茶壶枣离体多倍体诱导关键技术研究
|
摘要
【目的】离体多倍体育种是枣树育种的重要方法,对其关键技术的研究有利于建立和完善枣树离体多倍体育种体系,加快枣树多倍体种质的创制。【方法】以茶壶枣无菌叶片为材料,将预培养不同天数(7～20 d)的叶片固定后制成石蜡切片,进行细胞学观察以确定适宜的预培养时间。在此基础上,将叶片不定芽再生技术与秋水仙素染色体加倍技术相结合,在叶片预培养8、9、10、11 d后,分别用70和90 mg/L的秋水仙素处理48和72 h,并对处理后得到的不定芽进行流式细胞术倍性检测。【结果】通过对叶片最佳预培养时间的细胞学研究可以得出,茶壶枣无菌叶片在预培养8～9 d时,分生细胞的分裂能力最强,且未出现分化现象,在此时期用秋水仙素对叶片进行处理,可大大提高诱导率,并且可以有效避免嵌合体和混倍体的出现。染色体加倍试验发现,在预培养8 d后,用70 mg/L的秋水仙素处理72 h,其诱导率最高,达4.11%,并成功获得8株多倍体植株。通过流式细胞术确定这8株多倍体均为纯合四倍体。【结论】本研究探索出了将茶壶枣叶片不定芽再生体系与秋水仙素染色体加倍技术相结合,人工诱导茶壶枣多倍体的技术方法。
[Objective] In vitro polyploid breeding is an important method for jujube breeding. The study of its key technologies is conducive to the establishment and improvement of polyploid breeding system and the creation of polyploid germplasm of jujube. [Method] In this study, the leaves of Ziziphus jujuba Mill.cv. ‘Teapot' were used as materials, and the leaves of different days(7-20 day) were fixed and then made into paraffin sections for cytological observation. On this basis, the leaf adventitious bud regeneration technology was combined with the colchicine chromosome doubling technique. After the leaves were precultured for 8, 9, 10 and 11 days, they were treated with 70 mg/L and 90 mg/L colchicine for 48 and 72 hours,and the flow cytometry ploidy test was performed on the adventitious buds obtained after treatment.[Result] Through the cytological study on the optimal pre-culture time of the leaves, it can be concluded that the meristematic cells have the strongest division ability and no differentiation during the pre-culture of8-9 days. During this period, treatment of leaves with colchicine can greatly increase the induction rate and effectively avoid the appearance of chimeras and mixoploids. The chromosome doubling test found that after8 days of pre-culture, the induction rate was up to 4.11% after treatment with 70 mg/L colchicine for 72 hours, and 8 polyploid plants were successfully obtained. It was confirmed by flow cytometry that these 8 polyploids were pure tetraploids. [Conclusion] This study explores the technical method of artificially inducing polyploidy of Ziziphus jujuba Mill. cv. ‘Teapot' by adventitious bud regeneration technology and colchicine chromosome doubling technique.
[Objective] In vitro polyploid breeding is an important method for jujube breeding. The study of its key technologies is conducive to the establishment and improvement of polyploid breeding system and the creation of polyploid germplasm of jujube. [Method] In this study, the leaves of Ziziphus jujuba Mill.cv. ‘Teapot' were used as materials, and the leaves of different days(7-20 day) were fixed and then made into paraffin sections for cytological observation. On this basis, the leaf adventitious bud regeneration technology was combined with the colchicine chromosome doubling technique. After the leaves were precultured for 8, 9, 10 and 11 days, they were treated with 70 mg/L and 90 mg/L colchicine for 48 and 72 hours,and the flow cytometry ploidy test was performed on the adventitious buds obtained after treatment.[Result] Through the cytological study on the optimal pre-culture time of the leaves, it can be concluded that the meristematic cells have the strongest division ability and no differentiation during the pre-culture of8-9 days. During this period, treatment of leaves with colchicine can greatly increase the induction rate and effectively avoid the appearance of chimeras and mixoploids. The chromosome doubling test found that after8 days of pre-culture, the induction rate was up to 4.11% after treatment with 70 mg/L colchicine for 72 hours, and 8 polyploid plants were successfully obtained. It was confirmed by flow cytometry that these 8 polyploids were pure tetraploids. [Conclusion] This study explores the technical method of artificially inducing polyploidy of Ziziphus jujuba Mill. cv. ‘Teapot' by adventitious bud regeneration technology and colchicine chromosome doubling technique.
