滇东南濒危植物长梗杜鹃的花粉形态及其特性研究
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摘要
[目的]对滇东南特有濒危植物长梗杜鹃花粉形态及其特性进行系统研究,为长梗杜鹃进一步的遗传育种、花粉种质保存和传粉生物学等相关保护利用提供一些科学依据,并为系统分类研究作旁证。[方法]利用光学显微镜和扫描电子显微镜对长梗杜鹃花朵不同发育时期(花蕾膨大期、花瓣初开期和花瓣盛开期)的花粉形态进行了观察;通过TTC染色法对开花当天不同时段及开花后不同天数的花粉活力进行了快速检测;做L25(53)正交试验探讨10 g·L-1琼脂和不同浓度的蔗糖、H3BO3、Ca Cl2组成的培养基配方对长梗杜鹃花粉萌发的影响。同时,研究了其花粉在不同贮藏温度下活力随贮藏时间的变化情况。[结果]表明:从花蕾膨大期到花瓣盛开期,花粉逐渐成熟,为四合体花粉,呈正四面体排列,四合体直径为43.0 65.4μm,平均51.3μm;单粒花粉近球形且表面有粘丝,具三孔沟。在扫描电镜下,花粉粒外壁为均匀的颗粒状,孔沟周围颗粒细密呈波状突起;在光镜下,花粉粒外壁为内外2层,厚度约相等为0.8 1.2μm。长梗杜鹃整个单花花期花粉活力较强,于开花当天9:00达最高,为92.18%,至花末期(花后第9天)仍保持着48.5%的活力,适宜做父本开展相关杂交育种工作。长梗杜鹃开花当天9:00的花粉在10 g·L-1琼脂+100 g·L-1蔗糖+200 mg·L-1H3BO3+0 mg·L-1Ca Cl2的培养基中萌发率最高,达90.26%;极差分析表明,影响长梗杜鹃花粉萌发率高低的主次顺序为蔗糖→Ca Cl2→H3BO3,一定浓度的蔗糖和H3BO3均起到了很好的促萌效果,而Ca Cl2的添加则显著抑制了其花粉萌发。此外,适宜的低温有利于长梗杜鹃花粉的贮藏,-18℃条件下贮藏48 d仍有一定的萌发力。[结论]长梗杜鹃特殊的花粉外壁纹饰支持了前人对其系统位置划分的合理性。其较高的花粉活力、适宜的固体萌发培养基及贮藏温度的研究为今后以长梗杜鹃为父本的杂交育种提供了可靠的理论依据,对其他同属植物花粉形态及特性研究具有重要的指导意义。
[Objective]To provide some references for the studies on genetic breeding,pollen germplasm preservation and pollination biology of Rhododendron longipedicellatum by investigating the pollen grains morphology and its characteristics. [Method] The pollen grains of R. longipedicellatum at different development stages including alabastrum intumescence stage,petal loose stage and petal full bloom stage were observed by light microscope(LM)and scanning electron microscope(SEM). The pollen viability in the process of flowering was detected by TTC staining method. The effects of 10 g·L-1 agar and different concentrations of sucrose,H3 BO3 and Ca Cl2 on R. longi-pedicellatum pollen grains germination were studied by L25(53) orthogonal test. Simultaneously,the authors also studied the changes of pollen germination rate under different storage temperature. [Result]The pollen grains mature gradually from the alabastrum intumescence to the petal full bloom stage,and are arranged in tetrahedral tetrad.The tetrad diameter ranges from 43. 0 μm to 65. 4 μm with an average 51. 3 μm. A single pollen is subspheroidal with tricolporate aperture,and viscin threads were seen on the pollen grains surface. The pollen grains exine sculpture is uniform granular in SEM,and the granulate around the colpus are more compact. Exine is composed with two layers in LM. The pollen viability maintained a high level over single flowering period,which was the highest(92. 18%) at 9: 00 on the first day of anthesis,and still remained relatively high(48. 5%) until the end of flowering time(on the 9 days after flowering). The highest germination rate of R. longipedicellatum pollen grains at 9: 00 on the first day of anthesis was 90. 26% when cultured in the medium for 10 g·L-1 agar + 100 g·L-1 sucrose +200 mg·L-1 H3 BO3+ 0 mg·L-1 Ca Cl2. Range analysis showed that the important order of influence on pollen germination rate was sucrose,Ca Cl2 and H3 BO3,a certain concentration of sucrose and H3 BO3 had a good promote sprouting effect,while the addition of Ca Cl2 significantly inhibited the pollen grains germination. Furthermore,the appropriate low temperature was helpful to storage of R. longipedicellatum pollen,the germination rate maintained a certain level after 48 days storage with-18℃. [Conclusion]The special pollen grain exine sculpture of R. longipedicellatum supports the rationality of the division of its systematic position by predecessors. These findings of high pollen viability,suitable solid germination medium and storage temperature provide a reliable theoretical basis for hybridization breeding of R. longipedicellatum as the male parent.
