Research Article Identification of Chrysanthemum...

Research ArticleIdentification of Chrysanthemum(Chrysanthemum morifolium) Self-Incompatibility

Fan Wang12 Feng-Jiao Zhang12 Fa-Di Chen12 Wei-Min Fang12 and Nian-Jun Teng12

1 College of Horticulture Nanjing Agricultural University Nanjing 210095 China2 Jiangsu Province Engineering Lab for Modern Facility Agriculture Technology amp Equipment Nanjing 210095 China

Correspondence should be addressed to Nian-Jun Teng njtengnjaueducn

Received 24 August 2013 Accepted 27 November 2013 Published 27 January 2014

Academic Editors L Catuvelli and M Nikolic

Copyright copy 2014 Fan Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

There has been a heated argument over self-incompatibilityof chrysanthemum (Chrysanthemum morifolium) among chrysan-themum breeders In order to solve the argument we investigated pistil receptivity seed set and compatible index of 24chrysanthemum cultivars It was found that the 24 cultivars averagely had 37ndash363 pollen grains germinating on stigmas at 24hours after self-pollination through the fluorescence microscope using aniline blue staining method However only 10 of themproduced self-pollinated seeds and their seed sets and compatible indexes were 003ndash5650 and 004ndash8750 respectively Thecultivar ldquoQ10-33-1rdquo had the highest seed set (5650) and compatible index (8750) but ten of its progeny had a wide range ofseparation in seed set (0ndash3723) and compatible index (0ndash6865)The results indicated that most of chrysanthemum cultivars wereself-incompatible while a small proportion of cultivars were self-compatible In addition there is a comprehensive separation ofself-incompatibility among progeny from the same self-pollinated self-compatible chrysanthemum cultivarTherefore it is better toemasculate inflorescences during chrysanthemum hybridization breeding when no information concerning its self-incompatibilitycharacteristics is available However if it is self-incompatible and propagated by vegetative methods it is unnecessary to carry outemasculation when it is used as a female plant during hybridization breeding

1 Introduction

Chrysanthemum (Chrysanthemum morifolium) with a highornamental value is one of the ten most popular traditionalflowers in China and one of the fourmost popular cut flowersin the world therefore this flower occupies a very importantposition in the world flower industry [1ndash4] It is estimatedthat there are more than 20000 chrysanthemum cultivarsin the world and about 7000 cultivars in China Over 90of them were produced through traditional hybridizationbreeding technique andmany new chrysanthemum cultivarsare now being developed by this technique each year [4ndash6] Although the traditional hybridization breeding methodhas played a key role in developing chrysanthemum cultivarsby now there has been a heated argument over necessityof inflorescence emasculation a very complex and labori-ous process during chrysanthemum hybridization breedingamong chrysanthemum breeders [5ndash8] Some breeders think

that it is necessary to emasculate chrysanthemum inflores-cences before pollination during chrysanthemum hybridiza-tion breeding because they believe that chrysanthemumis self-compatible In contrast some other chrysanthemumbreeders feel that it is unnecessary to make inflorescencesemasculated during chrysanthemumhybridization breedingas they think that chrysanthemum is a self-incompatibleflower

Emasculation of chrysanthemum inflorescences is a verycomplex and time-consuming process as chrysanthemuminflorescence structure is not suitable for emasculation [3ndash5 9] Each chrysanthemum inflorescence consists of 20ndash30 peripheral ray florets with only pistil and 100ndash200 smallcentral disk florets with both pistil and stamen [3 4 9] Ifinflorescences need to be emasculated during chrysanthe-mum hybridization all the small central disk florets shouldbe completely removed by hands with tweezers at around 3days before anthesis Meanwhile the upper parts (tubiform

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 625658 9 pageshttpdxdoiorg1011552014625658

2 The Scientific World Journal

petals) of all peripheral ray florets should be entirely cut offby scissors until the stigmas are visibleThen the emasculatedinflorescences are bagged [3]Thus the emasculation of chry-santhemum inflorescences not only is very laborious butalso sometimes causes injuries to the inflorescences as aconsequence resulting in low breeding efficiency In additionthe emasculation process significantly reduces the numberof florets that can be used for production of hybrid seeds[9 10] For example if chrysanthemum is self-incompatiblethe small central disk florets can be also used for productionof hybrid seeds because it is unnecessary to remove themTherefore it is very urgent and necessary to reveal if chrysan-themum is self-incompatibility or not

