荻幼穗诱导不同类型愈伤组织的差异
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摘要
禾本科芒属植物荻(Miscanthus sacchariflorus)是原产我国的优良能源植物,本研究以采集自滨海盐碱地中的优质耐盐种质资源荻的幼穗为外植体,分析诱导出的不同类型愈伤组织在愈伤诱导率、分化率、生根率及移栽成活率等方面的差异,明确不同类型愈伤组织的分化和植株再生能力,从而建立荻高效再生体系,为荻优质种源的推广扩繁、体细胞筛选和遗传转化奠定基础。结果表明,最佳愈伤组织诱导培养基为MS+3.0 mg·L~(–1) 2,4-二氯苯氧乙酸(2,4-D)+0.1 mg·L~(–1) 6-苄氨基腺嘌呤(6-BA)+0.72 g·L~(–1)脯氨酸,以荻幼穗为外植体可以诱导出4种类型的愈伤,分别为黄色愈伤、白色愈伤、紫红色愈伤、透明水渍状愈伤,4种愈伤的诱导率分别为7.01%、61.92%、21.02%和9.72%;最佳分化培养基为MS+2.0 mg·L~(–1) 6-BA+0.5 mg·L~(–1)萘乙酸(NAA),其中黄色愈伤、白色愈伤和紫红色愈伤均为胚性愈伤组织,其分化率分别为93.16%、90.60%和29.91%;采用1/2MS+0.5 mg·L~(–1) NAA作为生根培养基时,3种胚性愈伤组织所分化出的再生植株生根率均达到100%;将再生植株经过炼苗后,移栽于以消毒蛭石为基质的小花盆中,20d后所有移栽苗全部成活。本研究不仅建立了荻高效再生体系,而且明确了黄色愈伤组织、白色愈伤组织和紫红色愈伤组织均可作为构建荻再生体系的愈伤类型,但白色愈伤组织更容易获得,且分化率显著高于紫红色愈伤组织,而与黄色愈伤组织差异不显著。因此,白色愈伤组织是构建荻高效再生体系的理想愈伤组织类型,同时也是构建高效遗传转化体系进行荻种质改良的基础和必备条件。
Miscanthus sacchariflorus is an excellent energy plant that originated in China, belonging to the genus Miscanthus of Gramineae. The immature inflorescences of the strongly salt-tolerant M. sacchariflorus accession collected from coastal saline-alkali soil were used as explants for callus induction in this study. To reveal the callus differentiation performance and plant regeneration ability of the different types of calli induced from immature inflorescences, the differences in callus induction, differentiation, rooting and, survival rate of seedlings were compared. A high frequency regeneration system of M.sacchariflorus was established based on this, which laid the foundation for the promotion, propagation, somatic cell screening, and genetic transformation of M. sacchariflorus germplasms. The optimum medium for callus induction was MS +3.0 mg·L~(–1) 2, 4-D + 0.1 mg·L~(–1) 6-BA + 0.72 g·L~(–1) proline. Four types of callus could be induced by using immature inflorescences as explants, namely, yellow callus, white callus, purple-red callus, and transparent watery stain callus, with induction rate of 7.01%, 61.92%, 21.02%, and 9.72%, respectively. The optimal differentiation medium was MS + 2.0 mg·L~(–1) 6-BA + 0.5 mg·L~(–1) NAA. The yellow, white, and purple-red calli were all embryonic, with differentiation rates of 93.16%,90.60%, and 29.91%, respectively. When 1/2 MS + 0.5 mg·L~(–1) NAA was used as the rooting medium, the rooting rates of the regenerated plants derived from three types of callus tissue reached 100%. After seedling hardening-off, the regenerated plants were transplanted into pots filled with sterile vermiculite. All transplanted plants were still alive after 20 days. Here,we not only established an efficient regeneration system for M. sacchariflorus, but also clarified that all three types of calli(yellow, white, and purple-red), can be used for establishment of regeneration systems. However, the white callus was more easily obtained, and its differentiation rate was significantly higher than that of the purple-red callus, but there was no significant difference with the yellow callus. Therefore, the white callus is an ideal callus type for constructing an efficient regeneration system for M. sacchariflorus and is also the basis and prerequisite for the construction of efficient genetic transformation systems for germplasm improvement of M. sacchariflorus.
