甘露醇和蔗糖对菊花低温离体保存的影响
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摘要
离体保存技术在植物种质资源保存方面具有独特优势和重要意义。为改善菊花离体保存技术,本研究以4个不同生长势的菊花品种为试验材料,在7±2℃条件下研究渗透调节物质甘露醇和蔗糖对不同菊花品种离体保存的影响,观察统计不同浓度蔗糖和甘露醇处理下菊花离体保存试管苗不同阶段的存活率和绿叶数,并对保存后试管苗的叶和茎段的组织结构、恢复生长能力和遗传稳定性进行了观察和鉴定。结果表明,保存12个月后,在MS培养基中添加15 g·L~(-1)甘露醇处理对南农橙乒乓和小洋菊试管苗的离体保存效果最佳,存活率分别达86.67%和93.33%;20 g·L~(-1)甘露醇处理对蒙娜丽莎黄和橙安娜保存效果最优,存活率分别达到80.00%和93.33%。60 g·L~(-1)蔗糖处理,南农橙乒乓和小洋菊保存12个月存活率均达到70%以上,但相比甘露醇处理,其绿叶数少、生长状况差;高浓度蔗糖(45～90 g·L~(-1))对蒙娜丽莎黄和橙安娜保存效果不佳。保存12个月后,20 g·L~(-1)甘露醇处理的蒙娜丽莎黄和橙安娜、15 g·L~(-1)甘露醇处理的小洋菊和南农橙兵兵的叶片和茎段细胞间隙减小,细胞密度增加,试管苗恢复正常培养45 d后不同品种菊花生长良好,株高、叶片数、茎粗、节间长等形态指标及SSR分子标记图谱与对照相比无显著差异,保持了良好的遗传稳定性。本研究为菊花种质资源的低温离体保存提供了理论依据与技术支撑。
In vitro techniques for conservation plant germplasm resources have unique advantages and important significance. To improve the conservation technology in chrysanthemum in vitro, four chrysanthemum cultivars with different growth potentials were as materials. The effects of osmotic substance mannitol and sucrose on chrysanthemum in vitro conversation were investigated at 7±2℃ in the present study. The survival rate and number of green leaves of plantlets treated with different concentrations of sucrose and mannitol were observed and counted at different stages. The histological structure, restoring growth ability and genetic stability of the leaf and stem segments were observed and identified. The results showed that MS medium with 15 g·L~(-1) mannitol had the best effects on Nannong Chengpingpang and Xiao yangju in vitro conservation with the survival rates of 86.67% and 93.33% after 12 months, respectively. The MS medium with 20 g·L~(-1) mannitol was the best for Monalisa Yellow and Anastasia Orange in vitro conservation, and the survival rate was 80.00% and 93.33%, respectively. The survival rate of Nannong Chengpingpang and Xiaoyangju were higher than 70% in MS medium with 60 g·L~(-1) sucrose, but the plantlets had less green leaves and were poor growth status compared with mannitol treatments. Supplement with high concentrations sucrose(45～90 g·L~(-1)) in MS medium didn′t work well for Monalisa Yellow and Anastasia Orange. The cell gap of leaves and stem segments of the 4 varieties, which Monalisa Yellow and Anastasia Orange treated by 20 g·L~(-1) mannitol and Xiao yangju and Nannong Chengpingpang treated by 15 g·L~(-1) mannitol, decreased and the cell density increased after 12 months in vitro conservation. The plantlets grew normally on normal condition after 45 days in vitro conservation. The regenerated plantlets height, number of leaf, stem diameter, internode length and SSR molecular marker profiles of vegenerated plantlets showed no significant difference from the control, indicated in vitro conservation kept the genetic stability. The results of this study provided a theoretical foundation and technical support for in vitro conservation of chrysanthemum.
