds, which were stored at a temperature of 10°C in dark condition after its storage period. As per the available literature, this is the first report pertaining to <i>in vitro</i> low-temperature storage of synthetic seeds of <i>P.</i> <i>zeylanica</i> and can further be utilized for the conservation of elite clones for the study of medicinally potent species.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140100003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effects of different healing agents on acclimatization success of in vitro rooted Garnem (Prunus dulcis × Prunus persica) rootstock","authors":"Heydem Ekinci, Necla Saskin, Bekir Erol Ak, Birgul Dikmetas Dogan","doi":"10.1007/s11627-024-10420-5","DOIUrl":"https://doi.org/10.1007/s11627-024-10420-5","url":null,"abstract":"<p>Continuing productivity of the acclimatization stage of plantlets means successful micropropagation. Due to the free water and high humidity in the culture container environment, poorly developed cuticle results in rapid water loss and drying of plantlets with watery stems and leaves, incomplete stomata, and large intercellular space. When plantlets are exposed to an environmental condition different from the culture medium, problems, such as rapid water loss and drying, may be encountered, and the survival rate of plantlets <i>in vitro</i> may be reduced. The aim of this study was to reduce the stress factors that occur during the acclimatization phase. For this reason, various healing agents have been used. Some of these compounds are ascorbic acid (AsA), salicylic acid (SA), and sodium nitroprusside (SNP). In the study, the response of AsA (100.0 and 200.0 mg L⁻¹), SA (100.0 and 200.0 mg L⁻¹), and SNP (100.0 and 200.0 µM) applications on growth parameters (survival rate (%), node count (pieces per plant), count of leaves (pieces per plant), shoot length (mm), and shoot diameter (mm)) and physiological variable (Soil Plant Analysis Development (SPAD)) were examined. The AsA100 (100.0 mg L⁻¹ ascorbic acid), AsA200 (200.0 mg L⁻¹ ascorbic acid), and SNP100 (100.0 µM sodium nitroprusside) applications resulted in an improvement in node count, leaf count per plant, shoot length, and shoot diameter parameters. The highest shoot length (60.50 ± 7.81 mm) and node count (16.83 ± 1.15 pieces per plantlet) were achieved with the AsA100 application. The maximum stem diameter (2.31 ± 0.37 mm) was determined with the SNP100 application. There were no statistically significant differences found in the survival rate, leaf count, and SPAD parameter. The current study determined that AsA, SA, and SNP applications were effective in regulating several growth parameters in Garnem plantlets and in reducing acclimation stress thereby facilitating adaptation to external conditions.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140100039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Silver nitrate mediated improvement in micropropagation and amelioration of micro-morpho-anatomical structures in Oxystelma esculentum (L.f.) Sm.","authors":"","doi":"10.1007/s11627-024-10417-0","DOIUrl":"https://doi.org/10.1007/s11627-024-10417-0","url":null,"abstract":"<h3>Abstract</h3> <p>The present study highlights the effect of silver nitrate (AgNO₃) in the promotion of <em>in vitro</em> regeneration and morpho-structural growth and developments in <em>Oxystelma esculentum</em> (L.f.) Sm. The incorporation of AgNO₃ into the Murashige and Skoog (MS) medium resulted in a favorable enhancement of shoot proliferation. Among the different concentrations of AgNO₃ screened, 0.5 mg L⁻¹ AgNO₃ along with 0.5 mg L⁻¹ <em>meta</em>-Topolin (<em>m</em>T) and 0.25 mg L⁻¹ indole-3-acetic acid (IAA) stimulated the highest count, and length of shoots (143 shoots and 14.0 cm length) after 3 mo of incubation and the 3rd subculture. The <em>in vitro</em>–induced deformities in the tissues of the control stem and foliages were improved when the shoots were grown in AgNO₃-enriched medium. Silver nitrate increased the photosynthetic pigments and improved the formation of functional stomata. The AgNO₃-supplemented medium-derived shoots exhibited accelerated adventitious root induction (100% rhizogenesis, 21.0 roots per shoot) when the cut ends of shoots were treated with 200.0 mg L⁻¹ indole-3-butyric acid (IBA) for 5 min under greenhouse conditions. The incorporation of AgNO₃ with optimal plant growth regulators in the medium promoted overall growth. It prevented <em>in vitro</em>–induced morphological and anatomical anomalies in the leaf and stem, which considerably improved the greenhouse and <em>in vivo</em> survival of micropropagated plantlets.