According to our knowledge there have been no studiessystematically investigating self-incompatibility of chrysan-themum so far We therefore carried out a systematicalinvestigation on pistil receptivity seed set and compatibleindex of 24 chrysanthemum cultivars with high ornamentalvalues in this study In addition we also examined pistilreceptivity seed set and compatible index of some progenyof one chrysanthemum cultivar The purpose of this studyis to solve the above-mentioned argument among chrysan-themum breeders through identifying chrysanthemum self-incompatibility and related cellular mechanisms In additionwe also hope that the expected results will provide valu-able information for increasing breeding efficiency duringchrysanthemum hybridization breeding in the future

2 Materials and Methods

21 Experimental Materials Twenty-four chrysanthemumcultivars with high ornamental values were used as materialsin this study They were grown in the ChrysanthemumGermplasm Resource Preserving Center Nanjing Agricul-tural University China (32∘051015840N 118∘901015840E)

22 Determination of Selfing Seed Set and Compatible Indexthrough Selfing Experiment In order to get some informationon seed set and compatible index of the 24 chrysanthemumcultivars we first carried out selfing experiment The selfingexperiment was performed in early November 2011 Twentyto thirty inflorescences were randomly selected from eachcultivar and then bagged with waterproof and breathablepaper bags at around 3 days before anthesis Cultivarsrsquonames date and the names of researchers were markedon the vegetable parchment tied with each inflorescenceAlthough the paper bags were waterproof and breathablerelative humidity sometimes was much higher inside thanoutside bags Therefore a small hole was made from eachbag to prevent mildew After two weeks all the bags wereremoved from the inflorescences because they had lost pistilreceptivity Around two months later (in the middle ofJanuary 2012) the inflorescences were cut off from plantsfor seed collection After that seed set (the average seednumber per inflorescencethe average floret number perinflorescence) and compatible index (the seed number ofall inflorescencesthe number of all inflorescence) were thencounted

23 Artificial Self-Pollinated Experiment Because it was dif-ficult to know the exact dates of anther dehiscence andpollen dispersal in the selfing experiment it was very difficultto determine pollen-stigma interaction or pistil receptivityWe therefore did artificial self-pollinated experiment as thepollination time can be controlled In the artificial self-pollinated experiment five inflorescences were randomlyselected from each cultivar at around 3 days before anthesisthen emasculated and bagged Around three days later whenthe shape of pistils became Y it meant that the pistils hadstrong receptivity [3 5] At this time artificial self-pollinatedexperiment was performed with fresh mature pollen grainsthat were collected from mature inflorescences on the sameplant or other plants of the same cultivar After artificialpollination the inflorescences were bagged again

24 Determination of Pistil Receptivity Because our previ-ous study indicated that the number of germinated pollengrains on chrysanthemum stigmas reached the peak at 24 hafter artificial pollination we thus here collected the self-pollinated inflorescences at 24 h after pollination in theartificial self-pollinated experimentThe self-pollinated inflo-rescences were cut off from each cultivar and immediatelyfixed and stored in FAA solution The pistils were strippedout softened in 1molL NaOH solution for 8 h at roomtemperature then washed with distilled water one time andimmersed in aniline blue staining fluid (01molL K

3PO4+

18 glycerol) for one to two hours in the dark [1 11] Afterthat the pistils were placed on the glass slides and gentlypressed with coverslips Then the germination behaviors ofpollen grains on stigmas and the growth of pollen tubes wereobserved under a fluorescence microscope (Zeiss Axioskop40) Good representative images were captured with anAxiocam MRC camera For each cultivar 30 pistils wereobserved and the numbers of germinated pollen grains oneach stigma were recorded