引文
[1]郭裕新,单公华.中国枣[M].上海:上海科学技术出版社,2010.Guo Y X,Shan G H.The Chinese jujube[M].Shanghai:Shanghai Scientific and Technical Publishers,2010.
[2]Jin X.Jujuba:Ziziphus jujuba[M].Ceará:Exotic Fruits,2018:263-269.
[3]Zou M,Chen Y L,Sun-Waterhouse D X,et al.Immunomodulatory acidic polysaccharides from Zizyphus jujube cv.Huizao:insights into their chemical characteristics and modes of action[J].Food Chemistry,2018,258(30):35-42.
[4]Shin M,Lee B M,Kim O,et al.Triterpenoids from Ziziphus jujuba induce apoptotic cell death in human cancer cells through mitochondrial reactive oxygen species production[J].Food Function,2018,9(7):3895-3905.
[5]Ninave P B,Patil S D.Antiasthmatic potential of Zizyphus jujuba Mill.and Jujuboside B:possible role in the treatment of asthma[J].Respiratory Physiology&Neurobiology,2019,260:28-36.
[6]刘孟军,王玖瑞,刘平,等.枣树免去雄杂交育种的设计与实践[J].园艺学报,2014,41(7):1495-1502.Liu M J,Wang J R,Liu P,et al.Design and practice of emasculation-free cross breeding in Chinese jujube[J].Horticultural Plant Journal,2014,41(7):1495-1502.
[7]Wang J R,Cui X M,Dai L,et al.Hybridization using a new malesterile germplasm as the female parents in Chinese jujube[J].Korean Journal of Horticultural Science&Technology,2015,33(3):396-402.
[8]常月梅.果树多倍体鉴定进展[J].山西林业科技,2000,3(1):1-4.Chang Y M.The advances in identification of fruit tree polyploidy[J].Shanxi Forestry Science and Technology,2000,3(1):1-4.
[9]康向阳.林木多倍体育种研究进展[J].北京林业大学大学学报,2003,25(4):70-74.Kang X Y.Advances in researches on polyploid breeding of forest trees[J].Journal of Beijing Forestry University,2003,25(4):70-74.
[10]石荫坪,王强生,贾元淑,等.枣胚乳三倍体的细胞学:中国果树资源细胞学研究之六[J].武汉植物学研究,1985,3(4):390-395.Shi Y P,Wang Q S,Jia Y S,et al.Cytology of endosperm-triploid in Zizyphus sativa[J].Wuhan Botanical Research,1985,3(4):390-395.
[11]严仁玲,王震星,杨恩芹,等.金丝小枣花药离体培养再生植株研究[J].河北果树,1996,3(3):9-10.Yan R L,Wang Z X,Yang E Q,et al.Study on in vitro cultured regenerated plants of Zizyphus jujuba Mill.cv.Jinsixiaozao[J].Hebei Fruit Tree,1996,3(3):9-10.
[12]王娜,刘孟军,代丽,等.秋水仙碱离体诱导冬枣和酸枣四倍体[J].园艺学报,2005,32(6):1008-1012.Wang N,Liu M J,Dai L,et al.In vitro tetraploid induction of Ziziphus jujuba‘Dongzao’and Z.acido jujuba(Z.spinosa Hu)with colchicine[J].Horticultural Plant Journal,2005,32(6):1008-1012.
[13]Gu X F,Yang A F,Meng H,et al.In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill.cv.Zhanhua[J].Plant Cell Reports,2005,24(11):671-676.