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140047687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of tissue culture regeneration system of Ficus tikoua","authors":"Xufei Li, Qianxia Li, Ying Cui, Tongfei Liu, Yanni Zhang","doi":"10.1007/s11627-024-10414-3","DOIUrl":"https://doi.org/10.1007/s11627-024-10414-3","url":null,"abstract":"<p><i>Ficus tikoua</i> Bureau is an ecologically functional perennial vine that belongs to the Moraceae family. Tissue culture is a common method for the rapid propagation of plants and is suitable for the rapid propagation of <i>F. tikoua</i>. The objective of the current study was to evaluate the process of <i>F. tikoua</i> organogenesis <i>in vitro</i> utilizing stem explants. An efficient tissue culture propagation method was developed by manipulating several plant growth regulators (PGRs) and their proportions, as well as evaluating alternative medium options for specific phases of differentiation. Explants were cultured on MS medium, supplemented with various thidiazuron (TDZ) concentrations with 6-benzylaminopurine (BA) and naphthaleneacetic acid (NAA), to investigate their efficacy during <i>in vitro</i> shoot organogenesis. The results demonstrated that the combination of 0.5 mg L⁻¹ BA, 1.0 mg L⁻¹ NAA, and 1.0 mg L⁻¹ TDZ was the most effective for callus formation (91.1%) and that the combination of 2.0 mg L⁻¹ BA and 0.5 mg L⁻¹ TDZ was the most suited for adventitious shoot induction (61.11%). The medium supplemented with 1.0 mg L⁻¹ BA, 1.0 mg L⁻¹ NAA, and 0.5 mg L⁻¹ TDZ yielded the highest bud multiplication number with a 6.05 proliferation factor. The cultured shoots rooted easily on half-strength MS medium containing 1.0 mg L⁻¹ IBA and 0.1 mg L⁻¹ NAA (68.52%). The subsequent regenerated plants were grown in a mixture of garden soil, perlite, and vermiculite in a ratio of 2:1:1, resulting in a survival rate of 86.17%.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140032521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation of endogenous U6 promoters for expanding the CRISPR toolbox in Nicotiana tabacum","authors":"Pan Luo, Siyuan Li, Lulu Li, Yanyan Li, Ying Qiao, Xianzhi Wu, Meiling Wu, Zhihao Song, Yong Yang, Ji Feng, Xingchun Tang","doi":"10.1007/s11627-024-10418-z","DOIUrl":"https://doi.org/10.1007/s11627-024-10418-z","url":null,"abstract":"","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140047591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of first protocol of hypocotyl-based regeneration and callus transformation in waterhemp (Amaranthus tuberculatus)","authors":"","doi":"10.1007/s11627-023-10408-7","DOIUrl":"https://doi.org/10.1007/s11627-023-10408-7","url":null,"abstract":"<h3>Abstract</h3> <p>Waterhemp (<em>Amaranthus tuberculatus</em> (Moq.) Sauer) is one of the most problematic weeds in the Midwestern agricultural systems in the USA. As a consequence of extensive herbicide selection pressure, many waterhemp populations across the USA evolved resistance to multiple herbicides, leading to limited options for chemical control. Genome editing tools, including CRISPR/Cas9, have great potential in weed science, especially when coupled with a gene drive system for a sustainable weed management. The application of modern biotechnological tools requires availability of an efficient regeneration and genetic transformation system. The objectives of this research were to develop an efficient protocol for successful regeneration of waterhemp <em>via</em> callus culture and optimize an <em>Agrobacterium-</em>mediated transformation method for waterhemp. Hypocotyl explants cultured on callus induction medium (CIM), C6 containing 1.0 mg L⁻¹ kinetin and 4.0 mg L⁻¹ 2,4-D for 2 wk followed by subculture into shoot induction medium (SIM), S8 containing 3.0 mg L⁻¹ BAP, 0.05 mg L⁻¹ 2,4-D, and 0.3 mg L⁻¹ GA₃ for 4 to 6 wk was successful in shoot regeneration. Leaf explants failed to respond to any treatment combinations tested for regeneration. Using the hypocotyl explant–based regeneration protocol optimized in this research, <em>Agrobacterium</em>-mediated transformation was performed using <em>uidA</em> and <em>hpt</em> genes as a proof of concept. Although transgenic plants were not regenerated, molecular characterization of the transformed calluses confirmed the presence of transgenic genes, for example <em>uidA</em> and <em>hpt</em>. This is the first report of the tissue-cultured-based regeneration and <em>Agrobacterium</em>-mediated transformation of hypocotyl derived calluses in waterhemp, which is a step forward in exploring the possibilities of genome editing research in weed science.