Miscanthus sacchariflorus is an excellent energy plant that originated in China, belonging to the genus Miscanthus of Gramineae. The immature inflorescences of the strongly salt-tolerant M. sacchariflorus accession collected from coastal saline-alkali soil were used as explants for callus induction in this study. To reveal the callus differentiation performance and plant regeneration ability of the different types of calli induced from immature inflorescences, the differences in callus induction, differentiation, rooting and, survival rate of seedlings were compared. A high frequency regeneration system of M.sacchariflorus was established based on this, which laid the foundation for the promotion, propagation, somatic cell screening, and genetic transformation of M. sacchariflorus germplasms. The optimum medium for callus induction was MS +3.0 mg·L~(–1) 2, 4-D + 0.1 mg·L~(–1) 6-BA + 0.72 g·L~(–1) proline. Four types of callus could be induced by using immature inflorescences as explants, namely, yellow callus, white callus, purple-red callus, and transparent watery stain callus, with induction rate of 7.01%, 61.92%, 21.02%, and 9.72%, respectively. The optimal differentiation medium was MS + 2.0 mg·L~(–1) 6-BA + 0.5 mg·L~(–1) NAA. The yellow, white, and purple-red calli were all embryonic, with differentiation rates of 93.16%,90.60%, and 29.91%, respectively. When 1/2 MS + 0.5 mg·L~(–1) NAA was used as the rooting medium, the rooting rates of the regenerated plants derived from three types of callus tissue reached 100%. After seedling hardening-off, the regenerated plants were transplanted into pots filled with sterile vermiculite. All transplanted plants were still alive after 20 days. Here,we not only established an efficient regeneration system for M. sacchariflorus, but also clarified that all three types of calli(yellow, white, and purple-red), can be used for establishment of regeneration systems. However, the white callus was more easily obtained, and its differentiation rate was significantly higher than that of the purple-red callus, but there was no significant difference with the yellow callus. Therefore, the white callus is an ideal callus type for constructing an efficient regeneration system for M. sacchariflorus and is also the basis and prerequisite for the construction of efficient genetic transformation systems for germplasm improvement of M. sacchariflorus.
引文
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[4]PIDLISNYUK V,STEFANOVSKA T,LEWIS E E,ERICKSON L E,DAVIS L C.Miscanthus as a productive biofuel crop for phytoremediation.Critical Reviews in Plant Sciences,2014,33(1):1-19.
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[6]薛帅,刘吉利,任兰天.不同繁殖技术芒草的种植成本估算与应用潜力评价.中国农业大学学报,2013,18(6):27-34.XUE S,LIU J L,REN L T.Benefit-cost analysis and utilization potential evaluation of different Miscanthus propagation systems.Journal of China Agricultural University,2013,18(6):27-34.
[7]王禹,易自力,易镇邪,王学华.芒属能源植物繁殖与栽培技术研究进展.可再生能源,2012(5):84-88.WANG Y,YI Z L,YI Z X,WANG X H.Research on breeding and cultivation technology of Miscanthus energy plant.Renewable Energy Resources,2012(5):84-88.
[8]ATKINSON C J.Establishing perennial grass energy crops in the UK:A review of current propagation options for Miscanthus.Biomass&Bioenergy,2009,33(5):752-759.
[9]DEFRA.Planting and Growing Miscanthus.Best Practice Guidelines for Applicants to Defra's Energy Crops Scheme.London:Department for Environment Food and Rural Affairs,2007.
[10]曾庆飞,韦鑫,陈锡,陈光洁,马培杰,吴佳海,王小利.多花黑麦草特高和多年生黑麦草四季高频组培再生体系的优化与建立.草业科学,2017,34(8):1649-1660.ZENG Q F,WEI X,CHEN X,CHEN G J,MA P J,WU J H,WANG X L.Optimisation and establishment of a high frequency regeneration system for Lolium multiflorum‘Tetragold’and L.perenne‘Four seasons’.Pratacultural Science,2017,34(8):1649-1660.
[11]HOLME I B,PETERSEN K K.Callus induction and plant regeneration from different explant types of Miscanthus×ogiformis Honda‘Giganteus’.Plant Cell,Tissue and Organ Culture,1996,45(1):43-52.
[12]PETERSEN K K,HAGBERG P,KRISTIANSEN K,FORKMANN G.In vitro chromosome doubling of Miscanthus sinensis.Plant Breeding,2010,121(5):445-450.
[13]PEPóP,TóTH S.The role of nitrogen and phosphorous source in Miscanthus in vitro cultures.Cereal research communications,2005,33(2-3):549-552.
[14]TRIEU A T N.Miscanthus transformation methods.Ceres Inc.World intellectual property organization,USA.Patent WO2010065534A3 Google Scholar,2010.
[15]ENGLER D,CHEN J.Transformation and engineered trait modification in Miscanthus species:U.S.Patent Application 12/988,789.2011-2-24.
[16]G?OWACKA K,JE?OWSKI S,KACZMAREK Z.Polyploidization of Miscanthus sinensis and Miscanthus×giganteus by plant colchicine treatment.Industrial Crops and Products,2009,30(3):444-446.
[17]G?OWACKA K,JE?OWSKI S,KACZMAREK Z.The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two Miscanthus species.Plant Cell,Tissue and Organ Culture,2010,102(1):79-86.
[18]CHAE W B,HONG S J,GIFFORD J M,RAYBURN A L,WIDHOLM J M,JUVIK J A.Synthetic polyploid production of Miscanthus sacchariflorus,Miscanthus sinensis,and Miscanthus×giganteus.Gcb Bioenergy,2013,5(3):338-350.
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