穿心莲花粉活力测定及离体萌发特性研究
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摘要
【目的】以穿心莲花粉为实验材料,明确穿心莲小花供体生长阶段、采集时间与穿心莲花粉活力之间的关系,建立穿心莲花粉体外萌发适宜的培养方法。【方法】采用碘(I2)—碘化钾(KI)染色法、红四氮唑(TTC)染色法、花粉管离体萌发法测定不同生长时期和采集时间的穿心莲花粉活力;采用离体培养法研究不同培养基组分和预处理对花粉管萌发的影响。【结果】I2-KI染色法结果显示,15:00采集的药丝比为1∶4的花粉染色率最高;TTC染色法结果显示,15:00采集的药丝比为1∶6的花粉染色率最高;花粉管萌发法测定结果显示,花粉活力随药丝比的减少而增高,其趋势与TTC法测定结果相同。穿心莲花粉离体萌发适宜培养基组合为100 g/L蔗糖+0.3 g/L氯化钙+1 g/L硼酸+150 g/L PEG6000,萌发率为82.5%。预处理条件对穿心莲花粉离体萌发有显著影响:4℃冷处理24 h能显著提高花粉萌发率(P <0.05);4℃冷处理48 h与30℃热处理24 h的穿心莲花粉管萌发率与对照组比较,均无显著性差异(P> 0.05);48 h内,随着热处理(30℃)时间的增长,花粉管萌发率降低。【结论】该研究可为穿心莲的杂交育种、花期调控等研究提供可靠的科学依据和技术资料,加速育种进程以及完善种质资源保存。
Objective With Andrographis paniculata pollen as the test materials,the relationship of Andrographis paniculata pollen viability with the growth stages and harvest time for Andrographis paniculata flowers was clarified,thus to establish an appropriate cultivation method for in vitro germination of Andrographis paniculata pollen. Methods We used iodine(I2)-potassium iodide(KI)staining method, 2,3,5-triphenyltetrazolium chloride(TTC) staining method, and in vitro pollen tube germination method to determine the viability of Andrographis paniculata pollen during different growth periods and at different harvest time. In vitro culture method was adopted to study the effects of the medium components and various pretreatments on the growth of pollen tube.Results I2-KI staining results showed that the staining rate of pollen with the ratio of anthers to filaments being 1∶4 harvested at 15:00 was the highest;TTC staining results showed that the staining rate of pollen with the ratio of anthers to filaments being 1∶6 harvested at 15:00 was the highest. The pollen tube germination test showed that the pollen viability was increased with the decrease of the ratio of anthers to filaments,the trend being the same as what showed by TTC method. The optimal medium for Andrographis paniculata pollen germination was the mixture consisting of 100 g/L of sucrose,0.3 g/L calcium chloride,1.0 g/L boric acid,and 150 g/L PEG6000,and the germination rate arrived at 82.5% under the above conditions. The pretreatment conditions had significant effects on the pollen germination in vitro:cold treatment for 24 h at 4 ℃ significantly increased the pollen germination rate(P < 0.05);the pollen tube growth rate in the group of cold treatment for 48 h at 4 ℃ and group of heat treatment for 24 h at 30 ℃ was no significantly different from that in the control group(P > 0.05);within 48 h,the pollentube growth rate was decreased with the prolongation of heat treatment time. Conclusion The results can provide reliable scientific basis and technical data for cross breeding and the regulation of flowering time for Andrographis paniculata(Burm. f.)Nees, and will accelerate breeding process and improve the conservation of germplasm resources.
