香榧幼胚发育与胚性感受态之间的相关性
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摘要
以突破种鳞第8周至第15周种子的香榧Torreya grandis ‘Merrillii’幼胚为外植体,研究了香榧幼胚胚龄、生长素等对香榧胚性愈伤组织以及体细胞胚直接发生的影响,筛选出高胚性感受态的香榧幼胚胚龄,并根据幼胚内源吲哚乙酸(IAA)和脱落酸(ABA)质量分数检测结果,初步确定了高胚性感受态的香榧幼胚生理特点。结果表明:在SH附加0.1 mg·L~(-1)NAA培养基中培养,第9周幼胚的胚性愈伤组织发生率最高,为48.3%,第10周幼胚的体胚直接发生率最高,为42.7%;而培养于SH基本培养基中的第14周幼胚直接萌发率最高,为65.1%。香榧不同胚龄幼胚内源ABA及IAA质量分数检测结果表明:不同发育时期香榧幼胚的内源IAA及ABA质量分数具显著差异(P<0.05)。其中香榧第8周幼胚内源IAA的质量分数最高,为53.90 ng·g~(-1),随着胚龄的逐渐增加,内源IAA质量分数呈下降趋势,第15周内源IAA质量分数最低,为35.60 ng·g~(-1)。内源ABA质量分数随着胚龄的增加呈先缓慢下降,再快速上升,后下降,其中第15周达到最大值,为160.20 ng·g~(-1)。通过对香榧幼胚离体培养及内源激素的测定,发现香榧第8~12周幼胚具有较高的胚性感受态。该研究结果对维持香榧幼胚高胚性感受态,开展香榧胚性愈伤组织及体胚发生培养条件的优化提供了技术指导。
To obtain high embryogenic frequency in the immature embryo developmental stage, immature embryos of Torreya grandis ‘Merrillii' from seeds(8 to 15 weeks after ovule breakthrough from the scales)were sampled as explants to optimize the best developmental stage for somatic embryogenesis. Endogenous Indole-3-acetic Acid(IAA) and Abscisic Acid(ABA) of immature embryos were also analyzed using enzymelinked immunosorbent assay(ELISA) method. Results showed that the embryogenic callus frequency(up to48.3%) and direct somatic embryogenesis frequency(up to 42.7%) were highest with immature embryos from seeds at 9 weeks after scale breakthrough on Schenk and Hilderbrandt(SH) basal medium supplemented with0.1 mg·L-11-Naphthaleneacetic acid(NAA). In vitro germination frequency was highest(up to 65.1%) with immature embryos from seeds at 14 weeks after scale breakthrough. Significant differences(P<0.05) among development stages of different immature embryos were found. With the development of immature embryos,endogenous IAA decreased gradually being highest with immature embryos from seeds at 8 weeks after scale breakthrough [up to 53.90 ng·g-1 fresh weight(FW)]. Meanwhile, endogenous ABA initially decreased followed by a rapid increase with the highest content being 160.20 ng·g-1 FW for immature embryos from seeds at15 weeks after scale breakthrough. This study revealed that with immature embryos of seeds from 8 to 12 weeks, embryogenic frequency was high which could help optimize embryogenic callus induction and somatic embryo genesis in Torreya grandis ‘Merrillii'.
