花楸花粉超低温保存及再生
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摘要
【目的】探讨花楸(Sorbus pohuashanensis)花粉超低温保存及再生的方法,以延长其花粉保存时间,为科研、生产提供理论和实践依据。【方法】分别设置不同培养基配方、不同光照、不同采集时间处理,进行花楸花粉离体萌发试验,测定各试验处理的花粉萌发率;设置不同冷冻方式和化冻方式处理,测定两种方式不同处理的花粉萌发率;制备不同含水率的花粉,分别测定其在超低温保存前、后的萌发率。【结果】(1)培养基为100g·L~(-1)蔗糖+100 mg·L~(-1)硼酸的处理,花粉萌发率最高,达72.42%;(2)黑暗处理花粉萌发率为77.24%,比光照处理高8.96个百分点;(3)一天中,10∶00和14∶00采集的花粉,其活力显著高于其他时间采集的花粉;(4)-20℃预冷冻培养30min后投入液氮保存24h的处理效果最好,花粉萌发率达43.15%;(5)超低温保存后40℃水浴解冻5min处理比其他解冻方式处理的花粉活力高,萌发率为35.70%;(6)花楸花粉超低温保存最适含水率为7.72%,此含水率下花粉超低温保存后萌发率最高,为31.53%。【结论】花楸花粉采用超低温技术保存24h后仍具有活力,黑暗条件、花粉采集时间、花粉萌发培养基、花粉含水率、冷冻与化冻方式均会对花楸花粉超低温保存效果产生显著影响。
【Objective】Cryopreservation and regeneration of pollens of Sorbus pohuashanensis were investigatied.【Method】An in vitro method was developed with experiments on medium formulation,light exposure,and pollen collection for the pollen germination rate determination.The germination rates of S.pohuashanensis pollens treated by various freezing and thawing procedures and different moisture contents before and after the treatments were compared.【Result】(1)The medium for optimum pollen germination was found to consist of sucrose 100 g·L~(-1) and boric acid 100 mg·L~(-1) that delivered a rate of 72.42%.(2)Incubation in darkness yielded a germination rate of 77.24%,which was 8.96 percentage point higher than that under light exposure.(3)The vitality of pollens collected at 10 a.m.or 2 p.m.was significantly higher than other time in a day.(4)Among all cryopreservation methods tested,pre-freezing pollens at-20℃for 30 min prior to submerging in liquid nitrogen for 24 hprovided the greatest germination rate of 43.15%after thawing.(5)For thawing,soaking the frozen pollens in a 40℃ water bath for 5 min rendered the highest germination rate of 35.70%.(6)Moisture content of the pollens for the cryopreservation was determined to be 7.72% to result in the best germination rate of 31.53% after thawing.【Conclusion】The cryopreservation conditions for S.pohuashanensis pollens as established would result in high viability and germination rate after storage.
【Objective】Cryopreservation and regeneration of pollens of Sorbus pohuashanensis were investigatied.【Method】An in vitro method was developed with experiments on medium formulation,light exposure,and pollen collection for the pollen germination rate determination.The germination rates of S.pohuashanensis pollens treated by various freezing and thawing procedures and different moisture contents before and after the treatments were compared.【Result】(1)The medium for optimum pollen germination was found to consist of sucrose 100 g·L~(-1) and boric acid 100 mg·L~(-1) that delivered a rate of 72.42%.(2)Incubation in darkness yielded a germination rate of 77.24%,which was 8.96 percentage point higher than that under light exposure.(3)The vitality of pollens collected at 10 a.m.or 2 p.m.was significantly higher than other time in a day.(4)Among all cryopreservation methods tested,pre-freezing pollens at-20℃for 30 min prior to submerging in liquid nitrogen for 24 hprovided the greatest germination rate of 43.15%after thawing.(5)For thawing,soaking the frozen pollens in a 40℃ water bath for 5 min rendered the highest germination rate of 35.70%.(6)Moisture content of the pollens for the cryopreservation was determined to be 7.72% to result in the best germination rate of 31.53% after thawing.【Conclusion】The cryopreservation conditions for S.pohuashanensis pollens as established would result in high viability and germination rate after storage.
引文
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[13]BREWBAKER J,MAJUMDER S K.Cultural studies of the pollen population effect and the self-incompatibility inhibition[J].American Journal of Botany,1961,48(6):457-464.