[14]宁强,刘晓光,刘孟军.枣多倍体育种研究进展[J].河北林果研究,2009,24(3):305-308.Ning Q,Liu X G,Liu M J.Research progress in polyploid breeding of Ziziphus jujuba[J].Hebei Journal of Forestry and Orchard Research,2009,24(3):305-308.
[15]张源源.橡胶树座果规律及三倍体诱导研究[D].北京:北京林业大学,2013.Zhang Y Y.Disciplines of fruit-set success and triploid induction of Hevea brasiliensis[D].Beijing:Beijing Forestry University,2013.
[16]Cui Y H,Hou L,Li X,et al.In vitro induction of tetraploid Ziziphus jujuba Mill.var.spinosa plants from leaf explants[J].Plant Cell,Tissue and Organ Culture,2017,131(1):175-182.
[17]Chauvin J E,Label A,Kermarrec M P.In vitro chromosomedoubling in tulip(Tulipa gesneriana L.)[J].The Journal of Horticultural Science and Biotechnology,2005,80(6):693-698.
[18]Ade R,Rai M K.Review:colchicine,current advances and future prospects[J].Nusantara Bioscience,2010,2(2):90-96.
[19]Eng W H,Ho W S.Polyploidization using colchicine in horticultural plants:a review[J].Scientia Horticulturae,2019,246:604-617.
[20]马光跃,陈红玉,申仲妹,等.茶壶枣果形多样性形态学观察[J].山西农业科学,2009,37(2):9-12.Ma G Y,Chen H Y,Shen Z M,et al.Morphological observation of diversity fruit shape in‘Chahuzao’variety of Chinese jujube[J].Journal of Shanxi Agricutural Sciences,2009,37(2):9-12.
[21]金琪.枣树再生体系的建立及离体加倍的初步研究[D].北京:北京林业大学,2014.Jin Q.Preliminary study on the system of regeneration and doubling in vitro of Chinese jujube(Ziziphus jujuba Mill.)[J].Beijing:Beijing Forestry University,2014.
[22]李和平.植物显微技术[M].北京:科学出版社,2009.Li H P.Plant microscopy[M].Beijing:Science Press,2009.
[23]马春华.枣叶片再生途径的组织学研究及影响遗传转化因素的分析[D].郑州:河南农业大学,2011.Ma C H.Histological studies of leaf regeneration and factors influencing the efficiency Agrobacterium-mediated transformation in Zizyphus jujube Mill.[D].Zhengzhou:Henan Agricultural University,2011.
[24]Ye Y M,Tong J,Shi X P,et al.Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle(Lagerstroemia indica L.)[J].Scientia Horticulturae,2010,124(1):95-101.
[25]Ma C,Ye X,Chen Y,et al.Anatomical observations of adventitious bud regeneration from leaf explants of Ziziphus jujuba Mill.‘Huizao’[J].Horticulture,Environment,and Biotechnology,2012,53(4):316-319.
[26]Cai X,Kang X Y.In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag[J].Plant Cell Reports,2011,30(9):1771-1778.
[27]G?owacka K,Je?owski S,Kaczmarek Z.In vitro induction of polyploidy by colchicine treatment of shoots and preliminary characterisation of induced polyploids in two Miscanthus species[J].Industrial Crops&Products,2010,32(2):88-96.
[28]Zhang Z,Dai H Y,Xiao M,et al.In vitro induction of tetraploids in Phlox subulata L.[J].Euphytica,2008,159(1-2):59-65.
[29]V??n?l?A.Polyploidization and early screening of Rhododendron hybrids[J].Euphytica,2000,112(3):239-244.
[30]Dhooghe E,Van Laere K,Eeckhaut T,et al.Mitotic chromosome doubling of plant tissues in vitro[J].Plant Cell,Tissue and Organ Culture,2011,104(3):359-373.
[31]Kermani M J,Sarasan V,Roberts A V,et al.Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability[J].Theoretical and Applied Genetics,2003,107(7):1195-1200.
[32]Petersen K K,Hagberg P,Kristiansen K.Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis[J].Plant Cell,Tissue and Organ Culture,2003,73(2):137-146.