To obtain high embryogenic frequency in the immature embryo developmental stage, immature embryos of Torreya grandis ‘Merrillii' from seeds(8 to 15 weeks after ovule breakthrough from the scales)were sampled as explants to optimize the best developmental stage for somatic embryogenesis. Endogenous Indole-3-acetic Acid(IAA) and Abscisic Acid(ABA) of immature embryos were also analyzed using enzymelinked immunosorbent assay(ELISA) method. Results showed that the embryogenic callus frequency(up to48.3%) and direct somatic embryogenesis frequency(up to 42.7%) were highest with immature embryos from seeds at 9 weeks after scale breakthrough on Schenk and Hilderbrandt(SH) basal medium supplemented with0.1 mg·L-11-Naphthaleneacetic acid(NAA). In vitro germination frequency was highest(up to 65.1%) with immature embryos from seeds at 14 weeks after scale breakthrough. Significant differences(P<0.05) among development stages of different immature embryos were found. With the development of immature embryos,endogenous IAA decreased gradually being highest with immature embryos from seeds at 8 weeks after scale breakthrough [up to 53.90 ng·g-1 fresh weight(FW)]. Meanwhile, endogenous ABA initially decreased followed by a rapid increase with the highest content being 160.20 ng·g-1 FW for immature embryos from seeds at15 weeks after scale breakthrough. This study revealed that with immature embryos of seeds from 8 to 12 weeks, embryogenic frequency was high which could help optimize embryogenic callus induction and somatic embryo genesis in Torreya grandis ‘Merrillii'.
引文
[1]由香玲,谭啸,戴金玲,等.胁迫诱导植物体细胞发生的研究进展[J].西北植物学报, 2010, 30(9):1929-1934.YOU Xiangling, TAN Xiao, DAI Jinling, et al. Progress on the stress induction of plant somatic embryogenesis[J].Acta Bot Boreal-Occident Sin, 2010, 30(9):1929-1934.
[2]ARNOLD S, SABALA I, BOZHKOV P, et al. Developmental pathways of somatic embryogenesis[J]. Plant Cell, Tissue Organ Cult, 2002, 69(3):233-249.
[3]GUAN Yuan, LI Shuigen, FAN Xiaofen, et al. Application of somatic embryogenesis in woody plants[J]. Front Plant Sci, 2016, 7:938. doi:10.3389/fpls. 2016. 00938.
[4]NIC-CAN G I, GALAZ-VALOS R M, DE-LA-PE譙A C, et al. Somatic embryogenesis:identified factors that lead to embryogenic repression:a case of speciesof the same genus[J]. PLoS One, 2015. doi:10.1371/journal. pone. 0126414.
[5]汤浩茹,王永清,任正隆.核桃体细胞胚发生与转基因研究进展[J].林业科学, 2000, 36(3):102-110.TANG Haoru, WANG Yongqing, REN Zhenglong. An overview of progress on somatic emrbyogenesis and transformation in walnut[J]. Sci Silv Sin, 2000, 36(3):102-110.
[6]刘淑芳,续海红.不同发育期梨胚的再生能力[J].江苏农业科学, 2012, 40(7):63-64.LI Shufang, XU Haihong. Regeneration frequency of embryo in different developmental stages[J]. Jiangsu Agric Sci,2012, 40(7):63-64.
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[8]姜新兵,陈力耕,何新华.香榧体细胞胚发生的研究[J].园艺学报, 2004, 31(5):654-656.JIANG Xinbing, CHEN Ligeng, HE Xinhua. Studies on the somatic embryogenesis of Torreya grandis[J]. Acta Hortic Sin, 2004, 31(5):654-656.
[9]刘海琳,陈立耕,童品璋,等.香榧茎段离体培养再生植株的研究[J].果树学报, 2007, 24(4):477-482.LIU Hailin, CHEN Ligeng TONG Pinzhang, et al. In vitro organogenesis and plant regeneration from stem segments of Torreya grandis[J]. J Fruit Sci, 2007, 24(4):477-482.
[10]姚进,黄坚钦,胡恒康,等.香榧体细胞胚发生的初步研究[J].浙江农林大学学报, 2013, 30(1):129-135.YAO Jin, HUANG Jianqin, HU Hengkang, et al. Somatic emryogenesis of Torreya grandis ‘Merrillii’[J]. J Zhejiang A&F Univ, 2013, 30(1):129-135.
[11]项伟波,赵金凯,吴家胜,等.香榧体细胞胚发生、发育的形态与细胞学观察[J].园艺学报, 2015, 42(8):1477-1486.XIANG Weibo, ZHAO Jinkai, WU Jiasheng, et al. Morphological and cytological observation of somatic emryogenesis and development in Torreya grandis ‘Merrillii’[J]. Acta Hortic Sin, 2015, 42(8):1477-1486.
