Lei WEI

Department of Biological Science and Biotechnology,Shaanxi Xueqian Normal University,Xi’an 710100,China

Cytoplasmic male sterility(CMS)is the most important type of male sterility.It is a genetic phenomenon co-controlled by nuclear and cytoplasmic genes.Cytoplasmic male sterility is the results of interaction between nucleus and cytoplasm[1].According to statistics,CMS has been found in more than 150 species and 270 hybrids among species[2-3].CMS has important practical and theoretical values.At present,the hybrids of rice,corn,canola and other economic crops that are widely used in agricultural production are all bred with cytoplasmic male sterile materials.So CMS is the basis of crop heterosis utilization to some extent[4].CMS is a maternally inherited trait cocontrolled by nucleus and cytoplasm.It provides an excellent material for researches on cytoplasmic genetics and nucleo-cytoplasmic interaction.

There are rich cytoplasmic male sterile line resources in China.According to incomplete statistics,more than 60 kinds of male sterile cytoplasms have been found in China.Currently,10 kinds of male sterile cytoplasms have been applied in production,including wild abortive cytoplasm(WA),dwarf abortive cytoplasm (DA),Honglian cytoplasm(HL),G-type cytoplasm (G),Maxie-type cytoplasm,D-type cytoplasm,Indonesia Paddy cytoplasm,K-type cytoplasm,Yunnan cytoplasm and BT-type cytoplasm(BT)[5].Yuan Longping,the father of hybrid rice,realized the three-line hybrid rice seed production by using wild abortive male sterile rice,making tremendous contributions to China’s and the world’s food production.

Zidao type cytoplasmic male ster-ile line A(ZL 99 1 20003.9)is characterized by early and thorough abortion[6-8],and it plays an important role in enriching rice cytoplasm resources,guiding hybrid rice breeding practices and revealing the mechanism of cytoplasmic male sterility in rice.Yingxiang A is bred from Zidao type cytoplasmic male sterile line by nucleus replacement.At the same time,the fragrance genes are also transferred.Therefore,the study on cytoplasmic male sterile line,Yingxiang A and its maintainer line will help to reveal the mechanism of Zidao type cytoplasmic male sterility,thereby guiding the breeding of three-line hybrid rice.

Material and Methods

The test materials included Zidao type cytoplasmic male sterile line,Yingxiang A and its maintainer line,Yingxiang B;wild abortive male sterile line,Zhenshan 97A and its maintainer line,Zhenshan 97B;Honglian type male sterile line,Honglian A and its maintainer line,Honglian B.The anthers were all collected from an experimental field located in Jiufeng,Wuhan.One part of the anthers were stained with I2-KI;one part were stained with silver nitrate;another part were fixed in FAA fixative,stained with Ehrlich hematoxylin and prepared into paraffin sections

Results and Analysis

I2-KI staining

At the meiotic phase,a large number of dyads,but a tiny amount of aborted microspores were formed inYingxiang A,a Zidao type male sterile line.The abortion morphology was similar to that of WA.It indicated the abortion of Zidao type male sterile lines mostly occurred at the meiotic phase,and only a small part occurred at the phase of microspore.At the same time,the abortive pollen number per anther was counted.As shown in Table 1,Yingxiang male sterile line had the most aborted microspores,whichweremostlyformedatthe monokaryotic phase.It suggested that the abortion in the Yingxiang male sterile line was most thorough.

Table 1 Statistics of pollen number per flower in rice

Silver nitrate staining

The staining results showed the aborted microspores were transformed into golden yellow transparent and irregular triangles after stained(Fig.1).This was similar to the typical abortion morphology.In the corresponding maintainerline,the microspores were round and filled with starch.Only a tiny amount of aborted microspores were found per anther in Yingxiang A,which was consistent with the I2-KI staining results.

Paraffin sections

Observation on cytoplasmic male sterile line,Yingxiang AAbnormal meiosis,represented by unequal division,occurred at the meiotic phase(Fig.2 B,C).The abortion arose at the meiotic phase and was basically completed tillthe phase ofmonomicrospore.Then only a very small number of double-nucleus abortive pollens were found.At maturity phase,none normal functional pollens could be ob-served(Fig.2 D).So the abortion was complete and thorough in Yingxiang A.The morphology of abortive pollens was similar to that of Zidao sterile line,butthe numberwas significantly greater than that of Zidao sterile line.In addition,the anther chambers had not been merged.There were still tapetal cells existing in anthers from the microsporocyte stage to the maturity stage(Fig.2 D).

Observation on maintainerline,Yingxiang BIn Yingxiang B,none abnormalities were found in meiosis(Fig.2 F).The tapetal cells were existed in anthers from the microsporocyte stage to the tetrad stage.When the pollens were mature,the tapetal cells disappeared(Fig.2 G,H).

Discussion

Considering the utilization of heterosis in rice,three-line breeding is an important way.However,the threeline breeding requires a stable rice male sterile line[9].Huet al.[8]and Weiet al.[6-7,10]considered that the Zidao type male sterility belonged to pollenfree abortion,and it is a new cytoplasm resource.The construction of new cytoplasm resources has important significance in developing multicytoplasm hybrid rice,preventing the large-scale occurrence of pests and diseases caused by genetic vulnerability and promoting the sustainable development of hybrid rice[11].