[Objective]To provide some references for the studies on genetic breeding,pollen germplasm preservation and pollination biology of Rhododendron longipedicellatum by investigating the pollen grains morphology and its characteristics. [Method] The pollen grains of R. longipedicellatum at different development stages including alabastrum intumescence stage,petal loose stage and petal full bloom stage were observed by light microscope(LM)and scanning electron microscope(SEM). The pollen viability in the process of flowering was detected by TTC staining method. The effects of 10 g·L-1 agar and different concentrations of sucrose,H3 BO3 and Ca Cl2 on R. longi-pedicellatum pollen grains germination were studied by L25(53) orthogonal test. Simultaneously,the authors also studied the changes of pollen germination rate under different storage temperature. [Result]The pollen grains mature gradually from the alabastrum intumescence to the petal full bloom stage,and are arranged in tetrahedral tetrad.The tetrad diameter ranges from 43. 0 μm to 65. 4 μm with an average 51. 3 μm. A single pollen is subspheroidal with tricolporate aperture,and viscin threads were seen on the pollen grains surface. The pollen grains exine sculpture is uniform granular in SEM,and the granulate around the colpus are more compact. Exine is composed with two layers in LM. The pollen viability maintained a high level over single flowering period,which was the highest(92. 18%) at 9: 00 on the first day of anthesis,and still remained relatively high(48. 5%) until the end of flowering time(on the 9 days after flowering). The highest germination rate of R. longipedicellatum pollen grains at 9: 00 on the first day of anthesis was 90. 26% when cultured in the medium for 10 g·L-1 agar + 100 g·L-1 sucrose +200 mg·L-1 H3 BO3+ 0 mg·L-1 Ca Cl2. Range analysis showed that the important order of influence on pollen germination rate was sucrose,Ca Cl2 and H3 BO3,a certain concentration of sucrose and H3 BO3 had a good promote sprouting effect,while the addition of Ca Cl2 significantly inhibited the pollen grains germination. Furthermore,the appropriate low temperature was helpful to storage of R. longipedicellatum pollen,the germination rate maintained a certain level after 48 days storage with-18℃. [Conclusion]The special pollen grain exine sculpture of R. longipedicellatum supports the rationality of the division of its systematic position by predecessors. These findings of high pollen viability,suitable solid germination medium and storage temperature provide a reliable theoretical basis for hybridization breeding of R. longipedicellatum as the male parent.
引文
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[3]Fang M Y,Fang R Z,He M Y,et al.Ericaceae[M]//Wu Z Y,Raven P H,Hong D Y.Flora of China.Beijing:Science Press&St.louis:Missouri Botanical Garden,2015,14:260-445.
[4]Cai L,Neilsen J,Dao Z L,et al.Rhododendron longipedicellatum(Ericaceae),a new species from Southeastern Yunnan,China[J].Phytotaxa,2016,282(4):296-300.
[5]Wasson E,Rodd T.Tree&shrubs[M].Beijing:China Forestry Publishing House,2004:612-627.
[6]张长芹,高连明,薛润光,等.中国杜鹃花的保育现状和展望[J].广西科学,2004,11(4):354-359.
[7]兰熙,张乐华,张金政,等.杜鹃花属植物育种研究进展[J].园艺学报,2012,39(9):1829-1838.
[8]李太强,刘雄芳,万友名,等.滇东南濒危植物长梗杜鹃转录组微卫星特征分析[J].林业科学研究,2017,30(4):533-541.
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[11]张艳红.中国杜鹃花属植物分类的研究进展[J].辽东学院学报:自然科学版,2011,18(3):198-202.
[12]张超仪,耿兴敏.六种杜鹃花属植物花粉活力测定方法的比较研究[J].植物科学学报,2012,30(1):92-99.
[13]李玉萍,陈堃,王燕青,等.测定杜鹃花花粉萌发力的液体培养基和贮藏方法研究[J].金陵科技学院学报,2012,28(3):56-61.
[14]陆琳,彭绿春,宋杰,等.不同高山杜鹃品种花粉活力测定及贮藏方法研究[J].山西农业科学,2016,44(2):175-178.