25 Examination of Embryo Development after Chrysanthe-mum Selfing Because low seed set during selfing experi-ments may be caused by two factors that is self-incom-patibility (pistil receptivity or pollen-stigma interaction) andembryo abortion we examined embryo development afterchrysanthemum selfing for the purpose of ruling out thepossibilities that low seed set was caused by embryo abortionAt two weeks after the inflorescences were bagged in theselfing experiment five inflorescences were sampled fromeach of 24 cultivars and immediately fixed in FAA solution(95 ethanol distilled water formalin glacial acetic acid =63 27 5 5) until use Ovaries were collected form the floretsof each inflorescence and subjected to dehydration througha graded series of ethanol solutions and then immersed inparaffin wax After that sections were cut to a thicknessof 8ndash10 120583m and stained with Heidenhainrsquos hematoxylin andthen observed and photographed under an Olympus BX41microscope [1 3] Because the cultivar ldquoQX-118rdquo did notproduce any selfing seeds and there were a very high numberof pollen grains germinated on its stigmas at 24 h after

The Scientific World Journal 3

Table 1 Pistil receptivity seed set and compatible index of 24 cultivars

Cultivar Number of pollen grains germinating per stigma Seed set () Compatible indexldquoQ10-22-2rdquo 37 plusmn 11 0 0ldquoNannonghongherdquo 60 plusmn 12 0 0ldquoQ07-26-3rdquo 67 plusmn 16 0 0ldquoQX-113rdquo 90 plusmn 13 0 0ldquoQ08-4-2rdquo 91 plusmn 13 0 0ldquoQX-004rdquo 98 plusmn 14 0 0ldquo260Shanghairdquo 132 plusmn 24 0 0ldquoQ10-6-9rdquo 150 plusmn 13 0 0ldquoQ10-17-3rdquo 170 plusmn 17 0 0ldquoQX-148rdquo 179 plusmn 15 0 0ldquoQX-145rdquo 198 plusmn 17 0 0ldquoQX-097rdquo 206 plusmn 23 0 0ldquoQX-149rdquo 228 plusmn 26 0 0ldquoQX-002rdquo 240 plusmn 29 0 0ldquoQX-118rdquo 296 plusmn 26 0 0ldquoQX-081rdquo 57 plusmn 13 003 plusmn 000 004 plusmn 000

ldquoQX-006rdquo 78 plusmn 13 003 plusmn 000 004 plusmn 000


ldquoQ10-33-2rdquo 102 plusmn 15 008 plusmn 002 013 plusmn 002

ldquoNannongxiangbinrdquo 123 plusmn 23 018 plusmn 005 022 plusmn 006

ldquoNannonghongchengrdquo 91 plusmn 15 050 plusmn 008 097 plusmn 012


ldquoNannongjinherdquo 363 plusmn 33 124 plusmn 032 173 plusmn 024


Values given are mean plusmn standard deviation

artificial pollination embryo development of this cultivarwasin particular investigated

26 Investigation on Self-Incompatibility of ldquoQ10-33-1rdquos Prog-eny The cultivar ldquoQ10-33-1rdquo produced a lot of seeds afterselfing in 2011 indicating that this cultivar is self-compatibleHowever we did not know if its progeny inherited its charac-teristics of self-compatibility We therefore also investigatedseed set compatible index and pistil receptivity of 10 ldquoQ10-33-1rdquos progeny in 2012 In the middle of March 2012 theselfing seeds of 9 cultivars were sown in a 1 2 (vv) mixture ofvermiculite and garden soil in the greenhouseThe daynighttemperature regime and the relative humidity were around2518∘C and 60ndash80 respectively [2] Two weeks later seedgermination rate was calculated At 5 weeks after sowing theseedlings at 6-leaf stage were transplanted to the Chrysan-themum Germplasm Resource Preserving Center NanjingAgricultural University China From early November 2012seed set compatible index and pistil receptivity of 10 ldquoQ10-33-1rdquos progeny were investigated according to the above-mentioned methods

27 Statistical Analysis The data were analyzed using theSPSS software 160 (SPSS Inc Chicago Ill USA) and wereshown as mean plusmn standard deviation

3 Results

31 Selfing Seed Set and Compatible Index of 24 Chrysanthe-mum Cultivars There are large differences in selfing seedset and compatible index among the 24 chrysanthemumcultivars (Table 1) Among them 15 cultivars did not produceany seeds after selfing As a consequence their seed set andcompatible index were 0 indicating that the 15 cultivars werecompletely self-incompatible Although the other 9 cultivarsproduced selfing seeds seed sets and compatible indexes offive cultivars (ldquoQX-081rdquo ldquoQX-006rdquo ldquoQX-003rdquo ldquoQ10-33-2rdquo andldquoNannongxiangbinrdquo) were very low much less than 05showing that the five cultivars were nearly self-incompatibleFor ldquoNannonghongchengrdquo ldquoQX-001rdquo and ldquoNannongjinherdquotheir seed sets and compatible indexes ranged from 050 to173 demonstrating that the three cultivars were partly self-compatible For ldquoQ10-33-1rdquo it was a highly self-compatiblecultivar as this cultivar had a very high seed set (5650) andcompatible index (8750) The results suggested that mostof chrysanthemum cultivars are self-incompatible or nearlyself-incompatible and only a small proportion of cultivars areself-compatible

32 The Germination Behavior of Pollen Grains on Stigmas of24 Chrysanthemum Cultivars The average number of pollengrains germinating on each stigma greatly varied amongthe 24 chrysanthemum cultivars (Table 1 Figures 1(a)ndash1(i))


(a) (b) (c)

(d) (e) (f)

Pg Pt

St

Sty

St

(g) (h) (i)

Figure 1 Germination behaviour of pollen grains on chrysanthemum stigmas at 24 hours after self-pollination ((a) (b) and (c)) Threerepresentative cultivars (ldquoQX-081rdquo ldquoNannonghongherdquo ldquoQX-006 2f-3rdquo and ldquoNannongxiangbinrdquo)with lownumber of pollen grains germinatingonper stigma ((d) (e) and (f))Three representative cultivars (ldquoNannongxiangbinrdquo ldquoQ10-6-9rdquo and ldquoQ10-17-3rdquo)withmediumnumber of pollengrains germinating on per stigma ((g) (h) and (i)) Three representative cultivars (ldquoQX-097rdquo ldquoQ10-33-1rdquo and ldquoQX-149rdquo) with high numberof pollen grains germinating on per stigma Abbreviations Pg pollen grain Pt pollen tube St stigma and Sty style All the figures havethe same bar (100120583m)

The lowest number was 37 in ldquoQ10-22-2rdquo and the highestwas 363 in ldquoNannongjinherdquo In addition the number ofpollen grains germinating on each stigma was not positivelyproportional to selfing seed set and compatible index Forexample ldquoQ10-33-1rdquo had the highest seed set and compatibleindex among these cultivars but the average number ofpollen grains germinating on each stigma was 219 thatwas lower than those of ldquoQX-149rdquo ldquoQX-002rdquo ldquoQX-118rdquo andldquoNannongjinherdquo Many pollen grains germinated on stigmasof ldquoQX-149rdquo ldquoQX-002rdquo ldquoQX-118rdquo but their seed sets andcompatible indexes were 0 One of the possible reasonsis that the pollen tubes of the three cultivars germinatedabnormally and failed to transfer sperms into embryo sacsfor double fertilization In contrast although ldquoQX-001rdquo hadonly 86 pollen grains that germinated on each stigma at24 h after artificial pollination it still produced some selfingseeds Therefore self-compatible cultivars usually have high

pistil receptivity or the average number of pollen grainsgerminating on each stigma However if chrysanthemumcultivars have high pistil receptivity it does not mean that thecultivars are self-compatible

33 Embryogenesis after Selfing Our previous studies indi-cated that chrysanthemum embryo development usuallyreaches the stage of globular embryo at around 10ndash15 dafter pollination if double fertilization occurs normally [13] However we did not observe any embryos from theinflorescences that were collected at two weeks after theywere bagged in the selfing experiment Instead we foundthat double fertilization did not happen in chrysanthemumembryo sacs as the embryo sacs were degenerating and thedegradation residues of synergids egg cells or central nucleicould be easily observed in the embryo sacs (Figures 2(a)and 2(b)) Such results ruled out the possibilities that low


Cc

Ec

Ds

(a)

Des

In

(b) (c)

Co

(d) (e) (f)

Figure 2 Ovary anatomy seeds and seedlings of chrysanthemum (a) An unfertilized embryo sac at two weeks after self-pollination (b) Adegenerative embryo sac at twoweeks after self-pollination (c) Self-pollinated seeds of ldquoQ10-33-1rdquo ((d) (e) and (f)) Progeny of self-pollinatedldquoQ10-33-1rdquo at different growth stages Abbreviations Cc central cell Co cotyledon Des degenerative embryo sac Ds degenerativesynergid Ec egg cell and In integument Bars 100 120583m ((a) and (b)) and 1 cm ((c)ndash(f))

seed set resulted from embryo abortion and confirmed self-incompatibility was the reason that chrysanthemum cultivarsdid not produce any selfing seeds

34 Germination of Selfing Seeds and Their Growth Selfingseeds began to germinate at around 5 days after sowing andmost of seeds germinated within two weeks after sowing(Figures 2(c) and 2(d)) Thereafter no seeds germinatedand germination rate of selfing seeds for each cultivar wascalculated The results indicated that selfing seeds from ldquoQX-081rdquo ldquoQX-006rdquo ldquoQX-003rdquo and ldquoNannongxiangbinrdquo did notgerminate (Table 2)The results combinedwith their seed setsand compatible indexes firmly confirmed that the four culti-vars were also self-incompatible Seed germination rates forother five cultivars (ldquoQ10-33-2rdquo ldquoNannonghongchengrdquo ldquoQX-001rdquo ldquoNannongjinherdquo and ldquoQ10-33-1rdquo) ranged from 239 to667 further confirming they were self-compatible Mostof seedlings reached 4-leaf stage and 6-leaf stage at around 3and 5 weeks after sowing respectively (Figures 2(e) and 2(f))Then progeny of ldquoQ10-33-1rdquo at 6-leaf stage were transplantedto the field for investigation on their self-incompatibility

35 Self-Incompatibility of ldquoQ10-33-1rdquos Progeny ldquoQ10-33-1rdquowas high self-compatible while there was comprehensiveseparation of self-incompatibility characteristics among its

Table 2 Germination rate of selfing seeds

Cultivars Seed germination rate ()ldquoQX-081rdquo 0ldquoQX-006rdquo 0ldquoQX-003rdquo 0ldquoQ10-33-2rdquo 667 plusmn 83

ldquoNannongxiangbinrdquo 0ldquoNannonghongchengrdquo 571 plusmn 65

ldquoQX-001rdquo 667 plusmn 72

ldquoNannongjinherdquo 275 plusmn 47

ldquoQ10-33-1rdquo 239 plusmn 43


progeny For example seed set and compatible index of ldquoQ10-33-1rdquo were 5650 and 8750 respectively (Table 1) How-ever seed set and compatible index of ten ldquoQ10-33-1rdquos progenywere 0ndash3723 and 0ndash6865 respectively (Table 3) Theaverage number of pollen grains germinating on each stigmaamong the ten progeny ranged from 140 to 3043 whichwas nearly positively proportional to seed set and compatibleindex (Table 3 Figures 3(a)ndash3(i)) The results suggested thatself-compatible chrysanthemum cultivars can produce bothself-compatible and self-incompatible progeny which is the


Table 3 Pistil receptivity seed set and compatible index of ten ldquoQ10-33-1rdquos progeny

Progeny Number of pollen grainsgerminating per stigma Seed set () Compatible index

ldquoQ10-33-1Ardquo 3043 plusmn 221 3723 plusmn 317 6865 plusmn 542

ldquoQ10-33-1Brdquo 1173 plusmn 160 3632 plusmn 265 5535 plusmn 386

ldquoQ10-33-1Crdquo 857 plusmn 104 2677 plusmn 224 4048 plusmn 351

ldquoQ10-33-1Drdquo 533 plusmn 124 797 plusmn 093 1464 plusmn 112

ldquoQ10-33-1Erdquo 547 plusmn 148 535 plusmn 071 425 plusmn 054

ldquoQ10-33-1Frdquo 140 plusmn 062 026 plusmn 004 041 plusmn 006

ldquoQ10-33-1Grdquo 273 plusmn 078 006 plusmn 002 008 plusmn 003

ldquoQ10-33-1Hrdquo 147 plusmn 068 003 plusmn 000 006 plusmn 000


ldquoQ10-33-11rdquo 720 plusmn 142 000 000Values given are mean plusmn standard deviation

Pg Pt

St

Sty

St

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 3 Germination behaviour of pollen grains on stigmas of nine ldquoQ10-33-1rdquos progeny at 24 hours after self-pollination ((a)ndash(i)) Q10-33-1Andash0 Abbreviations Pg pollen grain Pt pollen tube St stigma and Sty style All the figures have the same bar (100 120583m)


reason why there is wide separation of self-incompatibilityamong chrysanthemum The comprehensive separation ofself-incompatibility characteristics among selfing progenymay be mainly attributed to complex genetic background ofchrysanthemum cultivars that are allohexaploid species withan average chromosome number of 54

4 Discussion

Self-incompatibility is an important genetic mechanism toprevent plant inbreeding through preventing self-fertilization[12ndash15] When a viable pollen grain of a plant with self-incompatibility lands on a stigma of the same plant oranother plant with a similar genotype it fails to germinateor pollen tube growth is inhibited and consequently fertil-ization does not germinate and no seeds are produced [12]Self-incompatibility has been found in many families suchas Solanaceae Papaveraceae Rosaceae ScrophulariaceaeBrassicaceae Asteraceae and Convolvulaceae [12 16ndash21] Inthe present study although 15 chrysanthemum cultivars didnot produce selfing seeds it did not mean that they were self-incompatible Because seed production is closely related tothe events including pollen germination pollen tube growthovule fertilization and embryo development any abnormalevents can result in a total failure in seed production [22ndash25] Therefore in order to rule out the possibilities thatembryo abortion was not a factor leading to the failurein seed production of these chrysanthemum cultivars weinvestigated their embryo development at two weeks afterthe inflorescences were bagged The results indicated thatthe ovules were not fertilized at all and the embryo sacsunderwent degradation and thus embryo abortion was notthe reason for the failure in production of selfing seeds Inother words self-incompatibility was the reason of failurein production of selfing seeds and these cultivars wereconfirmed to be self-incompatible

Pollen germination and pollen tube growth on stigmasare closely related to self-incompatibility characteristics [1222 26] When a viable pollen grain of a plant reaches astigma of the plant and does not germinate the plant canbe easily regarded to be self-incompatible For example noviable pollen grains germinated on stigmas of Viburnummacrocephalum f keteleeri after self-pollination but the plantproduced many seeds after pollination with fresh viablepollen grains from another plant thus this species wasconsidered to be self-incompatible [27 28] However it isdifficult to identify whether a plant is self-incompatible ornot immediately if a pollen grain germinates on its stigmaas sometimes abnormal pollen tube growth is an indicatorof self-incompatibility and it is difficult to examine behaviorsof pollen tube growth in some species [22 29] Such a casehappened in chrysanthemum In this study it was found therewas no close relationship between self-incompatibility andthe number of pollen grains germinating on chrysanthemumstigma as selfing seed set of the 24 chrysanthemum cultivarswas not positively proportional to the number of pollengrains that germinated on stigmas For instance ldquoQX-118rdquohad a higher number of pollen grains that germinated on

stigmas compared with ldquoQ10-33-1rdquo while ldquoQX-118rdquo did notproduce selfing seeds and selfing seed set of ldquoQ10-33-1rdquowas as high as 565 (Table 1) Because it was difficultto discriminate morphological features of pollen tubes onstigmas between ldquoQ10-33-1rdquo and ldquoQX-118rdquo a possible reasonfor this discrepancy is that the growth of ldquoQX-118rdquo pollentubes was abnormal or inhibited and few pollen tubes couldgrow toward the embryo sac along the transmitting tissuesof style and as a consequence the ovules were not fertilizedIn contrast ldquoQ10-33-1rdquo pollen tubes grew normally andtransferred the sperms into embryo sacs for fertilization

There has been a heated argument over self-incom-patibility of chrysanthemum among chrysanthemum breed-ers Some of them think that chrysanthemum is completelyself-incompatible while the others believe that chrysanthe-mum is self-compatible [5ndash7]The results of the current studyindicated there was a wide separation of self-incompatibilityin chrysanthemum Most of chrysanthemum cultivars wereself-incompatible but a small portion of cultivars wereself-compatible demonstrating that chrysanthemum self-incompatibility is cultivar dependent Similar phenomenawere also found in some other species including Japaneseapricot peach and cabbage [30ndash32] Taken Japanese apri-cot as an example most of cultivars are self-incompatibleand some cultivars are self-compatible [30] The resultspresented here provide evidence that chrysanthemum self-incompatibility is cultivar dependentTherefore chrysanthe-mum breeders are suggested to emasculate inflorescencesduring chrysanthemum hybridization breeding in the futurethough emasculation is very laborious and sometime causesinjuries to inflorescences However if information shows thatchrysanthemum cultivars are self-incompatible then it isunnecessary to perform emasculation when these cultivarsare used as female plants The results in this study showthat fifteen cultivars without self-pollinated seeds and fourcultivars without germination of their self-pollinated seedsare self-incompatible (Tables 1 and 3)When the nineteen cul-tivars are used as maternal plants in hybridization breedingthey need not be emasculated

Self-pollinated seeds of four chrysanthemum cultivarsfailed to germinate many self-pollinated seeds of five culti-vars did not germinated and germination rate of ldquoQ10-33-1rdquo seeds was only 239 Tang once considered that somechrysanthemum ldquoseedsrdquo were not the real seeds becausethey did not contain embryos Such ldquoseedsrdquo were possiblyproduced from ovary tissues after they were stimulated bypollen-pistil interaction [33] Another possible reason is thatsome self-pollinated seeds did not contain normal embryosfor some previous studies showed that embryos frequentlydegenerated at different development stages in chrysanthe-mum hybridization breeding [1 3 34]We recently examinedanatomical features of some chrysanthemum self-pollinatedseeds and found that some of them really did not containany embryos and some others had abnormal embryos inmorphology (data no shown) Such results support the twoexplanations mentioned above In addition it was foundthat self-incompatibility separated widely among the self-ing progeny some progeny were self-incompatible whilesome were self-compatible A possible explanation for this


phenomenon is that chrysanthemum is an allohexaploidspecies with an average chromosome number of 54 andits genetic background is very complex [4 5] Becauseself-incompatibility of plants is genetically controlled by amultiallelic S locus [12] it is more possible that there is awide separation in self-incompatibility characteristics amongchrysanthemum selfing progeny There have been no stud-ies reporting molecular mechanism of chrysanthemum bynow and no information is available on chrysanthemumpollen self-incompatibility determinant and pistil S locuscomponent therefore it is impossible now to explain theunderlying reason of the separation in self-incompatibilityamong chrysanthemum selfing progeny

In summary we carried out a systematical investigationon pistil receptivity seed set and compatible index of 24chrysanthemum cultivars and ten selfing progeny of onecultivar in the current study Two finding are worth notingThe first one is that most of chrysanthemum cultivars wereself-incompatible and a small proportion of chrysanthe-mum cultivars were self-compatible The second one is thatthere is a comprehensive separation of self-incompatibilityamong progeny from the same self-pollinated self-compatiblechrysanthemum cultivar The findings suggest that emas-culation should be done during chrysanthemum hybridiza-tion breeding when information on its self-incompatibilitycharacteristics is unavailable However if a chrysanthemumcultivar is self-incompatible and is reproduced by vegetativepropagation it is unnecessary to do emasculation when usedas a female plant during hybridization breeding

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Fan Wang and Feng-Jiao Zhang contributed equally to thiswork

Acknowledgments

The authors thank Professor Xi-Jin Mu in Institute of BotanyChinese Academy of Sciences for his valuable discussions atearly stages of these experiments This study was supportedby the Programs for New Century Excellent Talents in Uni-versities Ministry of Education of China (NCET-11-0669)theNational Natural Science Foundation of China (31171983)the Fundamental Research Funds for the Central Universities(KYZ201308) and the Natural Science Foundation of JiangsuProvince (BK2010447)

