秋水仙素诱导月季品种‘月月粉’2n花粉的研究
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摘要
为获得二倍体古老月季品种‘月月粉’人工诱导2n花粉用于现代月季杂交育种,该研究在了解‘月月粉’花粉母细胞发育过程的基础上,使用秋水仙素注射结合包裹法处理幼嫩花蕾,成功获得了可正常萌发的2n花粉,并通过观察诱导获得的花粉离体及其在雌蕊上的萌发特征,初步评估2n花粉用于杂交育种的性能。结果表明:(1)秋水仙素药棉包裹造成部分花蕾在采粉前死亡,花蕾存活率随秋水仙素浓度提高和处理时间延长均极显著降低。(2)2n花粉诱导率在秋水仙素浓度为2.5、5.0、7.5和10.0 g/L间存在显著差异,但在处理时间为24、48和72 h间无显著差异;最佳诱导处理为5.0 g/L秋水仙素处理24 h,诱导率可达15.83%,该处理获得的2n花粉经亚历山大染色检验活力为27.2%。(3)‘月月粉’花粉母细胞在减数分裂时存在异常落后染色体和平行纺锤体,在四分体时期形成染色体数目加倍的二分体和三分体,最终产生2n花粉。(4)所诱导的2n花粉的体外萌发率和花粉管长度与天然花粉无显著差异,且都可以在母本柱头上萌发并长出花粉管进入子房,表明可用于进一步的杂交育种。
To obtain the artificially induced 2n pollen from the diploid Chinese ancient rose ‘Old Blush', which is to be used for the crossbreeding of modern rose and to effectively make use of its genetic resources, based on the understanding of the development of pollen mother cell of ‘Old Blush', this study used the method of colchicine injection combined with wrapping to treat the tender buds, and we successfully obtained the 2 n pollen which could be normally germinated. The performance of induced 2 n pollen for cross breeding was also evaluated by observing the germination characteristics in vitro and on the pistil. The results showed that:(1) the colchicine absorbent cotton wrapping caused the death of part of the buds before the pollination, and the survival rate of flower bud decreased equally significantly with the increase of colchicine concentration and treatment time.(2) The 2 n pollen induction rate differed significantly between the four colchicine concentrations of 2.5 g/L, 5.0 g/L, 7.5 g/L, and 10.0 g/L, but did not differ significantly between the three treatment periods of 24 h, 48 h, and 72 h. The best treatment was 24-hour 5.0 g/L colchicine, which can produce 15.83% viable 2 n pollen. The 2 n pollen vitality of this treatment was 27.2% detected by Alexander staining.(3) The mother cell of ‘Old Blush' pollen had abnormal lagging chromosomes and parallel spindle during meiosis. During the tetrad period, dyads and triads were formed whose number of chromosomes are doubled, and the 2 n pollen was produced eventually.(4) The in vitro germination rate and pollen tube length of induced 2 n pollen were insignificantly different from those of natural pollen, and both of them could germinate on the stigma of female parent and grow pollen tubes in the ovary, so they could be used for a further crossbreeding.
To obtain the artificially induced 2n pollen from the diploid Chinese ancient rose ‘Old Blush', which is to be used for the crossbreeding of modern rose and to effectively make use of its genetic resources, based on the understanding of the development of pollen mother cell of ‘Old Blush', this study used the method of colchicine injection combined with wrapping to treat the tender buds, and we successfully obtained the 2 n pollen which could be normally germinated. The performance of induced 2 n pollen for cross breeding was also evaluated by observing the germination characteristics in vitro and on the pistil. The results showed that:(1) the colchicine absorbent cotton wrapping caused the death of part of the buds before the pollination, and the survival rate of flower bud decreased equally significantly with the increase of colchicine concentration and treatment time.(2) The 2 n pollen induction rate differed significantly between the four colchicine concentrations of 2.5 g/L, 5.0 g/L, 7.5 g/L, and 10.0 g/L, but did not differ significantly between the three treatment periods of 24 h, 48 h, and 72 h. The best treatment was 24-hour 5.0 g/L colchicine, which can produce 15.83% viable 2 n pollen. The 2 n pollen vitality of this treatment was 27.2% detected by Alexander staining.(3) The mother cell of ‘Old Blush' pollen had abnormal lagging chromosomes and parallel spindle during meiosis. During the tetrad period, dyads and triads were formed whose number of chromosomes are doubled, and the 2 n pollen was produced eventually.(4) The in vitro germination rate and pollen tube length of induced 2 n pollen were insignificantly different from those of natural pollen, and both of them could germinate on the stigma of female parent and grow pollen tubes in the ovary, so they could be used for a further crossbreeding.