[15]郭红艳,王森,晏巢,等.华中五味子的雄花特征与花粉特性[J].林业科学,2016,52(3):112-120.
[16]Wang Y G,Fritsch P W,Shi S H,et al.Phylogeny and infrageneric classification of Symplocos(Symplocaceae)inferred from DNA sequence data[J].American Journal of Botany,2004,91(11):1901-1914.
[17]毛子军,杨永富,侯丽君.东北杜鹃花属植物花粉形态的研究[J].植物研究,2000,20(1):58-62,126-131.
[18]高连明,张长芹,李德铢,等.杜鹃花族花粉形态及其系统学意义[J].云南植物研究,2002,24(4):471-482.
[19]高连明,张长芹,李德铢,等.杜鹃属马银花亚属花粉形态的研究[J].武汉植物学研究,2002,20(3):177-181,245-247.
[20]王玉国,李光照,漆小雪,等.杜鹃花属植物花粉形态及其分类学意义[J].广西植物,2006,26(2):113-119.
[21]Cullen J.A revision of Rhododendron.I.Subgenus Rhododendron sections Rhododendron and Pogonanthum[J].Notes from the Royal Botanic Garden Edinburgh,1980,39(1):207.
[22]周凌瑜,刘群录,邵邻相.虫媒花与风媒花花粉形态的比较[J].上海交通大学学报:农业科学版,2008,26(3):177-182.
[23]刘林德,张萍,张丽,等.锦带花的花粉活力、柱头可授性及传粉者的观察[J].西北植物学报,2004,24(8):1431-1434.
[24]胡适宜.被子植物胚胎学[M].北京:人民教育出版社,1982:51-58.
[25]张敬丽.杜鹃花属迷人杜鹃的自然杂交起源研究[D].北京:中科院研究生院,2007.
[26]白天,关文灵,宋杰,等.锈叶杜鹃的开花特性及繁育系统研究[J].西部林业科学,2014,43(1):47-53.
[27]田晓玲.大喇叭杜鹃繁殖生物学研究[D].南京:南京林业大学,2011.
[28]张双双,奚晓军,张金凤.黑杨花粉活力和柱头可授性研究[J].安徽农业科学,2010,38(20):10550-10552.
[29]林锐,彭绿春,李世峰,等.常绿杜鹃品种‘XXL’的花粉活力及柱头可授性观察[J].西部林业科学,2016,45(6):115-120.
[30]Pierson E S,Li Y Q,Zhang H Q,et al.Pulsatory growth of pollen tubes:investigation of a possible relationship with the periodic distribution of cell wall components[J].Acta Botanica Neerlandica,1995,44(2):121-128.
[31]卜志国,杜绍华,张晓曼.映山红开花习性与花粉生活力研究[J].安徽农业科学,2011,39(8):4562-4563,4686.
[32]Fernando D D,Lazzaro M D,Owens J N.Growth and development of conifer pollen tubes[J].Sexual Plant Reproduction,2005,18(4):149-162.
[33]左丹丹,明军,刘春,等.植物花粉生活力检测技术进展[J].安徽农业科学,2007,35(16):4742-4745.
[34]姚成义,赵洁.钙和硼对蓝猪耳花粉萌发及花粉管生长的影响[J].武汉植物学研究,2004,22(1):1-7.
[35]Nepi M,Franchi G G,Padni E.Pollen hydration status at dispersal:cytophysiological features and strategies[J].Protoplasma,2001,216(3-4):171-180.
[2]Chamberlain D,Hyam R,Argent G,et al.The Genus Rhododendron:Its Classification and Synonymy[M].Edinburgh:Royal Botanic Garden Edinburgh,1996:181.
[3]Fang M Y,Fang R Z,He M Y,et al.Ericaceae[M]//Wu Z Y,Raven P H,Hong D Y.Flora of China.Beijing:Science Press&St.louis:Missouri Botanical Garden,2015,14:260-445.
[4]Cai L,Neilsen J,Dao Z L,et al.Rhododendron longipedicellatum(Ericaceae),a new species from Southeastern Yunnan,China[J].Phytotaxa,2016,282(4):296-300.
[5]Wasson E,Rodd T.Tree&shrubs[M].Beijing:China Forestry Publishing House,2004:612-627.
[6]张长芹,高连明,薛润光,等.中国杜鹃花的保育现状和展望[J].广西科学,2004,11(4):354-359.
[7]兰熙,张乐华,张金政,等.杜鹃花属植物育种研究进展[J].园艺学报,2012,39(9):1829-1838.