In vitro techniques for conservation plant germplasm resources have unique advantages and important significance. To improve the conservation technology in chrysanthemum in vitro, four chrysanthemum cultivars with different growth potentials were as materials. The effects of osmotic substance mannitol and sucrose on chrysanthemum in vitro conversation were investigated at 7±2℃ in the present study. The survival rate and number of green leaves of plantlets treated with different concentrations of sucrose and mannitol were observed and counted at different stages. The histological structure, restoring growth ability and genetic stability of the leaf and stem segments were observed and identified. The results showed that MS medium with 15 g·L~(-1) mannitol had the best effects on Nannong Chengpingpang and Xiao yangju in vitro conservation with the survival rates of 86.67% and 93.33% after 12 months, respectively. The MS medium with 20 g·L~(-1) mannitol was the best for Monalisa Yellow and Anastasia Orange in vitro conservation, and the survival rate was 80.00% and 93.33%, respectively. The survival rate of Nannong Chengpingpang and Xiaoyangju were higher than 70% in MS medium with 60 g·L~(-1) sucrose, but the plantlets had less green leaves and were poor growth status compared with mannitol treatments. Supplement with high concentrations sucrose(45～90 g·L~(-1)) in MS medium didn′t work well for Monalisa Yellow and Anastasia Orange. The cell gap of leaves and stem segments of the 4 varieties, which Monalisa Yellow and Anastasia Orange treated by 20 g·L~(-1) mannitol and Xiao yangju and Nannong Chengpingpang treated by 15 g·L~(-1) mannitol, decreased and the cell density increased after 12 months in vitro conservation. The plantlets grew normally on normal condition after 45 days in vitro conservation. The regenerated plantlets height, number of leaf, stem diameter, internode length and SSR molecular marker profiles of vegenerated plantlets showed no significant difference from the control, indicated in vitro conservation kept the genetic stability. The results of this study provided a theoretical foundation and technical support for in vitro conservation of chrysanthemum.
引文
[1] 李辛雷, 陈发棣. 菊花种质资源与遗传改良研究进展[J]. 植物学通报, 2004, 21(4): 392-401
[2] 王小光, 房伟民, 陈发棣, 滕年军. 切花小菊 41 个品种花粉量的测定与散粉特性分析[J]. 园艺学报, 2013, 40(4): 703-712
[3] 伊华林, 邓秀新. 植物种质离体保存技术研究进展[J]. 植物学通报, 1999, 16(5): 574-581
[4] 韩丽, 张金梅, 卢新雄, 王玲, 陈晓玲. 菊芋耐性胁迫及种质保存研究进展[J]. 植物遗传资源学报, 2014, 15(5):999-1005
[5] 王艳芳. 菊花缓慢生长离体保存研究[D]. 南京: 南京农业大学, 2007
[6] 王蒂. 植物组织培养[M]. 北京:中国农业出版社, 2005:198-207
[7] Bekheet S A, Matter M A, Taha H S, El-Ashry A A. In vitro conservation of jojoba (Simmondsia chinensis) shootlet cultures using osmotic stress and low temperature[J]. Middle East Journal of Agriculture Research, 2016,5(4):396-492