[33]王岩.酸枣和茶壶枣不定芽再生体系的建立及多倍体诱导技术研究[D].北京:北京林业大学,2015.Wang Y.Optimization of high efficient shoot regeneration system and polyploidy induction in Ziziphus jujuba Mill.[D].Beijing:Beijing Forestry University,2015.
[2]Jin X.Jujuba:Ziziphus jujuba[M].Ceará:Exotic Fruits,2018:263-269.
[3]Zou M,Chen Y L,Sun-Waterhouse D X,et al.Immunomodulatory acidic polysaccharides from Zizyphus jujube cv.Huizao:insights into their chemical characteristics and modes of action[J].Food Chemistry,2018,258(30):35-42.
[4]Shin M,Lee B M,Kim O,et al.Triterpenoids from Ziziphus jujuba induce apoptotic cell death in human cancer cells through mitochondrial reactive oxygen species production[J].Food Function,2018,9(7):3895-3905.
[5]Ninave P B,Patil S D.Antiasthmatic potential of Zizyphus jujuba Mill.and Jujuboside B:possible role in the treatment of asthma[J].Respiratory Physiology&Neurobiology,2019,260:28-36.
[6]刘孟军,王玖瑞,刘平,等.枣树免去雄杂交育种的设计与实践[J].园艺学报,2014,41(7):1495-1502.Liu M J,Wang J R,Liu P,et al.Design and practice of emasculation-free cross breeding in Chinese jujube[J].Horticultural Plant Journal,2014,41(7):1495-1502.
[7]Wang J R,Cui X M,Dai L,et al.Hybridization using a new malesterile germplasm as the female parents in Chinese jujube[J].Korean Journal of Horticultural Science&Technology,2015,33(3):396-402.
[8]常月梅.果树多倍体鉴定进展[J].山西林业科技,2000,3(1):1-4.Chang Y M.The advances in identification of fruit tree polyploidy[J].Shanxi Forestry Science and Technology,2000,3(1):1-4.
[9]康向阳.林木多倍体育种研究进展[J].北京林业大学大学学报,2003,25(4):70-74.Kang X Y.Advances in researches on polyploid breeding of forest trees[J].Journal of Beijing Forestry University,2003,25(4):70-74.
[10]石荫坪,王强生,贾元淑,等.枣胚乳三倍体的细胞学:中国果树资源细胞学研究之六[J].武汉植物学研究,1985,3(4):390-395.Shi Y P,Wang Q S,Jia Y S,et al.Cytology of endosperm-triploid in Zizyphus sativa[J].Wuhan Botanical Research,1985,3(4):390-395.
[11]严仁玲,王震星,杨恩芹,等.金丝小枣花药离体培养再生植株研究[J].河北果树,1996,3(3):9-10.Yan R L,Wang Z X,Yang E Q,et al.Study on in vitro cultured regenerated plants of Zizyphus jujuba Mill.cv.Jinsixiaozao[J].Hebei Fruit Tree,1996,3(3):9-10.
[12]王娜,刘孟军,代丽,等.秋水仙碱离体诱导冬枣和酸枣四倍体[J].园艺学报,2005,32(6):1008-1012.Wang N,Liu M J,Dai L,et al.In vitro tetraploid induction of Ziziphus jujuba‘Dongzao’and Z.acido jujuba(Z.spinosa Hu)with colchicine[J].Horticultural Plant Journal,2005,32(6):1008-1012.
[13]Gu X F,Yang A F,Meng H,et al.In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill.cv.Zhanhua[J].Plant Cell Reports,2005,24(11):671-676.
[14]宁强,刘晓光,刘孟军.枣多倍体育种研究进展[J].河北林果研究,2009,24(3):305-308.Ning Q,Liu X G,Liu M J.Research progress in polyploid breeding of Ziziphus jujuba[J].Hebei Journal of Forestry and Orchard Research,2009,24(3):305-308.