引文
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[20]丁一鸣,赵兰勇,张玲,等.玫瑰花柱提取液对玫瑰及月季花粉管离体生长的影响[J].中国农业科学,2012,45(9):1 791-1 797.DING Y M,ZHAO L Y,ZHANG L,et al.Effect of the extracted solution from the style of Rosa rugosa on the growth in vitro of pollen tube of R.rugosa and R.chinensis[J].Scientia Agricultura Sinica,2012,45(9):1 791-1 797.
[21]周旭红,桂敏,王继华,等.不同倍性香石竹杂交花粉管生长荧光显微观察及结实研究[J].西北植物学报,2012,32(1):67-74.ZHOU X H,GUI M,WANG J H,et al.Pollen tube growth after crossing between different ploids Dianthus caryophyllus by fluorescence microscopy and seed setting[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(1):67-74.
[22]MOKADEM HEL,CRESPEL L,MEYNET1J,et al.The occurrence of 2n-pollen and the origin of sexual polyploids in dihaploid roses(Rosa hybrida L.)[J].Euphytica,2002,125:169-177.
[23]WANG J,LI D,SHANG F.High temperature-induced production of unreduced pollen and its cytological effects in Populus[J].Scientific Reports,2017,7(1):52-81.
[24]张磊.高温诱导杜仲花粉染色体加倍技术研究[D].北京:北京林业大学,2010.
[25]赖杭桂.木薯2n配子途径诱导多倍体的研究[D].海口:海南大学,2014.
[26]高鹏,林威,康向阳.秋水仙碱诱导杜仲花粉染色体加倍的研究[J].北京林业大学学报,2004,26(4):39-42.GAO P,LIN W,KANG X Y.Pollen chromosome doubling of Eucommia ulmoides induced by colchicine[J].Journal of Beijing Forestry University,2004,26(4):39-42.
[27]杨珺.桉树生殖生物学基础与染色体加倍技术研究[D].北京:北京林业大学,2015.
[28]MASTERSON J.Stomatal size in fossil plants:Evidence for polyploid in majority of angiosperms[J].Science,1994,264(5 157):421-424.
[29]ZLESAK D C.Pollen diameter and guard cell length as predictors of ploidy in diverse rose cultivars,species,and breeding lines[J].Floriculture&Ornamental Biotechnology,2009,3(Special Issue 1):53-70.
[30]YONG W R,BEUSELINCK P R.Cytology of 2npollen formation and pollen morphology in diploid Lotus tenuis(Fabaceae)[J].American Journal of Botany,1996,83(8):1 057-1 062.
[31]ADAMS K L,WENDEL J F.Polyploidy and genome evolution in plants[J].Current Opinion in Plant Biology,2005,8(2):135-141.
[2]EILEEN W.ERLANSON,D S.Phylogeny and polyploidy in Rosa[J].New Phytologist,1938,37(1):72-81.
[3]范莉娟,周燕,高述民.月季育种技术研究进展[J].中国农学通报,2015,31(13):130-134.FAN L J,ZHOU Y,GAO S M.Research advances on breeding technology of Rosa hybrida L.[J].Chinese Agricultural Science Bulletin,2015,31(13):130-134.
[4]冯慧,常卫民,从日晨.远缘杂交在现代月季育种中的作用[J].广东农业科学,2009,(12):72-74.FENG H,CHANG W M,CONG R C.Application of distant hybridization in breeding of modern rose[J].Guangdong Agricultural Sciences,2009,(12):72-74.
[5]孙宪芝,赵惠恩.月季育种研究现状分析[J].西南林学院学报,2003,(4):65-69.SUN X Z,ZHAO H E.Review of research measures and development[J].Journal of Southwest Forestry College,2003,(4):65-69.
[6]ZLESAK D C,THILL C A,ANDERSON N O.Trifluralinmediated polyploidization of Rosa chinensis minima,(Sims)Voss seedlings[J].Euphytica,2004,141(3):281-290.
[7]SUGIMOTO H,FUKUI H,AOKI Y,et al.Tetraploid induction by colchicines in Rosa banksiae[C]//V International Symposium on Rose Research and Cultivation,Gifu,Japan,24-28May 2009.2010.
[8]CRESPEL L,GUDIN S,MEYNET J,et al.AFLP-based estimation of 2ngametophytic heterozygosity in two parthenogenetically derived dihaploids of Rosa hybrida L.[J].Theoretical&Applied Genetics,2002,104(2/3):451-456.
[9]CRESPEL L,RICCI S C,GUDIN S.The production of 2n pollen in rose[J].Euphytica,2006,151(2):155-164.
[10]MCHALE N A.Environmental induction of high frequency2npollen formation in diploid Solanum[J].Genome,2011,25(6):609-615.