[14]徐进,陈天华,王章荣,等.不同贮藏方法及光照对马尾松花粉活力的影响[J].南京林业大学学报,1998(3):74-77.XU J,CHEN T H,WANG Z R,et al.Effects of different storage methods and light on pollen viability of Pinus massoniana[J]Journal of Nanjing Forestry University,1998(3):74-77.(in Chinese)
[15]彭向永,刘俊祥,李振坚,等.蒿柳花粉离体萌发及花粉管生长特性[J].北京林业大学学报,2017,39(3):81-86.PENG X Y,LIU J X,LI Z J,et al.Characteristics of pollen germination and pollen tube growth in Salix viminalis in vitro[J].Journal Of Beijing Forestry University,2017,39(3):81-86.(in Chinese)
[16]王伟伟,姚玲,何开跃,等.不同柳树花粉离体培养及活力测定方法比较研究[J].江苏林业科技,2018,45(4):1-7.WANG W W,YAO L,HE K Y,et al.Comparative study on in vitro pollen germination and viability evaluation of different willow species[J].Journal of Jiangsu Forestry Science &Technology,2018,45(4):1-7.(in Chinese)
[17]张洪伟,段一凡,李稚,等.不同贮藏方法对桂花花粉活力影响的研究[J].南京林业大学学报(自然科学版),2014,38(S1):7-12.ZHANG H W,DUAN Y F,LI Z,et al.Study on effects of different storage methods on pollen vitality of Osmanthus fragrans[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2014,38(S1):7-12.(in Chinese)
[18]李稚.桂花传粉生物学与杂交育种研究[D].南京:南京林业大学,2014.LI Z.Pollination biology and cross breeding of Osmanthus fragrans Lour[D].Nanjing:Nanjing Forestry University,2014.(in Chinese)
[19]常海龙,张垂明,张伟,等.甘蔗花粉活力和柱头可授性日变化研究[J].广东农业科学,2017,44(4):14-18.CHANG H L,ZHANG C M,ZHANG W,et al.Study on diumal variation of sugarcane′s pollen vitality and stigma energy[J].Guangdon Agricultura Sciences,2017,44(4):14-18.(in Chinese)
[20]崔光芬,杜文文,段青,等.不同倍性亚洲百合的花粉形态与活力变化规律分析[J].华北农学报,2015,30(S1):55-60.CUI G F,DU W W,DUAN Q,et al.Analysis on pollen morphology and variation of viability of liliumasiatic hybrids with different ploidy[J].Acta Agriculturae Boreali-Sinica,2015,30(S1):55-60.(in Chinese)
[21]赵剑颖,张华丽,张西西,等.万寿菊花粉活力及柱头可授性研究[J].中国农学通报,2012,28(19):159-163.ZHAO J Y,ZHANG L H,ZHANG X X,et al.Pollen viability and stigma receptivity of Tageteserecta[J].Chinese Agricultural Science Bulletin,2012,28(19):159-163.(in Chinese)
[22]时婷婷,张桂玲,温四民,等.百合花粉超低温保存方法研究[J].北方园艺,2013(3):64-66.SHI T T, ZHANG G L, WEN S M,et al. Pollen cryopreservation of Lilium[J].Northern Horticulture,2013(3):64-66.(in Chinese)
[23]王玉萍,张峰,王蒂.马铃薯花粉的超低温保存研究[J].园艺学报,2003(6):683-686.WANG Y P,ZHANG F, WANG D.Study on pollen cryopreservation of Solanum tuberosum L.[J].Acta HorticulturaeSinica,2003(6):683-686.(in Chinese)
[2]KNOWLTON H E.Studies in pollen with special reference to longevity[J].Cornell Univ Agr Exp Sta Mem,1922,52:751-793.
[3]李秉玲,王荷,刘燕.日本牡丹品种花粉超低温保存[J].北京林业大学学报,2010,32(4):297-300.LI B L,WANG H,LIU Y.Pollen cryopreservation of Japanese tree peony cultivars[J]. Journal of Beijing Forestry University,2010,32(4):297-300.(in Chinese)
[4]刘芊,徐瑾,石印,等.丁香属12种植物花粉保存方法研究[J].浙江农业学报,2014,26(3):615-620.LIU Q,XU J,SHI Y,et al.Storage methods of 12species of lilac(Syringa L.)pollen[J]. Acta Agriculturae Zhejiangensis,2014,26(3):615-620.(in Chinese)
[5]常维霞,姚小华,龙伟.山茶属3种植物花药超低温保存研究[J].中国油料作物学报,2016,38(1):52-57.CHANG W X,YAO X H,LONG W.Anther cryopreservation of 3 Camellia species[J].Chinese Journal of Oil Crop Sciences,2016,38(1):52-57.(in Chinese)
[6]李万义,孔鑫,韩亚洁,等.桃叶卫矛花粉超低温保存[J].江苏农业科学,2018,46(10):144-148.LI W Y,KONG X,HAN Y J,et al.Pollen cryopreservation of Euonymus maackii Rupr.[J].Jiangsu Agricuitural Sciences,2018,46(10):144-148.(in Chinese)
[7]胡适宜.植物胚胎学实验方法(一)花粉生活力的测定[J].植物学通报,1993,10(2):60-62.HU S Y.Experimental methods in plant embryology(I)Determination of pollen viability[J].Chinese Bulletin of Botany,1993,10(2):60-62.(in Chinese)
[8]刘婷婷,代其林,奉斌,等.甘蓝型油菜花粉超低温保存及其花粉活力的研究[J].生物技术通报,2010(3):114-118.LIU T T,DAI Q L,FENG B,et al.Studies on the activity of pollens in Brassica napus L.cryopreserved under ultralow temperature[J].Biotechnology Bulletin,2010(3):114-118.(in Chinese)
[9]姜忠国,董丹,张琪,等.玉蝉花花粉生活力检测和超低温保存研究[J].安徽农业科学,2016,44(1):216-219,222.JIANG Z G,DONG D,ZHANG Q,et al.The viability detection and pollen cryopreservation of Iris ensata Thunb[J].Journal of Anhui Agri,2016,44(1):216-219,222.(in Chinese)
[10]王小敏,李海燕,吴文龙,等.蓬蘽花粉低温贮藏研究[J].江苏农业科学,2010(3):170-171.WANG X M,LI H Y,WU W L,et al.Study on storage of Rubus hirsutus Thunb.pollen at low temperature[J].Jiangsu Agricuitural Sciences,2010(3):170-171.(in Chinese)
[11]潘瑞炽.植物生理学[M].北京:高等教育出版社,2001:260.PAN R Z. Plant Physiology[M]. Beijing:Higher Education Press,2001:260.(in Chinese)
[12]杨晓冬,孙素琴,李一勤.硼缺乏导致花粉管细胞壁多糖分布的改变[J].植物学报,1999(11):1169-1176.YANG X D,Sun S Q,Li Y Q.Boron deficiency causes Changes in the distribution of Major polysaccharides of pollen tube wall[J].Acta Botanica Sinica,1999(11):1169-1176.(in Chinese)
[13]BREWBAKER J,MAJUMDER S K.Cultural studies of the pollen population effect and the self-incompatibility inhibition[J].American Journal of Botany,1961,48(6):457-464.