[12]SCHENK T, SCHENK R U, HILDEBRANDT A C. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures[J]. Can J Bot, 1972, 50(1):199-204.
[13]盛德策,李凤兰,刘忠华.植物体细胞胚发生的细胞生物学研究进展[J].西北植物学报, 2008, 28(1):204SHENG Dece, LI Fenglan, LIU Zhonghua. Advances in the cytobiology of plant somatic embryogenesis[J]. Acta Bot Boreal-Occident Sin, 2008, 28(1):204-215.
[14]STASOLLA C, YEUNG E C. Recent advances in conifer somatic embryogenesis:improving somatic embryo quality[J]. Plant Cell Tissue Organ Cult, 2003, 74(1):15-35.
[15]NISKANEN A, LU J, SEITZ S, et al. Effect of parent genotype on somatic embryogenesis in Scots pine(Pinus sylvestris)[J]. Tress Physiol, 2004, 24(11):1259-1265.
[16]LARA-CHAVEZ A, FLINN B S, EGERTSDOTTER U. Initiation of somatic embryogenesis from immature zygotic embryos of Oocarpa pine(Pinus oocarpa Schiede ex Schlectendal)[J]. Tree Physiol, 31(5):539-554.
[17]PULLMAN G S, CHOPRA R, CHASE K M. Loblolly pine(Pinus taeda L.)somatic embryogenesis:improvements in embryogenic tissue initiation by supplementation of medium with organic acids, vitamins B12 and E[J]. Plant Sci,2006, 170(3):648-658.
[18]PULLMAN G S, ZENG X Y, COPELAND-KAMP B, et al. Conifer somatic embryogenesis:improvements by supplementation of medium with oxidation-reduction agents[J]. Tree Physiol, 2015, 35(2):1-16.
[19]齐力旺,韩一凡,李玲,等.华北落叶松体细胞胚胎发生及遗传转化实验系统的建立[J].实验生物学报,2000, 33(4):357-365.QI Liwang, HAN Yifan, LI Ling, et al. The somatic embryogenesis and establishment of transformation experi ment system in Larix principis-rupprechth[J]. Acta Biol Exp Sin, 2000, 33(4):357-365.
[20]KROGSTRUP P. Embryolike structures from cotyledons and ripe embryos of Norway spruce(Picea abies)[J]. Can J For Res, 1986, 16(3):664-668.
[21]LELU-WALTER MA, BERNIER-CARDOU M, KLIMASZEWSKA K. Clonal plant production from self-and cross-pollinated seed families of Pinus sylvestris L. through somatic embryogenesis[J]. Plant Cell Tiss Org Cult, 2008, 92(1):31-45.
[22]BECWAR M R, NAGMANI R, WANN S R. Initiation of embryogenic cultures and somatic embryo development in loblolly pine(Pinus taeda)[J]. Can J For Res, 1990, 20(6):810-817.
[23]ZHANG Qixiang, HU Hengkang, HUANG Youjun, et al. The relationship between developmental stages of zygotic embryos at explanting and embryogenic frequency on hickory(Carya cathayensis Sarg.)[J]. Sci Hortic, 2012, 139:66-70.
[24]王丽,鲍晓明,黄百渠,等.香雪兰外植体形态学极性决定的体细胞胚胎发生[J].植物学报, 1998, 40(2):138-143.WANG Li, BAO Xiaoming, HUANG Baiqu, et al. Somatic embryogenic potential determined by the morphological polarity of the explant in tissue cultures of Freesia refracta[J]. Acta Bot Sin, 1998, 40(2):138-143.
[25]王颖,刘春朝,陈秀兰. ABA促进针叶树体细胞胚胎分化[J].植物生理学通讯, 2002, 3(6):273-278.WANG Ying, LIU Chunzhao, CHEN Xiulan. Somatic embryogenesis in conifers by ABA[J]. Plant Physiol Commun,2002, 3(6):273-278.