[8] Jain A, Hussain Z, Yadav R P, Bhardwaj R. Effect of carbendazim on in vitro conservation and genetic stability assessment in Curcuma longa. and Zingiber officinale[J]. Journal of Herbs Spices & Medicinal Plants, 2018,24(1):1-13
[9] 张志勇, 齐泽民, 黄作喜. 铁皮石斛生物技术研究进展[J]. 核农学报, 2014, 28(4):605-610
[10] 尹明华, 洪森荣. 黄独脱毒苗种质离体保存及其遗传稳定性和病毒变化的初步研究[J]. 植物研究, 2011, 31(5):579-584
[11] 慈惠婷, 汪阳, 周玉丽. 薄荷试管苗的离体保存[J]. 安徽科技学院学报, 2017, 31(4):39-44
[12] Trejgell A, Kamińska M, Tretyn A. In vitro slow growth storage of Senecio macrophyllus shoots[J]. Acta Physiologiae Plantarum, 2015, 37(11):234-243
[13] Budiarto K. Alternative in vitro media for medium-term conservation of chrysanthemum (Dendranthema grandiflora Tvelve)[J]. Bumi Lestari, 2009, 9(1):48-53
[14] 王艳芳, 房伟民, 陈发棣, 赵宏波, 刘灶长. ‘神马’菊花的离体保存及遗传稳定性[J]. 西北植物学报, 2007, 27(7): 1341-1348
[15] 王艳芳. 菊花缓慢生长离体保存研究[D]. 南京:南京农业大学, 2007
[16] 邹喻萍, 葛颂, 王晓东. 系统与进化植物学中的分子标记[M]. 北京:科学出版社, 2001: 3-9
[17] 陆敏佳, 蒋玉蓉, 陆国权, 陈国林, 毛前. 利用SSR标记分析藜麦品种的遗传多样性[J]. 核农学报, 2015, 29(2):260-269
[18] 王惠梅, 苏晓娜, 黄雪雯, 王圣子海, 熊琦哲, 江绍琳, 江绍玫, 吴建利. 利用SSR分析华中、华东五大湖区野生菰的遗传结构与多样性[J]. 核农学报, 2018,32(4):654-664
[19] Roussos P A. Growth and biochemical responses of jojoba [Simmondsia chinensis (Link) Schneid] explants cultured under mannitol-simulated drought stress in vitro[J]. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 2013, 147(2):272-284
[20] Holobiuc Ⅰ. Somatic Embryogenesis in Long-term Cultures of Gentiana lutea L. in the Presence of Osmotic Stress: The Gentianaceae-Volume 2: Biotechnology and Applications[M]. Berlin Heidelberg: Springer, 2015:139-161
[21] Thakur S, Tiwari K L, Jadhav S K. In vitro, approaches for conservation of Asparagus racemosus Willd[J]. In Vitro Cellular & Developmental Biology-Plant, 2015, 51(6):619-625
[22] 牛爱国, 张开春, 张晓明, 闫国华, 李文生, 姜立杰. 樱桃种质资源试管苗保存方法[J]. 园艺学报, 2005, 32(2): 298-300
[23] 兰伟, 徐培培, 许树成. 香青兰的离体保存研究[J]. 热带作物学报, 2013, 34(4):675-680
[24] 叶秀仙, 黄敏玲, 樊荣辉, 吴建设, 钟淮钦. 蝴蝶兰离体保存及其遗传稳定性研究[J]. 福建农业学报, 2014, 29(10):976-981
[25] 何克勤, 程晓紊, 胡能兵,周玉丽, 宋桂成, 崔广荣. 甜叶菊种质离体保存后的DNA甲基化研究[J]. 中药材, 2016, 39(10):2194-2197
[26] 林如. 福建柑桔种质试管苗离体保存及其生理与解剖学研究[D]. 福州:福建农林大学, 2006
[27] 叶秀仙, 黄敏玲, 吴建设. 甘露醇和生长抑制剂对文心兰离体保存的影响[J]. 福建农业学报, 2011, 26(1): 76-82
[28] 徐志微, 杨丽丽, 杜国强, 师校欣. 培养基渗透压和生长调节剂对葡萄种质离体保存的效应[J]. 分子植物育种, 2014, 12(4):720-725
[29] Gomes F, Clemente M, Figueiredo P, Plactio F, Machodo H, Santos C, Costa R L. Castanea spp. hybrid clones in vitro conservation: synthetic seeds vs. slow growth storage[J]. Acta Horticulturae, 2017(1155):37-44
[30] Chauhan R, Keshavkant S, Jadhav S K, Quraishi A. In vitro slow-growth storage of Chlorophytum borivilianum Sant. et Fernand: a critically endangered herb[J]. In Vitro Cellular & Developmental Biology-Plant, 2016, 52(3):315-321