[8]李太强,刘雄芳,万友名,等.滇东南濒危植物长梗杜鹃转录组微卫星特征分析[J].林业科学研究,2017,30(4):533-541.
[9]IUCN.IUCN Red List Categories and Criteria:version 3.1[M].Gland,Switzerland and Cambridge,United Kingdom:IUCN Species Survival Commission,2001:30.
[10]周兰英,王永清,张丽.26种杜鹃属植物花粉形态及分类学研究[J].林业科学,2008,44(2):55-63.
[11]张艳红.中国杜鹃花属植物分类的研究进展[J].辽东学院学报:自然科学版,2011,18(3):198-202.
[12]张超仪,耿兴敏.六种杜鹃花属植物花粉活力测定方法的比较研究[J].植物科学学报,2012,30(1):92-99.
[13]李玉萍,陈堃,王燕青,等.测定杜鹃花花粉萌发力的液体培养基和贮藏方法研究[J].金陵科技学院学报,2012,28(3):56-61.
[14]陆琳,彭绿春,宋杰,等.不同高山杜鹃品种花粉活力测定及贮藏方法研究[J].山西农业科学,2016,44(2):175-178.
[15]郭红艳,王森,晏巢,等.华中五味子的雄花特征与花粉特性[J].林业科学,2016,52(3):112-120.
[16]Wang Y G,Fritsch P W,Shi S H,et al.Phylogeny and infrageneric classification of Symplocos(Symplocaceae)inferred from DNA sequence data[J].American Journal of Botany,2004,91(11):1901-1914.
[17]毛子军,杨永富,侯丽君.东北杜鹃花属植物花粉形态的研究[J].植物研究,2000,20(1):58-62,126-131.
[18]高连明,张长芹,李德铢,等.杜鹃花族花粉形态及其系统学意义[J].云南植物研究,2002,24(4):471-482.
[19]高连明,张长芹,李德铢,等.杜鹃属马银花亚属花粉形态的研究[J].武汉植物学研究,2002,20(3):177-181,245-247.
[20]王玉国,李光照,漆小雪,等.杜鹃花属植物花粉形态及其分类学意义[J].广西植物,2006,26(2):113-119.
[21]Cullen J.A revision of Rhododendron.I.Subgenus Rhododendron sections Rhododendron and Pogonanthum[J].Notes from the Royal Botanic Garden Edinburgh,1980,39(1):207.
[22]周凌瑜,刘群录,邵邻相.虫媒花与风媒花花粉形态的比较[J].上海交通大学学报:农业科学版,2008,26(3):177-182.
[23]刘林德,张萍,张丽,等.锦带花的花粉活力、柱头可授性及传粉者的观察[J].西北植物学报,2004,24(8):1431-1434.
[24]胡适宜.被子植物胚胎学[M].北京:人民教育出版社,1982:51-58.
[25]张敬丽.杜鹃花属迷人杜鹃的自然杂交起源研究[D].北京:中科院研究生院,2007.
[26]白天,关文灵,宋杰,等.锈叶杜鹃的开花特性及繁育系统研究[J].西部林业科学,2014,43(1):47-53.
[27]田晓玲.大喇叭杜鹃繁殖生物学研究[D].南京:南京林业大学,2011.
[28]张双双,奚晓军,张金凤.黑杨花粉活力和柱头可授性研究[J].安徽农业科学,2010,38(20):10550-10552.
[29]林锐,彭绿春,李世峰,等.常绿杜鹃品种‘XXL’的花粉活力及柱头可授性观察[J].西部林业科学,2016,45(6):115-120.
[30]Pierson E S,Li Y Q,Zhang H Q,et al.Pulsatory growth of pollen tubes:investigation of a possible relationship with the periodic distribution of cell wall components[J].Acta Botanica Neerlandica,1995,44(2):121-128.
[31]卜志国,杜绍华,张晓曼.映山红开花习性与花粉生活力研究[J].安徽农业科学,2011,39(8):4562-4563,4686.
[32]Fernando D D,Lazzaro M D,Owens J N.Growth and development of conifer pollen tubes[J].Sexual Plant Reproduction,2005,18(4):149-162.
[33]左丹丹,明军,刘春,等.植物花粉生活力检测技术进展[J].安徽农业科学,2007,35(16):4742-4745.
[34]姚成义,赵洁.钙和硼对蓝猪耳花粉萌发及花粉管生长的影响[J].武汉植物学研究,2004,22(1):1-7.
[35]Nepi M,Franchi G G,Padni E.Pollen hydration status at dispersal:cytophysiological features and strategies[J].Protoplasma,2001,216(3-4):171-180.