[26]林鹿,傅家瑞. ABA对花生胚离体发育的调节[J].中国油料, 1996, 18(1):4-7.LIN Lu, FU Jiarui. Regulation by ABA on development of peanut embryos in vitro[J]. Chin Oil Sci, 1996, 18(1):4-7.
[27]IRAQI D, TREMBLAY F M. The role of sucrose during maturation of black spruce(Picea mariana)and white spruce(Picea glauca)somatic embryos[J]. Physiol Plant, 2001, 111:381-388.
[2]ARNOLD S, SABALA I, BOZHKOV P, et al. Developmental pathways of somatic embryogenesis[J]. Plant Cell, Tissue Organ Cult, 2002, 69(3):233-249.
[3]GUAN Yuan, LI Shuigen, FAN Xiaofen, et al. Application of somatic embryogenesis in woody plants[J]. Front Plant Sci, 2016, 7:938. doi:10.3389/fpls. 2016. 00938.
[4]NIC-CAN G I, GALAZ-VALOS R M, DE-LA-PE譙A C, et al. Somatic embryogenesis:identified factors that lead to embryogenic repression:a case of speciesof the same genus[J]. PLoS One, 2015. doi:10.1371/journal. pone. 0126414.
[5]汤浩茹,王永清,任正隆.核桃体细胞胚发生与转基因研究进展[J].林业科学, 2000, 36(3):102-110.TANG Haoru, WANG Yongqing, REN Zhenglong. An overview of progress on somatic emrbyogenesis and transformation in walnut[J]. Sci Silv Sin, 2000, 36(3):102-110.
[6]刘淑芳,续海红.不同发育期梨胚的再生能力[J].江苏农业科学, 2012, 40(7):63-64.LI Shufang, XU Haihong. Regeneration frequency of embryo in different developmental stages[J]. Jiangsu Agric Sci,2012, 40(7):63-64.
[7]黎章矩,戴文圣.中国香榧[M].北京:科学出版社, 2007.
[8]姜新兵,陈力耕,何新华.香榧体细胞胚发生的研究[J].园艺学报, 2004, 31(5):654-656.JIANG Xinbing, CHEN Ligeng, HE Xinhua. Studies on the somatic embryogenesis of Torreya grandis[J]. Acta Hortic Sin, 2004, 31(5):654-656.
[9]刘海琳,陈立耕,童品璋,等.香榧茎段离体培养再生植株的研究[J].果树学报, 2007, 24(4):477-482.LIU Hailin, CHEN Ligeng TONG Pinzhang, et al. In vitro organogenesis and plant regeneration from stem segments of Torreya grandis[J]. J Fruit Sci, 2007, 24(4):477-482.
[10]姚进,黄坚钦,胡恒康,等.香榧体细胞胚发生的初步研究[J].浙江农林大学学报, 2013, 30(1):129-135.YAO Jin, HUANG Jianqin, HU Hengkang, et al. Somatic emryogenesis of Torreya grandis ‘Merrillii’[J]. J Zhejiang A&F Univ, 2013, 30(1):129-135.
[11]项伟波,赵金凯,吴家胜,等.香榧体细胞胚发生、发育的形态与细胞学观察[J].园艺学报, 2015, 42(8):1477-1486.XIANG Weibo, ZHAO Jinkai, WU Jiasheng, et al. Morphological and cytological observation of somatic emryogenesis and development in Torreya grandis ‘Merrillii’[J]. Acta Hortic Sin, 2015, 42(8):1477-1486.
[12]SCHENK T, SCHENK R U, HILDEBRANDT A C. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures[J]. Can J Bot, 1972, 50(1):199-204.
[13]盛德策,李凤兰,刘忠华.植物体细胞胚发生的细胞生物学研究进展[J].西北植物学报, 2008, 28(1):204SHENG Dece, LI Fenglan, LIU Zhonghua. Advances in the cytobiology of plant somatic embryogenesis[J]. Acta Bot Boreal-Occident Sin, 2008, 28(1):204-215.