[11]PCRIX Y,RALLO G,FOLZER H,et al.Polyploidization mechanisms:temperature environment can induce diploid gamete formation in Rosasp.[J].Journal of Experimental Botany,2011,62(10):3 587-3 597.
[12]CRESPEL L,CAMILLE L B,RELION D,et al.Effect of high temperature on the production of 2npollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization[J].Plant Breeding,2015,134(3):356-364.
[13]ALLUM J F,BRINGLOE D H,ROBERTS A V.Chromosome doubling in a Rosa rugosa,Thunb.hybrid by exposure of in vitro nodes to oryzalin:the effects of node length,oryzalin concentration and exposure time[J].Plant Cell Reports,2007,26(11):1 977-1 984.
[14]PEGAH K,MARYAM J K,GHORBAN A N,et al.Role of mitotic inhibitors and genotype on chromosome doubling of Rosa[J].Euphytica,2008,160(2):267-275.
[15]DEWITTE A,EECKHAUT T,HUYLENBROECK J V,et al.Induction of 2npollen formation in Begonia by trifluralin and N2O treatments[J].Euphytica,2010,171(2):283-293.
[16]ZHOU X,MO X,GUI M,et al.Cytological,molecular mechanisms and temperature stress regulating production of diploid male gametes in Dianthus caryophyllus L.[J].Plant Physiology&Biochemistry Ppb,2015,97:255-263.
[17]WU J H,DATSON P M,MANAKO K I,et al.Meiotic chromosome pairing behaviour of natural tetraploids and induced autotetraploids of Actinidia chinensis[J].Theoretical&Applied Genetics,2014,127(3):549-557.
[18]OKAZAKI K,KURIMOTO K,MIYAJIMA I,et al.Induction of 2npollen in tulips by arresting the meiotic process with nitrous oxide gas[J].Euphytica,2005,143(1-2):101-114.
[19]BARBAGONZALEZ R,LOKKER A C,LIM K B,et al.Use of2ngametes for the production of sexual polyploids from sterile O-riental x Asiatic hybrids of lilies(Lilium)[J].Theoretical&Applied Genetics,2004,109(6):1 125-1 132.
[20]丁一鸣,赵兰勇,张玲,等.玫瑰花柱提取液对玫瑰及月季花粉管离体生长的影响[J].中国农业科学,2012,45(9):1 791-1 797.DING Y M,ZHAO L Y,ZHANG L,et al.Effect of the extracted solution from the style of Rosa rugosa on the growth in vitro of pollen tube of R.rugosa and R.chinensis[J].Scientia Agricultura Sinica,2012,45(9):1 791-1 797.
[21]周旭红,桂敏,王继华,等.不同倍性香石竹杂交花粉管生长荧光显微观察及结实研究[J].西北植物学报,2012,32(1):67-74.ZHOU X H,GUI M,WANG J H,et al.Pollen tube growth after crossing between different ploids Dianthus caryophyllus by fluorescence microscopy and seed setting[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(1):67-74.
[22]MOKADEM HEL,CRESPEL L,MEYNET1J,et al.The occurrence of 2n-pollen and the origin of sexual polyploids in dihaploid roses(Rosa hybrida L.)[J].Euphytica,2002,125:169-177.
[23]WANG J,LI D,SHANG F.High temperature-induced production of unreduced pollen and its cytological effects in Populus[J].Scientific Reports,2017,7(1):52-81.
[24]张磊.高温诱导杜仲花粉染色体加倍技术研究[D].北京:北京林业大学,2010.
[25]赖杭桂.木薯2n配子途径诱导多倍体的研究[D].海口:海南大学,2014.
[26]高鹏,林威,康向阳.秋水仙碱诱导杜仲花粉染色体加倍的研究[J].北京林业大学学报,2004,26(4):39-42.GAO P,LIN W,KANG X Y.Pollen chromosome doubling of Eucommia ulmoides induced by colchicine[J].Journal of Beijing Forestry University,2004,26(4):39-42.
[27]杨珺.桉树生殖生物学基础与染色体加倍技术研究[D].北京:北京林业大学,2015.
[28]MASTERSON J.Stomatal size in fossil plants:Evidence for polyploid in majority of angiosperms[J].Science,1994,264(5 157):421-424.
[29]ZLESAK D C.Pollen diameter and guard cell length as predictors of ploidy in diverse rose cultivars,species,and breeding lines[J].Floriculture&Ornamental Biotechnology,2009,3(Special Issue 1):53-70.
[30]YONG W R,BEUSELINCK P R.Cytology of 2npollen formation and pollen morphology in diploid Lotus tenuis(Fabaceae)[J].American Journal of Botany,1996,83(8):1 057-1 062.
[31]ADAMS K L,WENDEL J F.Polyploidy and genome evolution in plants[J].Current Opinion in Plant Biology,2005,8(2):135-141.