[31] Vettorazzi R G, Carvalho Ⅴ S, Sudré C P, Rodrigues R. Developing an in vitro optimized protocol to sweet potato landraces conservation[J]. Acta Scientiarum-Agronomy, 2017, 39(3):359
[32] 兰伟, 陈发棣. 纪伊潮菊离体保存及其遗传稳定性分析[J]. 西北植物学报, 2010, 30(3): 487-494
[33] Sintim H, Akromah R. Differing sucrose requirements for in-vitro conservation of cassava genotypes[J]. International Journal of Plant & Soil Science, 2015, 7(1):45-54
[34] Tahtamouni R, Shibli R, Al-Abdallat A, Al-Qudah T. Analysis of growth, oil yield, and carvacrol in Thymbra spicata L.after slow-growth conservation[J]. Turkish Journal of Agriculture & Forestry, 2016(40):213-221
[35] El-Bahr M K, El-Hamid A A, Matter M A, Shaltout A, Bekheet S A, EI-Ashry A A. In vitro conservation of embryogenic cultures of date palm using osmotic mediated growth agents[J]. Journal of Genetic Engineering & Biotechnology, 2016, 14(2):363-370
[36] 傅伊倩, 孔滢, 刘燕. 大花卷丹的组织培养及限制生长保存[J]. 植物生理学报, 2012, 48(3): 277-281
[37] 陈辉, 陈晓玲, 陈龙清, 卢新雄. 百合种质资源限制生长法保存研究[J]. 园艺学报, 2006, 33(4): 789-793
[38] 兰伟, 陈素梅, 尹冬梅, 陈发棣. 那贺川野菊的离体保存[J]. 园艺学报, 2010, 37(12): 2007-2016
[39] 潘学军, 李霞, 张文娥. 离体保存对野生毛葡萄试管苗生长及叶片抗氧化酶活性的影响[J]. 果树学报, 2013(4): 615-620
[2] 王小光, 房伟民, 陈发棣, 滕年军. 切花小菊 41 个品种花粉量的测定与散粉特性分析[J]. 园艺学报, 2013, 40(4): 703-712
[3] 伊华林, 邓秀新. 植物种质离体保存技术研究进展[J]. 植物学通报, 1999, 16(5): 574-581
[4] 韩丽, 张金梅, 卢新雄, 王玲, 陈晓玲. 菊芋耐性胁迫及种质保存研究进展[J]. 植物遗传资源学报, 2014, 15(5):999-1005
[5] 王艳芳. 菊花缓慢生长离体保存研究[D]. 南京: 南京农业大学, 2007
[6] 王蒂. 植物组织培养[M]. 北京:中国农业出版社, 2005:198-207
[7] Bekheet S A, Matter M A, Taha H S, El-Ashry A A. In vitro conservation of jojoba (Simmondsia chinensis) shootlet cultures using osmotic stress and low temperature[J]. Middle East Journal of Agriculture Research, 2016,5(4):396-492
[8] Jain A, Hussain Z, Yadav R P, Bhardwaj R. Effect of carbendazim on in vitro conservation and genetic stability assessment in Curcuma longa. and Zingiber officinale[J]. Journal of Herbs Spices & Medicinal Plants, 2018,24(1):1-13
[9] 张志勇, 齐泽民, 黄作喜. 铁皮石斛生物技术研究进展[J]. 核农学报, 2014, 28(4):605-610
[10] 尹明华, 洪森荣. 黄独脱毒苗种质离体保存及其遗传稳定性和病毒变化的初步研究[J]. 植物研究, 2011, 31(5):579-584
[11] 慈惠婷, 汪阳, 周玉丽. 薄荷试管苗的离体保存[J]. 安徽科技学院学报, 2017, 31(4):39-44
[12] Trejgell A, Kamińska M, Tretyn A. In vitro slow growth storage of Senecio macrophyllus shoots[J]. Acta Physiologiae Plantarum, 2015, 37(11):234-243
[13] Budiarto K. Alternative in vitro media for medium-term conservation of chrysanthemum (Dendranthema grandiflora Tvelve)[J]. Bumi Lestari, 2009, 9(1):48-53
[14] 王艳芳, 房伟民, 陈发棣, 赵宏波, 刘灶长. ‘神马’菊花的离体保存及遗传稳定性[J]. 西北植物学报, 2007, 27(7): 1341-1348
[15] 王艳芳. 菊花缓慢生长离体保存研究[D]. 南京:南京农业大学, 2007
[16] 邹喻萍, 葛颂, 王晓东. 系统与进化植物学中的分子标记[M]. 北京:科学出版社, 2001: 3-9
[17] 陆敏佳, 蒋玉蓉, 陆国权, 陈国林, 毛前. 利用SSR标记分析藜麦品种的遗传多样性[J]. 核农学报, 2015, 29(2):260-269
[18] 王惠梅, 苏晓娜, 黄雪雯, 王圣子海, 熊琦哲, 江绍琳, 江绍玫, 吴建利. 利用SSR分析华中、华东五大湖区野生菰的遗传结构与多样性[J]. 核农学报, 2018,32(4):654-664