Yingxiang sterile line is bred from the Zidao sterile line.It is also characterized by early and thorough abortion.The thorough abortion of abortive pollens can effectively prevent the emergence of false hybrids and ensure the purity of seeds,which have important guiding significance fordeveloping new crossing combinations and breeding new varieties.

In terms of cytological studies on cytoplasmic male sterility in rice,the relationship between tapetum and male sterility has aroused much attention.Studies have shown that the tapetal cells in maintainer line generally start to get apoptosis around the maturity of pollens.However,in sterile line,the tapetal cells may die before or after the normal apoptosis in tapetum.Chenet al.[12]studied the calcium distribution in the anther of Yunnan purple cytoplasmic male sterile rice during the development of anther by using potassium pyroantimonate precipitation method.They considered the abnormaldevelopmentoftapetum was closely related to the abortion of microspores,and it determined the abortion of microspores through influencing the nutrients in sporogenous tissues and the formation of pollen wall and providing identification substances.The further studies showed that the early programmed death of tapetal cells and the abnormal mitochondria prevent the formation of fertile pollens,resulting in the emergence of male sterility in plants.

The male sterility in Yingxiang line is characterized by abnormal meiosis and tapetum.The unequal division may lead to the occurrence of dysfunction and disrupt the normal development of separated cells,resulting in the apoptosis and degradation of cells,thereby resulting in the abortion of spores.The abnormalities in tapetal cells may affect the formation of fertile pollens,leading to male sterility in plants.

In summary,there is difference in abortion degree between Yingxiang sterile line and Zidao sterile line,which may be caused by different nuclear effects.However,the abortion of Yingxiang sterile line is as thorough as that of Zidao sterile line,and they all contain fragrance gene,which have important significance for hybrid rice breeding.The abortion of Yingxiang sterile line is thorough,so it has a good application prospect in the production practice.Meanwhile,the abnormal proliferation of tapetum cells may also be related to the male sterility.Cytological studies have revealed preliminarily physiological and biochemical characteristics of Zidao type rice,laying a foundation for further study on male sterile mechanism.

[1]PASCAL T,ETIENNE H.Cytoplasmic male sterility and mitochondrial metabolism in plants[J].Mitochondrion,2014,19:166-171.

[2]KAUL M.Male sterility in higher plants[M].Springer-Verlag Press,1988.

[3]SCHNABLE P,WISE R.The molecular basis of cytoplasmic male sterility and fertility restoration[J].Trends Plant Sci,1998,3:175-180.

[4]LI JX(李竟雄),ZHOU HS(周洪生).High yield is not a dream:basic research on cytoplasmic male sterility in grain,cotton and oil(高產(chǎn)不是夢—糧棉油雄性不育雜種優(yōu)勢的基礎(chǔ)研究)[M].Changsha:Hunan Science and Technology Press(長沙:湖南科學(xué)技術(shù)出版社),1995.

[5]LI ZB(李澤炳).A preliminary discussion about the classification of male sterile lines of rice in China(對我國水稻雄性不育系分類的初步探討)[J].Acta Agronomica Sinica(作物學(xué)報(bào)),1980,6(1):17-26.

[6]WEI L(魏磊),FEI ZJ(費(fèi)震江),DING Y(丁毅).Mitochondrial RNA editing of ATPaseatp6 gene transcripts of Yunnan purple rice(Oryza sativaL.)(紫稻(Oryza sativaL.)線粒體ATP合成酶atp6基因轉(zhuǎn)錄本RNA編輯 [J].Journal of Wuhan Botanical Research(武漢植物學(xué)研究),2010,28(3):251-256.

[7]WEI L,YAN Z,DING Y.Mitochondrial comparative proteomic analysis of sterile line and its maintain line of Purple cytoplasmic rice(Oryza sativaL.)[J].Advances in Bioscience and Biotechnology,2010,1(3):145-151.

[8]HU Y,WU Q,LIU S,et al.Study of rice pollen grains by Multispectral Imaging Microscopy[J].Microscope Research and Technique,2005,68(6):335-346.

[9]XU SH(徐樹華).Cytological observation on pollen development in the main male sterile types of rice(Oryza sativaL.)developed in China(我國水稻主要雄性不育類型花粉發(fā)育的細(xì)胞學(xué)觀察)[J].Scientia Agricultura Sinica(中國農(nóng)業(yè)科學(xué)),1982,2:9-14.

[10]WEI L(魏磊),DING Y(丁毅),LIU Y(劉義),et al.Microcalorimetric analysis of anthers of male sterile rice sterile line(水稻雄性不育系花藥微量熱分析)[J].Journal of Wuhan Botanical Research(武漢植物學(xué)研究),2002,20(4):308-310.

[11]CAI SX(蔡善信).Breeding of CMS line Y Huanong A with Y-type cytoplasm in rice(水稻Y型細(xì)胞質(zhì)雄性不育系Y華農(nóng)A的選育)[J].Hybrid Rice(雜交水稻),2001,16(6):9-10.

[12]CHEN XJ(陳小軍),LIU SN(劉樹楠),WEI L(魏磊),et al.The calcium distribution in the anther of Yunnan purple cytoplasmic male sterile rice during anther development(云南紫稻細(xì)胞質(zhì)雄性不育系花藥發(fā)育過程中Ca2+的分布)[J].Journal of Wuhan Botanical Research(武漢植物學(xué)研究),2005,23(2):101-106.