3种抗褐化剂影响水曲柳体胚发生的生理分析
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摘要
水曲柳体胚发生过程中往往伴随褐化现象的产生,并且大多体胚生长在褐化的外植体上,为解析外植体褐化和体胚发生之间的联系,本研究通过在培养基中添加PVP(聚乙烯毗咯烷酮)、L-Glu(L-谷氨酸)和AgNO_3(硝酸银),探究其对外植体褐化、体胚发生和生理生化的影响。研究结果表明:(1)低浓度(0. 1、0. 5 g·L~(-1)) PVP和100 mg·L~(-1)L-Glu处理加剧了外植体褐化,但显著促进了体胚发生,并且体胚发生率高达60%以上(分别比对照组提高了6. 59%、24. 08%和22. 88%);(2) 200 mg·L~(-1)L-Glu处理有效降低了外植体褐化,褐化率为68. 11%(相比对照组降低了5. 83%),但是体胚发生率降低,其发生率为46. 32%(相较于对照组降低了22. 8%);(3) 3种抗褐化剂处理后外植体细胞内的多酚氧化酶活性(PPO)和超氧化物歧化酶(SOD)活性均低于对照组,过氧化物酶(POD)活性和丙二醛(MDA)含量均高于对照组。因此,研究表明低浓度的抗褐化剂可以促进体胚发生,在这个过程中涉及到POD活性升高和MDA含量增加。本研究为解析水曲柳体胚发生伴随外植体褐化的生物学机理以及水曲柳体胚高频发生提供理论依据。
The somatic embryogenesis of Fraxinus mandshurica Rupr. is often accompanied by browning,and most somatic embryos grow on browning explants. We analyzed the relationship between explant browning and somatic embryogenesis,by adding PVP(polyvinylpyrrolidone),L-Glu(L-glutamic acid) and AgNO_3(silver nitrate) to the culture medium,and studied its effects on browning,somatic embryogenesis and physiological and biochemical characteristics of explants. The results showed that:(1) Low concentration(0. 1 and 0. 5 g·L~(-1)) PVP,and 100 mg·L~(-1) L-Glu treatment exacerbated browning of explants,but significantly promoted somatic embryogenesis,somatic embryogenesis rate exceeded 60%(increased by 6. 59%,24. 08% and22. 88% compared to the control,respectively).(2) The 200 mg·L~(-1) L-Glu treatment effectively reduced the browning of explants,the browning rate was 68. 11%(down 5. 83% compared with the control),but the somatic embryogenesis rate was reduced to 46. 32%(down 22. 8% compared with the control).(3) The activities of polyphenol oxidase(PPO) and superoxide dismutase(SOD) in explants were lower than those of control after treatment with three anti-browning agents,peroxidase(POD) activity and malondialdehyde(MDA)content were higher than the control. Therefore,it is believed that a low concentration of anti-browning agent can promote somatic embryogenesis,and in this process,an increase in POD activity and an increase in MDA content are involved. We analyzed the biological mechanism of somatic embryogenesis with explants browning and got the frequency of somatic embryogenesis of F. mandshurica.
The somatic embryogenesis of Fraxinus mandshurica Rupr. is often accompanied by browning,and most somatic embryos grow on browning explants. We analyzed the relationship between explant browning and somatic embryogenesis,by adding PVP(polyvinylpyrrolidone),L-Glu(L-glutamic acid) and AgNO_3(silver nitrate) to the culture medium,and studied its effects on browning,somatic embryogenesis and physiological and biochemical characteristics of explants. The results showed that:(1) Low concentration(0. 1 and 0. 5 g·L~(-1)) PVP,and 100 mg·L~(-1) L-Glu treatment exacerbated browning of explants,but significantly promoted somatic embryogenesis,somatic embryogenesis rate exceeded 60%(increased by 6. 59%,24. 08% and22. 88% compared to the control,respectively).(2) The 200 mg·L~(-1) L-Glu treatment effectively reduced the browning of explants,the browning rate was 68. 11%(down 5. 83% compared with the control),but the somatic embryogenesis rate was reduced to 46. 32%(down 22. 8% compared with the control).(3) The activities of polyphenol oxidase(PPO) and superoxide dismutase(SOD) in explants were lower than those of control after treatment with three anti-browning agents,peroxidase(POD) activity and malondialdehyde(MDA)content were higher than the control. Therefore,it is believed that a low concentration of anti-browning agent can promote somatic embryogenesis,and in this process,an increase in POD activity and an increase in MDA content are involved. We analyzed the biological mechanism of somatic embryogenesis with explants browning and got the frequency of somatic embryogenesis of F. mandshurica.