Objective With Andrographis paniculata pollen as the test materials,the relationship of Andrographis paniculata pollen viability with the growth stages and harvest time for Andrographis paniculata flowers was clarified,thus to establish an appropriate cultivation method for in vitro germination of Andrographis paniculata pollen. Methods We used iodine(I2)-potassium iodide(KI)staining method, 2,3,5-triphenyltetrazolium chloride(TTC) staining method, and in vitro pollen tube germination method to determine the viability of Andrographis paniculata pollen during different growth periods and at different harvest time. In vitro culture method was adopted to study the effects of the medium components and various pretreatments on the growth of pollen tube.Results I2-KI staining results showed that the staining rate of pollen with the ratio of anthers to filaments being 1∶4 harvested at 15:00 was the highest;TTC staining results showed that the staining rate of pollen with the ratio of anthers to filaments being 1∶6 harvested at 15:00 was the highest. The pollen tube germination test showed that the pollen viability was increased with the decrease of the ratio of anthers to filaments,the trend being the same as what showed by TTC method. The optimal medium for Andrographis paniculata pollen germination was the mixture consisting of 100 g/L of sucrose,0.3 g/L calcium chloride,1.0 g/L boric acid,and 150 g/L PEG6000,and the germination rate arrived at 82.5% under the above conditions. The pretreatment conditions had significant effects on the pollen germination in vitro:cold treatment for 24 h at 4 ℃ significantly increased the pollen germination rate(P < 0.05);the pollen tube growth rate in the group of cold treatment for 48 h at 4 ℃ and group of heat treatment for 24 h at 30 ℃ was no significantly different from that in the control group(P > 0.05);within 48 h,the pollentube growth rate was decreased with the prolongation of heat treatment time. Conclusion The results can provide reliable scientific basis and technical data for cross breeding and the regulation of flowering time for Andrographis paniculata(Burm. f.)Nees, and will accelerate breeding process and improve the conservation of germplasm resources.
引文
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[23]苏芸芸,王康才,薛启.不同产地藿香花粉活力与柱头可授性研究[J].草业学报,2016,25(9):189.
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[25]张涛,黄敏.蔗糖和PEG对山茶花花粉离体萌发的影响[J].北方园艺,2009(1):101.
[26]刘自刚,呼天明,杨亚丽.黄芩花粉离体萌发与花粉管生长研究[J].中国中药杂志,2011,36(19):2636.
[27]邓衍明,叶晓青,梁丽建,等.茉莉花粉离体培养萌发及花粉管生长特性研究[J].华北农学报,2014,29(5):107.
[28]周家杏,曾丽,陶懿伟,等.微型月季花粉生活力测定方法的研究[J].上海交通大学学报(农业科学版),2007,25(6):574.
[29]贾文庆,刘宇.紫薇花粉生活力的测定[J].陕西农业科学,2007(1):46.
[30]贾文庆,刘宇.金光杏梅的花粉生活力测定方法比较[J].安徽农业科学,2006,34(14):3275.
[31]刘保财,黄颖桢,赵云青,等.两面针花粉活性与形态特征研究[J].中草药,2016,47(9):1589.
[32]李国树,徐成东,李天星,等.几种杜鹃花花粉生活力研究[J].北方园艺,2010(22):80.
[33]曹芳,李志刚,李旭新,等.大豆花粉活力测定方法的比较研究[J].中国农学通报,2017,33(17):66.
[34]刘帮龙,张晓慧,干友民,等.野生马蹄金花粉生活力检测方法比较[J].草业科学,2011,28(11):1941.
[35]褚怡,范义荣,张韶伊,等.大花蕙兰与国兰花粉活力及柱头可授性分析[J].浙江农林大学学报,2013,30(6):950.
[2]国家药典委员会.中华人民共和国药典(一部)[S].北京:中国医药科技出版社,2015:268.
[3]陈丽霞,曲戈霞,邱峰.穿心莲二萜内酯类化学成分的研究[J].中国中药杂志,2006,31(19):1594.
[4]陈元生,罗战勇,郭尚志,等.穿心莲种质资源的评价与利用初报[J].广东农业科学,2005(1):5.
[5]陈元生,王振华,罗战勇,等.穿心莲优良种质筛选研究[J].现代中药研究与实践,2006,20(4):3.
[6]张婷,赵林,李刚波,等.苹果花粉离体萌发适宜蔗糖浓度的筛选[J].湖南农业科学,2018(8):24.
[7] Derksen J. Pollen tubes:a model system for plant cell growth[J].Bot Acta,1996,109:341.
[8] Mascarenhas J P. Molecular mechanisms of pollen tube growth and differentiation[J]. Plant Cell,1993,5(10):303.