[14]徐进,陈天华,王章荣,等.不同贮藏方法及光照对马尾松花粉活力的影响[J].南京林业大学学报,1998(3):74-77.XU J,CHEN T H,WANG Z R,et al.Effects of different storage methods and light on pollen viability of Pinus massoniana[J]Journal of Nanjing Forestry University,1998(3):74-77.(in Chinese)
[15]彭向永,刘俊祥,李振坚,等.蒿柳花粉离体萌发及花粉管生长特性[J].北京林业大学学报,2017,39(3):81-86.PENG X Y,LIU J X,LI Z J,et al.Characteristics of pollen germination and pollen tube growth in Salix viminalis in vitro[J].Journal Of Beijing Forestry University,2017,39(3):81-86.(in Chinese)
[16]王伟伟,姚玲,何开跃,等.不同柳树花粉离体培养及活力测定方法比较研究[J].江苏林业科技,2018,45(4):1-7.WANG W W,YAO L,HE K Y,et al.Comparative study on in vitro pollen germination and viability evaluation of different willow species[J].Journal of Jiangsu Forestry Science &Technology,2018,45(4):1-7.(in Chinese)
[17]张洪伟,段一凡,李稚,等.不同贮藏方法对桂花花粉活力影响的研究[J].南京林业大学学报(自然科学版),2014,38(S1):7-12.ZHANG H W,DUAN Y F,LI Z,et al.Study on effects of different storage methods on pollen vitality of Osmanthus fragrans[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2014,38(S1):7-12.(in Chinese)
[18]李稚.桂花传粉生物学与杂交育种研究[D].南京:南京林业大学,2014.LI Z.Pollination biology and cross breeding of Osmanthus fragrans Lour[D].Nanjing:Nanjing Forestry University,2014.(in Chinese)
[19]常海龙,张垂明,张伟,等.甘蔗花粉活力和柱头可授性日变化研究[J].广东农业科学,2017,44(4):14-18.CHANG H L,ZHANG C M,ZHANG W,et al.Study on diumal variation of sugarcane′s pollen vitality and stigma energy[J].Guangdon Agricultura Sciences,2017,44(4):14-18.(in Chinese)
[20]崔光芬,杜文文,段青,等.不同倍性亚洲百合的花粉形态与活力变化规律分析[J].华北农学报,2015,30(S1):55-60.CUI G F,DU W W,DUAN Q,et al.Analysis on pollen morphology and variation of viability of liliumasiatic hybrids with different ploidy[J].Acta Agriculturae Boreali-Sinica,2015,30(S1):55-60.(in Chinese)
[21]赵剑颖,张华丽,张西西,等.万寿菊花粉活力及柱头可授性研究[J].中国农学通报,2012,28(19):159-163.ZHAO J Y,ZHANG L H,ZHANG X X,et al.Pollen viability and stigma receptivity of Tageteserecta[J].Chinese Agricultural Science Bulletin,2012,28(19):159-163.(in Chinese)
[22]时婷婷,张桂玲,温四民,等.百合花粉超低温保存方法研究[J].北方园艺,2013(3):64-66.SHI T T, ZHANG G L, WEN S M,et al. Pollen cryopreservation of Lilium[J].Northern Horticulture,2013(3):64-66.(in Chinese)
[23]王玉萍,张峰,王蒂.马铃薯花粉的超低温保存研究[J].园艺学报,2003(6):683-686.WANG Y P,ZHANG F, WANG D.Study on pollen cryopreservation of Solanum tuberosum L.[J].Acta HorticulturaeSinica,2003(6):683-686.(in Chinese)