[19] Roussos P A. Growth and biochemical responses of jojoba [Simmondsia chinensis (Link) Schneid] explants cultured under mannitol-simulated drought stress in vitro[J]. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 2013, 147(2):272-284
[20] Holobiuc Ⅰ. Somatic Embryogenesis in Long-term Cultures of Gentiana lutea L. in the Presence of Osmotic Stress: The Gentianaceae-Volume 2: Biotechnology and Applications[M]. Berlin Heidelberg: Springer, 2015:139-161
[21] Thakur S, Tiwari K L, Jadhav S K. In vitro, approaches for conservation of Asparagus racemosus Willd[J]. In Vitro Cellular & Developmental Biology-Plant, 2015, 51(6):619-625
[22] 牛爱国, 张开春, 张晓明, 闫国华, 李文生, 姜立杰. 樱桃种质资源试管苗保存方法[J]. 园艺学报, 2005, 32(2): 298-300
[23] 兰伟, 徐培培, 许树成. 香青兰的离体保存研究[J]. 热带作物学报, 2013, 34(4):675-680
[24] 叶秀仙, 黄敏玲, 樊荣辉, 吴建设, 钟淮钦. 蝴蝶兰离体保存及其遗传稳定性研究[J]. 福建农业学报, 2014, 29(10):976-981
[25] 何克勤, 程晓紊, 胡能兵,周玉丽, 宋桂成, 崔广荣. 甜叶菊种质离体保存后的DNA甲基化研究[J]. 中药材, 2016, 39(10):2194-2197
[26] 林如. 福建柑桔种质试管苗离体保存及其生理与解剖学研究[D]. 福州:福建农林大学, 2006
[27] 叶秀仙, 黄敏玲, 吴建设. 甘露醇和生长抑制剂对文心兰离体保存的影响[J]. 福建农业学报, 2011, 26(1): 76-82
[28] 徐志微, 杨丽丽, 杜国强, 师校欣. 培养基渗透压和生长调节剂对葡萄种质离体保存的效应[J]. 分子植物育种, 2014, 12(4):720-725
[29] Gomes F, Clemente M, Figueiredo P, Plactio F, Machodo H, Santos C, Costa R L. Castanea spp. hybrid clones in vitro conservation: synthetic seeds vs. slow growth storage[J]. Acta Horticulturae, 2017(1155):37-44
[30] Chauhan R, Keshavkant S, Jadhav S K, Quraishi A. In vitro slow-growth storage of Chlorophytum borivilianum Sant. et Fernand: a critically endangered herb[J]. In Vitro Cellular & Developmental Biology-Plant, 2016, 52(3):315-321
[31] Vettorazzi R G, Carvalho Ⅴ S, Sudré C P, Rodrigues R. Developing an in vitro optimized protocol to sweet potato landraces conservation[J]. Acta Scientiarum-Agronomy, 2017, 39(3):359
[32] 兰伟, 陈发棣. 纪伊潮菊离体保存及其遗传稳定性分析[J]. 西北植物学报, 2010, 30(3): 487-494
[33] Sintim H, Akromah R. Differing sucrose requirements for in-vitro conservation of cassava genotypes[J]. International Journal of Plant & Soil Science, 2015, 7(1):45-54
[34] Tahtamouni R, Shibli R, Al-Abdallat A, Al-Qudah T. Analysis of growth, oil yield, and carvacrol in Thymbra spicata L.after slow-growth conservation[J]. Turkish Journal of Agriculture & Forestry, 2016(40):213-221
[35] El-Bahr M K, El-Hamid A A, Matter M A, Shaltout A, Bekheet S A, EI-Ashry A A. In vitro conservation of embryogenic cultures of date palm using osmotic mediated growth agents[J]. Journal of Genetic Engineering & Biotechnology, 2016, 14(2):363-370
[36] 傅伊倩, 孔滢, 刘燕. 大花卷丹的组织培养及限制生长保存[J]. 植物生理学报, 2012, 48(3): 277-281
[37] 陈辉, 陈晓玲, 陈龙清, 卢新雄. 百合种质资源限制生长法保存研究[J]. 园艺学报, 2006, 33(4): 789-793
[38] 兰伟, 陈素梅, 尹冬梅, 陈发棣. 那贺川野菊的离体保存[J]. 园艺学报, 2010, 37(12): 2007-2016
[39] 潘学军, 李霞, 张文娥. 离体保存对野生毛葡萄试管苗生长及叶片抗氧化酶活性的影响[J]. 果树学报, 2013(4): 615-620