[15]张源源.橡胶树座果规律及三倍体诱导研究[D].北京:北京林业大学,2013.Zhang Y Y.Disciplines of fruit-set success and triploid induction of Hevea brasiliensis[D].Beijing:Beijing Forestry University,2013.
[16]Cui Y H,Hou L,Li X,et al.In vitro induction of tetraploid Ziziphus jujuba Mill.var.spinosa plants from leaf explants[J].Plant Cell,Tissue and Organ Culture,2017,131(1):175-182.
[17]Chauvin J E,Label A,Kermarrec M P.In vitro chromosomedoubling in tulip(Tulipa gesneriana L.)[J].The Journal of Horticultural Science and Biotechnology,2005,80(6):693-698.
[18]Ade R,Rai M K.Review:colchicine,current advances and future prospects[J].Nusantara Bioscience,2010,2(2):90-96.
[19]Eng W H,Ho W S.Polyploidization using colchicine in horticultural plants:a review[J].Scientia Horticulturae,2019,246:604-617.
[20]马光跃,陈红玉,申仲妹,等.茶壶枣果形多样性形态学观察[J].山西农业科学,2009,37(2):9-12.Ma G Y,Chen H Y,Shen Z M,et al.Morphological observation of diversity fruit shape in‘Chahuzao’variety of Chinese jujube[J].Journal of Shanxi Agricutural Sciences,2009,37(2):9-12.
[21]金琪.枣树再生体系的建立及离体加倍的初步研究[D].北京:北京林业大学,2014.Jin Q.Preliminary study on the system of regeneration and doubling in vitro of Chinese jujube(Ziziphus jujuba Mill.)[J].Beijing:Beijing Forestry University,2014.
[22]李和平.植物显微技术[M].北京:科学出版社,2009.Li H P.Plant microscopy[M].Beijing:Science Press,2009.
[23]马春华.枣叶片再生途径的组织学研究及影响遗传转化因素的分析[D].郑州:河南农业大学,2011.Ma C H.Histological studies of leaf regeneration and factors influencing the efficiency Agrobacterium-mediated transformation in Zizyphus jujube Mill.[D].Zhengzhou:Henan Agricultural University,2011.
[24]Ye Y M,Tong J,Shi X P,et al.Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle(Lagerstroemia indica L.)[J].Scientia Horticulturae,2010,124(1):95-101.
[25]Ma C,Ye X,Chen Y,et al.Anatomical observations of adventitious bud regeneration from leaf explants of Ziziphus jujuba Mill.‘Huizao’[J].Horticulture,Environment,and Biotechnology,2012,53(4):316-319.
[26]Cai X,Kang X Y.In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag[J].Plant Cell Reports,2011,30(9):1771-1778.
[27]G?owacka K,Je?owski S,Kaczmarek Z.In vitro induction of polyploidy by colchicine treatment of shoots and preliminary characterisation of induced polyploids in two Miscanthus species[J].Industrial Crops&Products,2010,32(2):88-96.
[28]Zhang Z,Dai H Y,Xiao M,et al.In vitro induction of tetraploids in Phlox subulata L.[J].Euphytica,2008,159(1-2):59-65.
[29]V??n?l?A.Polyploidization and early screening of Rhododendron hybrids[J].Euphytica,2000,112(3):239-244.
[30]Dhooghe E,Van Laere K,Eeckhaut T,et al.Mitotic chromosome doubling of plant tissues in vitro[J].Plant Cell,Tissue and Organ Culture,2011,104(3):359-373.
[31]Kermani M J,Sarasan V,Roberts A V,et al.Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability[J].Theoretical and Applied Genetics,2003,107(7):1195-1200.
[32]Petersen K K,Hagberg P,Kristiansen K.Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis[J].Plant Cell,Tissue and Organ Culture,2003,73(2):137-146.
[33]王岩.酸枣和茶壶枣不定芽再生体系的建立及多倍体诱导技术研究[D].北京:北京林业大学,2015.Wang Y.Optimization of high efficient shoot regeneration system and polyploidy induction in Ziziphus jujuba Mill.[D].Beijing:Beijing Forestry University,2015.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |