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1、African Journal of Biotechnology Vol.10(64),pp.14061-14066,19 October,2011 Available online at http:/www.academicjournals.org/AJB DOI:10.5897/AJB11.1297 ISSN 16845315 2011 Academic Journals Full Length Research Paper Cytological evidences of pollen abortion in Ornithogalum caudatum Ait 1Department o

2、f Chemistry and Life Science,Chuzhou University,Anhui,239000,China.2Institute of Crop Science,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou,310058,China.3College of Environmental and Resource Sciences,Zhejiang University,Hangzhou,310058,China.4Institute of Nuclear Agricultura

3、l Science,Zhejiang University,Hangzhou,310029,China.5Wuxing District of Huzhou Agriculture and Forestry Development Bureau,Huzhou,313000,China.6Department of Soil Science,PMAS Arid Agriculture University,Rawalpindi,46300,Pakistan.7School of Modern Agricultural Technology,Huzhou City,313023,China.Acc

4、epted 11 August,2011 Ornithogalum caudatum Ait exhibits strong physiological adaptability;however,it suffers a serious menace of pollen abortion,as almost no seed is formed in the plants.In the current study,chromosome karyotype of root-tip cells(RTC)during mitosis,and chromosome behavior of pollen

5、mother cells(PMC)during meiosis in O.caudatum were investigated.We recorded 18 euchromosomes with variable number(2836)of B chromosomes in the somatic cells genome.In leptonema of meiotic prophase I,the synapsis between euchromosomes was normal,but there were several darker granular nodular bodies i

6、n the cells,which could be the precursor for B chromosomes.Some of the cells were observed as duplication loop in zygotene,indicating that the PMCs had gone through a chromosome structure variation with duplication,which could reduce the fertility of pollens.Numerous chromosome bridges,fragments and

7、 lagged chromosomes were observed in meiosis anaphase I.It reflects that these cells might have undergone paracentric inversion,which is one of the important reasons of pollen abortion.In meiosis anaphase II,the sister chromatids separate unusually forming the triad,which leads to gamete sterility.T

8、hese findings conclude that pollen abortion in O.caudatum mainly results from the variation in chromosome structure,which could be related to the B chromosomes in somatic cells.Key words:Chromosome structure variation,B chromosome,root-tip cells,pollen mother cells,gamete sterility.INTRODUCTION Genu

9、s Ornithogalum caudatum Ait,with common name as Star of Bethlehem or Christmas Star,is a member of the Liliaceae family.It is native to southern Africa,and called as false sea onion or pregnant onion(Tilton and Horner,1980;Kariukia and Kako,1999).It is occasionally grown as a window or greenhouse pl

10、ant,with an alias of *Corresponding author.E-mail:biotechnologist15yahoo.ca.Tel.+86-13757132428.Abbreviations:m,Metacentric;sm,submetacentric;st,subtelocentric.Urginea maritima.Now,it is widely cultivated as a medicinal value just like aloe vera(Tang et al.,2002;Chen et al.,2010).Recently,saponins i

11、solated from O.caudatum are being used to cure lung cancer,breast cancer,and their anticancer activity has been docu-mented.The root system of O.caudatumis developed and the lamina is strong,with many physiological adaptations,such as drought,freezing and stress tolerance etc.However,pollen abortion

12、 is serious in O.caudatum,therefore,only the developed bulbs are its main propagulum.Phenomenon of pollen abortion in O.caudatum is very common,and it could be related to abnormality of euchromosome pairing in meiosis;further,B 14062 Afr.J.Biotechnol.chromosomes may affect the pollen fertility(Wiega

13、s,1980;Parker et al.,1990;Gonzlez-Snchez et al.,2004;Kumar and Srivastava,2010).The B chromosomes are accessory genomic elements to the so-called A or standard genome.They have been found in many species of plants and animals.Populations usually show a polymorphism for the number and morphology of B

14、 chromosomes(Camacho et al.,2000;Basheva et al.,2010).The B chromosome also called as supernumerary chromosome,additional chromosome or extra chromosome,is a kind of particular chromosome outside the genome chromosome.Genetic pattern of B chromosome is transmitted in a non-Mendelian fashion due to t

15、heir unstable mitotic and/or meiotic behavior,and is irregular,its genetic viability is generally inert,and B chromosome is smaller than euchromosomes.The B chromosomes have a special distribution pattern of heterochromatin,and have some impact on the adaptability and fertility in plants(Camacho et

16、al.,2000;Jimnez et al.,2000;Bidau et al.,2004;Perfectti et al.,2004;Basheva et al.,2010).In this study,we report the results of studies based upon B chromosomes characteristic and behavior in somatic cell and two kinds of chromosomes structure variation,such as duplication and paracentric inversion

17、in meiosis,which may lead to the pollen abortion in O.caudatum.MATERIALS AND METHODS Plant materials This study was carried out from 2009 to 2010 in the province of Anhui,located in the eastern China.The materials used for this study were fresh young root tips and pollen mother cells(PMC)in anthers

18、of O.caudatum.Karyotyping of root-tip cells Somatic chromosomes were studied in the root tip cell(RTC)meristems of growing plants,which were pretreated in ice-cold distilled water(0 C)for 24 h.Then fixed in Carnoys solution(absolute ethyl alcohol and glacial acetic acid in 3:1,v/v)at 4 C for 24 h an

19、d stored in 70%aqueous ethanol(Ostergren and Haneen,1962).Root tips were macerated and dissociated with 1 M HCl at 60 C for 5 min,stained according to the improved carbol fuchsin technique before squashing(Darlington and La Cour,1976).Slides were made permanent using Canadian balsam as the mounting

20、medium.At least 10 metaphase cells were drawn using a Sony camera lucida and selecting the five best for measurements.The nomenclature used for the description of chromosome morphology is that proposed by Levan et al.(1964).The karyotype classification followed was that from Stebbins(1971).Idiograms

21、 were drawn based on mean centromeric index and arranged in the order of decreasing size.Chromosome characterization of PMCs The pollen mother cells(PMCs)were studied using the same method as for RTC except acid dissociation.They were characterised by terminal and interstitial knobs at the pachytene

22、 (Jagathesan and Sreenivasan,1971)and were identified through geimsa banding technique(Mirzaghaderi,2010).A double stain that differentially stains the filled and unfilled pollen grains was used to assess the pollen fertility(Alexander,1969).RESULTS B chromosomes and karyotype analysis Total number

23、of chromosomes in O.caudatum species is not fixed,but they range from 46 to 54.Generally,each cell includes 18 large chromosomes and 28 to 36 small ones(Figure 1).The difference between chromosome numbers is caused by the unequal number of these small chromosomes.These small chromosomes are signific

24、antly shorter than the 18 large ones.The number and morphology are multifarious in different cells,and there is no congruent relationship among the 18 large chromosomes,so the probability of satellite is excluded.With the definition and characteristics of B chromosomes by Beukeboom(1994),Camacho et

25、al.(2002)and Jones and Houben(2003),we consider that these small chromosomes are B chromosomes.Karyotype formulae and parameters for the studied species are summarized in Tables 1 and 2,and karyogram is shown in Figure 1,which illustrate the karyogram of mitotic metaphases.As a whole,karyo-ypes of t

26、he analyzed species showed a predominance of submetacentric(sm)chromosomes.The karyotype formula is 2n=18=4 m+14 sm+(28 to 36)Bs.The morphologic difference of all 18 euchromosomes was inconspicuous;the ratio of the longest and the shortest chromosome was 1.35.In general,the karyotype was moderately

27、symmetrical in the Stebbins 2A category,and there was no satellite chromosome in the genome.B chromosomes in meiosis During PMC meiosis,the characteristics of behavior and change of B chromosomes were significantly different than that of A chromosomes.Generally,B chromosomes were small granular corp

28、uscle,and most of them showed the lagged chromosome phenomenon.For this,some examples at different stages of meiosis with typical pictures of A and B chromosomes are shown in Figure 2 and described subsequently.Figure 2A to E shows that the synapsis of A chromosomes are normal from leptonema period

29、to early diplonema in meiosis prophase I.There is no obvious B chromosome,but many nodule shapes.Deep dye granular corpuscles can be observed on the fibers of A chromosome,which might be the precursor of B chromosomes.Figure 2E and F shows the diplonema period,where the synapsis of A chromosomes rea

30、ched the maximum,and simultaneously,the cross knots are obvious.Here,B chromosomes begin to come off from A Cai et al.14063 Figure 1.Chromosome karyotype picture of Ornithogalum caudatum Ait.A.2n=18+34 BS;B.2n=18+28 BS;C.2n=18+30 BS;D.2n=18+32 BS;E.2n=18+36 BS.Table 1.Characteristics of chromosomes

31、in Ornithogalum caudatum Ait.Sample Relative length(%)Index of relative length Centromere index(%)Arm ratio(long/short)Type of chromosome Short Long Total 1 1.38 2.63 4.01 1.15 34.41 1.91 sm 2 1.35 2.54 3.89 1.12 34.70 1.88 sm 3 1.30 2.46 3.76 1.08 34.57 1.89 sm 4 1.33 2.17 3.45 0.99 38.55 1.63 m 5

32、1.21 2.50 3.68 1.06 32.88 2.07 sm 6 1.19 2.15 3.34 0.96 35.63 1.81 sm 7 1.13 2.11 3.24 0.93 34.88 1.87 sm 8 1.14 1.89 3.03 0.87 37.63 1.66 m 9 1.09 1.87 2.96 0.85 37.58 1.72 sm chromosomes gradually,pairing one another,forming independent bivalents as A chromosomes.Figure 2G and H shows that B chrom

33、osomes move closer to the equatorial plate as A chromosomes in meiosis meta-phase I,there is obvious cytological difference between A and B chromosomes.A chromosome in meiosis We observed a clear loop structure in a pachytene cell(Figure 2D),which was formed out of one chromosome from a pair of syna

34、psis A chromosomes,which could have resulted from the deletion or duplication of 14064 Afr.J.Biotechnol.Table 2.Comparison of karyotype in Ornithogalum caudatum Ait.Karyotype formula Chromosome length ratio(L/S)Proportion of AR2 Index of asymmetry(%)Karyotype type 2n=18=4 m+14 sm+(28?36)Bs 1.35 0.11

35、 64.80 2A Figure 2.Ornithogalum caudatum Ait PMC meiosis chromosome characteristics at different periods.A:leptonema;B:zygotene;C:pachytene;D:pachytene(arrow indicates the duplication loop);E:early diplonema;F:diplonema(arrows indicate cross knots and B chromosomes);G:diakinesis;H:metaphase I;I:anap

36、hase I(arrow indicates lagging chromosomes);J:Late anaphase I(arrows indicate lagging chromosome fragments and chromosome bridges);K:anaphase II(arrow indicates lagging chromosome);L:telophase II(triad).chromosome structure variation.In our opinion,if it belongs to deletion,then the plant with this

37、kind of chromosomal aberration should be aborting fully,and it should be so with the pollens;but in fact,in addition to the fertility of pollens in O.caudatum,its floral organs and other parts of the nutritive organs are very developed,a variety of physiological adaptation are also very strong.There

38、fore,we believe,deletion mutation in O.caudatum chromosomes may be impossible,this type of variation should belong to duplication.Several chromosome bridges and chromosome frag-ments were observed in some cells during meiosis anaphase I of PMCs(Figure 2I and J),suggesting that these PMCs have underg

39、one paracentric inversion.Because inversion can induce delete or duplicate chromatids,and the spores grown from them would be abortive.In addition,the lagged B chromosomes can still be observed in meiosis anaphase II(Figure 2K),and we also found triad in some PMCs(Figure 2L),the chromosomal inversio

40、n and the triad can be the most important cytological evidence of pollen abortion in O.caudatum.DISCUSSION The role of B chromosomes in plants is obvious during the growth and development processes;and the harmfulness is mainly due to variation in the number of B chromosomes.Our study shows that in

41、addition to containing the 18 A chromosomes,there are 28 to 36 B chromosomes in the somatic cells of O.caudatum.But there were not so much B chromosomes in meiosis stages of PMCs,which could relate to chromosome loss because of pair error among B chromosomes during meiotic process.This phenomenon is

42、 similar to that described earlier in the B chromosomes of rye(Wiegas,1980;Houben et al.,2001).In maize,B chromosomes also affect the pollen viability;they could make A chromosomes,which show more complex genetic behavior,the frequency of univalent in PMCs is increased significantly in meiosis metap

43、hase I(Rusche et al.,1997;Gonzlez-Snchez et al.,2004;Masonbrink and Birchler,2010).Such behavior of B chromosomes may cause pollen abortion.The phenomenon of pollen abortion is common in O.caudatum,suggesting that pollen abortion of O.caudatum may be related to the B chromosomes,which can lead to po

44、llen sterility directly.It also affects the pollen abortion through A chromosomes indirectly,such as causing the structure variation of A chromosomes.In this study,we found that B chromosome of O.caudatum showed the characteristics and behavior as defined by Beukeboom(1994).However,the cytologic fea

45、tures and genetic behaviors were significantly different to A chromosomes,for example,B chromosomes showed heterochromatinization activity during leptonema,zygo-nema and pachytene period,and showed lagging chromosomes in meiosis anaphase I and II,etc.These Cai et al.14065 features were similar to th

46、e B chromosomes of rye.The triad structure was also reported in the study of B chromosomes during meiosis telophase II in garden pea(Berdnikov et al.,2003;Gonzlez-Snchez et al.,2003;Teruel et al.,2007).Triad and anaphase bridges structure are important cytological evidence in pollen abortion in O.ca

47、udatum;however,the causality between chromosome structure variation and B chromosome needs to be explored.Duplication loop,anaphase bridges and lagging chromosomes were found in meiosis anaphase I and II of O.caudatum,which indicated that duplication and inver-sion occurred between two homologous A

48、chromosomes.This event produces some distorted chromosome fragments,such as chromatid without centromere,chromosome pieces in the inversion heterozygote.Therefore,we can speculate that these chromosome fragments will enter the spores randomly in meiosis anaphase II,and continue to pass on,and would

49、develop into B chromosomes eventually.Our results support the hypothesis that A chromosomes develop from the B chromosomes(Jamilena et al.,1994;Lamb et al.,2005;Basheva et al.,2010).From leptonema to early diplonema period in meiosis prophase I,there was no significant number of B chromosomes.Instea

50、d,we observed many nodule shaped,fast color granular corpuscles on the fiber of A chromosomes.The B chromosomes appeared after early diplonema period in PMCs,and especially,it was very easy to differentiate the B and A chromosomes in this period.Therefore,we speculate that B chromosomes could be der