[9]田惠桥,朱学艺.被子植物生殖生物学[M].北京:科学出版社,2012:16.
[10] Kumchai J, Wei Y C, Lee C Y, et al. Production of interspecific hybrids between commercial cultivars of the eggplant(Solanum melongena L.)and its wild relative S. torvum[J].Genet Mol Res,2013,12(1):755.
[11] Padilla F,Soria N,Oleas A,et al. The effects of pesticides on morphology, viability, and germination of Blackberry(Rubus glaucus Benth.)and Tree tomato(Solanum betaceum Cav.)pollen grains[J]. 3 Biotech,2017,7(3):154.
[12]朴锦,具红光.正交设计优化PEG引发关苍术种子萌发的最优处理方案[J].中国农学通报,2015,31(7):138.
[13]詹妮,黄烈健.大叶相思花粉离体萌发适宜条件及活力检测方法[J].林业科学,2016,52(2):67.
[14]李旭新,巴根那,杨恒山,等.肋柱花花粉萌发及其活力测定方法比较[J].安徽农业科学,2017,45(13):127.
[15]高露璐,李林芳,马育珠,等.毛叶铁线莲花粉活力测定方法[J].分子植物育种,2017,15(11):4667.
[16]席延坡,林辰壹,刘俊霞,等.二倍体棱叶蒜花粉活性的变化特征[J].草业科学,2015,32(12):2057.
[17]潘瑞炽.植物生理学[M].北京:高等教育出版社,2004:260.
[18]尤扬,贾文庆.西府海棠花粉生活力测定[J].安徽农业科学,2011,39(6):3423.
[19] Ma L,Sun D. The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth[J].Planta,1997,202(3):336.
[20]田慧桥,远彤.钙在被子植物受精过程中的作用[J].植物生理学报,2000,26(25):369.
[21]杜纪红,叶正文,苏明申,等.桃花粉离体萌发和花粉管生长特性研究[J].西北植物学报,2011,31(1):64.
[22]储立民,金爱红,徐冬青,等.钙离子浓度对丝瓜花粉萌发和花粉管生长的影响[J].河南农业科学,2005,34(4):56.
[23]苏芸芸,王康才,薛启.不同产地藿香花粉活力与柱头可授性研究[J].草业学报,2016,25(9):189.
[24]赵宏波,陈发棣,房伟民.栽培小菊和几种菊属植物花粉离体萌发研究[J].南京农业大学学报,2005,28(2):22.
[25]张涛,黄敏.蔗糖和PEG对山茶花花粉离体萌发的影响[J].北方园艺,2009(1):101.
[26]刘自刚,呼天明,杨亚丽.黄芩花粉离体萌发与花粉管生长研究[J].中国中药杂志,2011,36(19):2636.
[27]邓衍明,叶晓青,梁丽建,等.茉莉花粉离体培养萌发及花粉管生长特性研究[J].华北农学报,2014,29(5):107.
[28]周家杏,曾丽,陶懿伟,等.微型月季花粉生活力测定方法的研究[J].上海交通大学学报(农业科学版),2007,25(6):574.
[29]贾文庆,刘宇.紫薇花粉生活力的测定[J].陕西农业科学,2007(1):46.
[30]贾文庆,刘宇.金光杏梅的花粉生活力测定方法比较[J].安徽农业科学,2006,34(14):3275.
[31]刘保财,黄颖桢,赵云青,等.两面针花粉活性与形态特征研究[J].中草药,2016,47(9):1589.
[32]李国树,徐成东,李天星,等.几种杜鹃花花粉生活力研究[J].北方园艺,2010(22):80.
[33]曹芳,李志刚,李旭新,等.大豆花粉活力测定方法的比较研究[J].中国农学通报,2017,33(17):66.
[34]刘帮龙,张晓慧,干友民,等.野生马蹄金花粉生活力检测方法比较[J].草业科学,2011,28(11):1941.
[35]褚怡,范义荣,张韶伊,等.大花蕙兰与国兰花粉活力及柱头可授性分析[J].浙江农林大学学报,2013,30(6):950.