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140004427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vivo regeneration efficiency of enset (Ensete ventricosum (Welw.) Cheesman) using indigenous shoot induction method developed in Ethiopia","authors":"Bizuayehu Tesfaye, Bewuketu Haile","doi":"10.1007/s11627-024-10411-6","DOIUrl":"https://doi.org/10.1007/s11627-024-10411-6","url":null,"abstract":"<p>Enset (<i>Ensete ventricosum</i> [Welw.] Cheesman) is native to Ethiopia, valued for its versatility and drought tolerance. Farmers have developed a vegetative method for regenerating multiple shoots that produce plantlets derived from wound callus, a process similar to shoot production in <i>in vitro</i> tissue culture. This <i>in vivo</i> regeneration method, however, is a low-cost, tissue culture-free technique that is executed in a field context. The objectives of this study were to investigate the efficiency of <i>in vivo</i> regeneration in different enset landraces using the indigenous method of shoot induction and to compare the capability of wild and domesticated enset landraces to regenerate shoots. Three corm treatments (whole, halved, and quartered) from wild and from three domesticated landraces (<i>Gudiro</i>, <i>Bosso</i>, and <i>Yekko</i>), each of which corm or corm pieces with the apical meristem was removed, were buried in a randomized complete block design with three replicates. The study demonstrated that enset plants can be easily propagated <i>via</i> mechanical cuttings of their corms, giving rise to plantlets deriving from wound callus. This can be done in soil; for example, it is not necessary to use costly <i>in vitro</i> techniques. All domesticated enset landraces showed 100% regeneration frequency in every condition of corm treatment employed and produced an average of 51 to 501 shoots per corm depending on landraces and corm treatments used. In wild enset, 50 to 95% regeneration and an average of 6 to 85 shoots per corm were noted depending on the corm treatments. In general, the regeneration capacity of enset was strongly influenced by the landrace and corm treatment. Splitting the corm has remarkably enhanced shoot induction efficiency in every enset landrace including in wild enset: the more pieces the corm was split into, the more sprouts per corm. Furthermore, this study revealed that domesticated enset has a significantly higher capacity for shoot regeneration compared to wild enset, which could provide some insights into the evolution of this clonal crop. Clonal propagation likely conditioned the pathways of enset evolution under domestication.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139988327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct organ regeneration from apical shoot buds of adult Pinus massoniana Lamb","authors":"","doi":"10.1007/s11627-024-10415-2","DOIUrl":"https://doi.org/10.1007/s11627-024-10415-2","url":null,"abstract":"<h3>Abstract </h3> <p>Direct organogenesis is an important technique for plant rapid propagation, which is mainly controlled by the balance of auxin and cytokinin. <em>Pinus massoniana</em> (Lamb.) is a perennial tree species with high application value. Previous studies have shown that direct organ regeneration of young <em>P. massoniana</em> is feasible. However, there are few reports on direct organogenesis of adult <em>P. massoniana</em>. This research studied the effects of apical shoot bud disinfection, plant growth regulators of axillary bud induction, and adventitious root induction of adult <em>P. massoniana</em>. The results showed that the survival rate could reach 50% after removing the outer coat scales and soaking in 500.0 mg L⁻¹ carbendazim solution for 10 min, 75% alcohol for 30 s, and 2.0% NaClO with one drop of Tween for 25 min. The addition of 0.2% plant preservative mixture (PPM) to the medium effectively inhibited contamination of endogenous bacteria, and the survival rate was 90%. The suitable medium for axillary