引文
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16.Liu C P,Yang L,Shen H L.Proteomic analysis of immature Fraxinus mandshurica cotyledon tissues during somatic embryogenesis:effects of explant browning on somatic embryogenesis[J].International Journal of Molecular Sciences,2015,16(6):13692-13713.
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19.赵世杰,许长成,邹琦,等.植物组织中丙二醛测定方法的改进[J].植物生理学通讯,1994,30(3):207-210.Zhao S J,Xu C C,Zou Q,et al.Improvement of method for measurement of malondialdehyde in plant tissues[J].Plant Physiology Communications,1994,30(3):207-210.
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21.高红兵,杜凤国,王欢.抗褐化剂对天女木兰芽外植体褐化与酚酸氧化的影响[J].林业科学研究,2017,30(3):525-532.Gao H B,Du F G,Wang H.Effects of browning inhibitors on bud explants browning and phenolic acids oxidation of Magnolia sieboldii K.Koch[J].Forest Research,2017,30(3):525-532.
22.夏亚男,蒋建雄,易自力,等.3种抗褐化剂对南荻外植体褐变及愈伤诱导率的影响[J].北方园艺,2014(17):93-96.Xia Y N,Jiang J X,Yi Z L,et al.Effect of three antioxidants on browning of explant and callus induction rate of Miscanthus lutarioriparius[J].Northern Horticulture,2014(17):93-96.
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24.陈爱萍,冯维卫,刘允康.3种豆科牧草愈伤组织继代培养抗褐化的研究[J].中国农学通报,2017,33(22):138-144.Chen A P,Feng W W,Liu Y K.Anti-browning agents affecting callus subculture of three legume forage species[J].Chinese Agricultural Science Bulletin,2017,33(22):138-144.
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2.杨玲,刘虹男,张冬严,等.生长调节剂和渗透调节物质对水曲柳体胚发生的影响[J].植物研究,2017,37(5):682-689.Yang L,Liu H N,Zhang D Y,et al.Effect of plant growth regulators and osmoticums on somatic embryogenesis of Fraxinus mandshurica Rupr.[J].Bulletin of Botanical Research,2017,37(5):682-689.
3.Smertenko A,Bozhkov P V.Somatic embryogenesis:life and death processes during apical-basal patterning[J].Journal of Experimental Botany,2014,65(5):1343-1360.
4.Us-Camas R,Rivera-Solís G,Duarte-AkéF,et al.In vitro culture:an epigenetic challenge for plants[J].Plant Cell,Tissue and Organ Culture,2014,118(2):187-201.
5.Lelu-Walter M A,Teyssier C,Guérin V,et al.Vegetative propagation of larch species:somatic embryogenesis improvement towards its integration in breeding programs[M].//Park Y S,Bonga J M,Moon H K.Vegetative propagation of forest trees.Seoul,Korea:National Institute of Forest Science,2016:551-571.
6.Lelu-Walter M A,Thompson D,Harvengt L,et al.Somatic embryogenesis in forestry with a focus on Europe:state-ofthe-art,benefits,challenges and future direction[J].Tree Genetics&Genomes,2013,9(4):883-899.
7.Kong D M,Preece J E,Shen H L.Somatic embryogenesis in immature cotyledons of Manchurian ash(Fraxinus mandshurica Rupr.)[J].Plant Cell,Tissue and Organ Culture,2012,108(3):485-492.
8.Yang L,Bian L,Shen H L,et al.Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash(Fraxinus mandshurica Rupr.)[J].Plant Cell,Tissue and Organ Culture,2013,115(2):115-125.
9.丛建民,沈海龙,李玉花,等.水曲柳体胚发生过程中不同状态类型外植体的生理生化状态[J].华南农业大学学报,2012,33(1):48-52.Cong J M,Shen H L,Li Y H,et al.Physiological and biochemical status of different-types of explants in somatic embryogenesis of Fraxinus mandshurica[J].Journal of South China Agricultural University,2012,33(1):48-52.
10.孙倩,杨玲,沈海龙,等.PPO处理对水曲柳合子胚子叶外植体褐化和体胚发生的影响[J].东北林业大学学报,2012,40(11):1-5,9.Sun Q,Yang L,Shen H L,et al.Effect of PPO treatments on zygotic cotyledon explant browning and somatic embryogenesis of Fraxinus mandshurica[J].Journal of Northeast Forestry University,2012,40(11):1-5,9.
11.刘艳,沈海龙,丛建民.5-氮胞苷对水曲柳合子胚外植体状态及体胚发生的影响[J].东北林业大学学报,2011,39(8):25-27,32.Liu Y,Shen H L,Cong J M.Effect of 5-azacytidine on zygotic cotyledon explants and somatic embryogenesis of Fraxinus mandshurica[J].Journal of Northeast Forestry U-niversity,2011,39(8):25-27,32.