[14]STASOLLA C, YEUNG E C. Recent advances in conifer somatic embryogenesis:improving somatic embryo quality[J]. Plant Cell Tissue Organ Cult, 2003, 74(1):15-35.
[15]NISKANEN A, LU J, SEITZ S, et al. Effect of parent genotype on somatic embryogenesis in Scots pine(Pinus sylvestris)[J]. Tress Physiol, 2004, 24(11):1259-1265.
[16]LARA-CHAVEZ A, FLINN B S, EGERTSDOTTER U. Initiation of somatic embryogenesis from immature zygotic embryos of Oocarpa pine(Pinus oocarpa Schiede ex Schlectendal)[J]. Tree Physiol, 31(5):539-554.
[17]PULLMAN G S, CHOPRA R, CHASE K M. Loblolly pine(Pinus taeda L.)somatic embryogenesis:improvements in embryogenic tissue initiation by supplementation of medium with organic acids, vitamins B12 and E[J]. Plant Sci,2006, 170(3):648-658.
[18]PULLMAN G S, ZENG X Y, COPELAND-KAMP B, et al. Conifer somatic embryogenesis:improvements by supplementation of medium with oxidation-reduction agents[J]. Tree Physiol, 2015, 35(2):1-16.
[19]齐力旺,韩一凡,李玲,等.华北落叶松体细胞胚胎发生及遗传转化实验系统的建立[J].实验生物学报,2000, 33(4):357-365.QI Liwang, HAN Yifan, LI Ling, et al. The somatic embryogenesis and establishment of transformation experi ment system in Larix principis-rupprechth[J]. Acta Biol Exp Sin, 2000, 33(4):357-365.
[20]KROGSTRUP P. Embryolike structures from cotyledons and ripe embryos of Norway spruce(Picea abies)[J]. Can J For Res, 1986, 16(3):664-668.
[21]LELU-WALTER MA, BERNIER-CARDOU M, KLIMASZEWSKA K. Clonal plant production from self-and cross-pollinated seed families of Pinus sylvestris L. through somatic embryogenesis[J]. Plant Cell Tiss Org Cult, 2008, 92(1):31-45.
[22]BECWAR M R, NAGMANI R, WANN S R. Initiation of embryogenic cultures and somatic embryo development in loblolly pine(Pinus taeda)[J]. Can J For Res, 1990, 20(6):810-817.
[23]ZHANG Qixiang, HU Hengkang, HUANG Youjun, et al. The relationship between developmental stages of zygotic embryos at explanting and embryogenic frequency on hickory(Carya cathayensis Sarg.)[J]. Sci Hortic, 2012, 139:66-70.
[24]王丽,鲍晓明,黄百渠,等.香雪兰外植体形态学极性决定的体细胞胚胎发生[J].植物学报, 1998, 40(2):138-143.WANG Li, BAO Xiaoming, HUANG Baiqu, et al. Somatic embryogenic potential determined by the morphological polarity of the explant in tissue cultures of Freesia refracta[J]. Acta Bot Sin, 1998, 40(2):138-143.
[25]王颖,刘春朝,陈秀兰. ABA促进针叶树体细胞胚胎分化[J].植物生理学通讯, 2002, 3(6):273-278.WANG Ying, LIU Chunzhao, CHEN Xiulan. Somatic embryogenesis in conifers by ABA[J]. Plant Physiol Commun,2002, 3(6):273-278.
[26]林鹿,傅家瑞. ABA对花生胚离体发育的调节[J].中国油料, 1996, 18(1):4-7.LIN Lu, FU Jiarui. Regulation by ABA on development of peanut embryos in vitro[J]. Chin Oil Sci, 1996, 18(1):4-7.
[27]IRAQI D, TREMBLAY F M. The role of sucrose during maturation of black spruce(Picea mariana)and white spruce(Picea glauca)somatic embryos[J]. Physiol Plant, 2001, 111:381-388.