bud induction was Murashige and Skoog (MS) basal medium with 6-benzyl aminopurine (6-BA, 2.0 mg L⁻¹) and indolebutyric acid (IBA, 0.1 mg L⁻¹), and the induction rate reached 15%. A single apical shoot bud induced up to seven axillary buds, and the axillary buds grew vigorously. In addition, 6-BA (2.0 mg L⁻¹) was suitable for needle bundle axillary bud induction. Quarter Douglas-fir cotyledon revised medium (DCR; Gupta and Durzan 1985) with naphthaleneacetic acid (NAA, 0.5 mg L⁻¹) and IBA (0.1 mg L⁻¹) was the most suitable for adventitious root induction. This study preliminarily constructed a regeneration system for direct organogenesis of adult <em>P. massoniana</em>, which was expected to provide key technical support for vegetative propagation of excellent breeding materials for adult <em>P. massoniana</em>.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139924246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro micropropagation of a novel non-flowering radiation mutant of Platanus × acerifolia Willd.","authors":"","doi":"10.1007/s11627-023-10409-6","DOIUrl":"https://doi.org/10.1007/s11627-023-10409-6","url":null,"abstract":"<h3>Abstract</h3> <p>An efficient <em>in vitro</em> micropropagation system for <em>Platanus</em> × <em>acerifolia</em> ‘F03’, a radiation mutant that has not flowered for 19 yr, has been established. Axillary buds were used as explants. After sprouting on Murashige and Skoog medium (MS) supplemented with 0.3 mg·L⁻¹ 6-benzylaminopurine (6-BA) together with 0.05 mg·L⁻¹ α-naphthaleneacetic acid (NAA), the best medium for multiplication and elongation was MS basal medium supplemented with 0.1 mg·L⁻¹ 6-benzylaminopurine and with a multiplication rate of 3.43 ± 0.57. Plantlets over 1.5 cm could root on McCown Woody Plant Medium (WPM) supplemented with 0.01 mg·L⁻¹ α-naphthaleneacetic acid and 7.0 g·L⁻¹ agar with an ideal rooting rate and transplanting survival rate of 100 ± 0%. The optimum survival rate was possible when trays were covered with a thin plastic lid for more than 14 d. After transplanting, air humidity and temperature were crucial for young plant survival. The system established here could serve as a pathway for commercial production of the line and provide sterile leaf explants for regeneration and further genetic improvement of the species.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139924056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Status and prospects of yam somatic embryogenesis: a pathway for biotechnology applications","authors":"Chukwunalu O. Ossai, Morufat O. Balogun, Norbert G. Maroya","doi":"10.1007/s11627-024-10413-4","DOIUrl":"https://doi.org/10.1007/s11627-024-10413-4","url":null,"abstract":"<p>Yam is an important staple in sub-Saharan Africa, but the availability of quality seed yam is majorly constrained by the low propagation ratio. This is because the propagating explant is limited to the tuber and nodal parts as yam rarely flowers. There are several reports of the use of somatic embryogenesis (SE) in the rapid propagation of different crop species and as a regenerative pathway in plant genetic engineering. However, SE deployment in yam is still at the protocol development stage. This review thus exploits the status of SE application in improving the yam propagation rate. This article reviews the potential of the various yam propagation techniques in rapidly multiplying disease-free yam with their propagating explants. The advantages SE offers are rapidly propagating yam, the factors to consider in the protocol optimization of SE application in rapidly multiplying different yam varieties, and as a platform for full utilization of genetic engineering in yam. The findings so far show that SE potentially offers a faster rate of propagating yam varieties. However, due to the differences in varietal endogenous hormonal and gene products, response to SE in yam is constrained by varietal specificity. Hence, the applicability of SE in yam is still at the protocol development state. This review, thus, presents the need for more research efforts to elucidate the molecular and phytochemical controlling mechanisms of SE in yam to improve the yam multiplication rate and lay an efficient platform for the exploitation of other biotechnological advancements in improving yam species.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139924061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}