12.李官德,肖娟丽,罗晓丽,等.不同棉花愈伤组织状态与胚胎发生及其植株再生的关系[J].山西农业科学,2006,34(1):29-31.Li G D,Xiao J L,Luo X L,et al.Somatic embryogenesis and plant regeneration of calli derived from cotton(Gossypium hirsutum L.)depending on their quality[J].Journal of Shanxi Agricultural Sciences,2006,34(1):29-31.
13.Tapia E,Sequeida,Castro,et al.Development of grapevine somatic embryogenesis using an air-lift bioreactor as an efficient tool in the generation of transgenic plants[J].Journal of Biotechnology,2009,139(1):95-101.
14.Ko T S,Nelson R L,Korban S S.Screening multiple soybean cultivars(MG 00 to MG VIII)for somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons[J].Crop Science,2004,44(5):1825-1831.
15.Find J I,Hargreaves C L,Reeves C B.Progress towards initiation of somatic embryogenesis from differentiated tissues of radiata pine(Pinus radiata D.Don)using cotyledonary embryos[J].In Vitro Cellular&Developmental BiologyPlant,2014,50(2):190-198.
16.Liu C P,Yang L,Shen H L.Proteomic analysis of immature Fraxinus mandshurica cotyledon tissues during somatic embryogenesis:effects of explant browning on somatic embryogenesis[J].International Journal of Molecular Sciences,2015,16(6):13692-13713.
17.刘春苹,水曲柳体胚发生伴随外植体褐化的生理机制及差异蛋白研究[D].哈尔滨:东北林业大学,2009.Liu C P.Physiologymeehanismand differential protein of Fraxinus mandshuriea somatic embry genes is a ompanied explant browning[D].Harbin:Northeast Forestry University,2009.
18.杨和平,程井辰,周吉源,等.石刁柏体细胞胚胎发生过程中超氧物歧化酶活性的变化[J].植物学报,1993,35(6):490-493.Yang H P,Cheng J C,Zhou J Y,et al.Change of superoxide dismutase activities during somatic embryogenesis in Asparagus officinalis L.[J].Acta Botanica Sinica,1993,35(6):490-493.
19.赵世杰,许长成,邹琦,等.植物组织中丙二醛测定方法的改进[J].植物生理学通讯,1994,30(3):207-210.Zhao S J,Xu C C,Zou Q,et al.Improvement of method for measurement of malondialdehyde in plant tissues[J].Plant Physiology Communications,1994,30(3):207-210.
20.叶睿华,吕享,李小兰,等.五种抗褐化剂对杜鹃兰原球茎增殖培养的作用效果[J].植物生理学报,2018,54(6):1103-1110.Ye R H,LüX,Li X L,et al.Effects of five browning inhibitors on protocorms proliferation culture of Cremastra appendiculata[J].Plant Physiology Journal,2018,54(6):1103-1110.
21.高红兵,杜凤国,王欢.抗褐化剂对天女木兰芽外植体褐化与酚酸氧化的影响[J].林业科学研究,2017,30(3):525-532.Gao H B,Du F G,Wang H.Effects of browning inhibitors on bud explants browning and phenolic acids oxidation of Magnolia sieboldii K.Koch[J].Forest Research,2017,30(3):525-532.
22.夏亚男,蒋建雄,易自力,等.3种抗褐化剂对南荻外植体褐变及愈伤诱导率的影响[J].北方园艺,2014(17):93-96.Xia Y N,Jiang J X,Yi Z L,et al.Effect of three antioxidants on browning of explant and callus induction rate of Miscanthus lutarioriparius[J].Northern Horticulture,2014(17):93-96.
23.饶慧云,邵祖超,柳海宁,等.抗褐化剂对葡萄愈伤组织继代培养过程中酚类物质、相关酶及其基因表达的影响[J].植物生理学报,2015,51(8):1322-1330.Rao H Y,Shao Z C,Liu H N,et al.Effect of browning inhibitors on callus subculture of phenolic compounds,enzyme and gene expression of grape[J].Plant Physiology Journal,2015,51(8):1322-1330.
24.陈爱萍,冯维卫,刘允康.3种豆科牧草愈伤组织继代培养抗褐化的研究[J].中国农学通报,2017,33(22):138-144.Chen A P,Feng W W,Liu Y K.Anti-browning agents affecting callus subculture of three legume forage species[J].Chinese Agricultural Science Bulletin,2017,33(22):138-144.
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