References

[1] C Q Sun Z Z Huang Y L Wang et al ldquoOvercoming pre-fertilization barriers in the wide cross between Chrysanthemumgrandiflorum (Ramat) Kitamura and C nankingense (Nakai)Tzvel by using special pollination techniquesrdquo Euphytica vol178 no 2 pp 195ndash202 2011

[2] C Q Sun F D Chen N J Teng Z L Liu W M Fangand X L Hou ldquoInterspecific hybrids between Chrysanthemumgrandiflorum (Ramat) Kitamura andC indicum (L) DesMouland their drought tolerance evaluationrdquo Euphytica vol 174 no1 pp 51ndash60 2010

[3] C Q Sun F D Chen N J Teng Z L Liu W M Fang andX L Hou ldquoFactors affecting seed set in the crosses betweenDendranthema grandiflorum (Ramat) Kitamura and its wildspeciesrdquo Euphytica vol 171 no 2 pp 181ndash192 2009

[4] N O Anderson ldquoChrysanthemum Dendranthema grandifloraTzvelvrdquo in Flower Breeding and Genetics N O Anderson Edpp 389ndash437 Springer Amsterdam The Netherlands 2007

[5] H J Li Chrysanthemums in China Jiangsu Scientific andTechnical Press Nanjing China 1993

[6] J Y Chen Classification System for Chinese Flower CultivarsChina Forestry Press Beijing China 2001

[7] J Y Chen S Q Wang X C Wang and P W Wang ldquoThirtyyearsrsquo studies on breeding ground-cover chrysanthemum newcultivarsrdquo Acta Horticulturae vol 404 pp 30ndash36 1995

[8] C Q Sun F D Chen W M Fang Z L Liu and N J TengldquoAdvances in research on distant dybridization of chrysanthe-mumrdquo Scientia Agricultura Sinica vol 43 no 12 pp 2508ndash25172010

[9] X G Wang W M Fang F D Chen and N J Teng ldquoDetermi-nation of pollen quantity and features of pollendispersal for 41spray cut chrysanthemum cultivarsrdquo Acta Horticulturae Sinicavol 40 no 4 pp 703ndash712 2013

[10] Y Deng N Teng S Chen et al ldquoReproductive barriersin the intergeneric hybridization between Chrysanthemumgrandiflorum (Ramat) Kitam and Ajania przewalskii Poljak(Asteraceae)rdquo Euphytica vol 174 no 1 pp 41ndash50 2010

[11] N J Teng Y L Wang C Q Sun W M Fang and F D ChenldquoFactors influencing fecundity in experimental crosses of waterlotus (Nelumbo nuciferaGaertn) cultivarsrdquo BMC Plant Biologyvol 12 article 82 2012

[12] V E Franklin-Tong Ed Self-Incompatibility in FloweringPlantsmdashEvolution Diversity and Mechanisms Springer BerlinGermany 2008

[13] M Iwano and S Takayama ldquoSelfnon-self discrimination inangiosperm self-incompatibilityrdquo Current Opinion in PlantBiology vol 15 no 1 pp 78ndash83 2012

[14] BH J deGraaf S Vatovec J A Juarez-Dıaz et al ldquoThePapaverself-incompatibility pollen S-determinant PrpS functions inArabidopsis thalianardquo Current Biology vol 22 no 2 pp 154ndash159 2012

[15] M J Wheeler B H J de Graaf N Hadjiosif et al ldquoIdentifica-tion of the pollen self-incompatibility determinant in Papaverrhoeasrdquo Nature vol 459 no 7249 pp 992ndash995 2009

[16] CWang G Xu X Jiang et al ldquoS-RNase triggers mitochondrialalteration and DNA degradation in the incompatible pollentube of Pyrus pyrifolia in vitrordquo Plant Journal vol 57 no 2 pp220ndash229 2009

[17] Y Tarutani H Shiba M Iwano et al ldquoTrans-acting smallRNA determines dominance relationships in Brassica self-incompatibilityrdquo Nature vol 466 no 7309 pp 983ndash986 2010

[18] S Ikeda J B Nasrallah R Dixit S Preiss and M E Nas-rallah ldquoAn aquaporin-like gene required for the Brassica self-incompatibility responserdquo Science vol 276 no 5318 pp 1564ndash1566 1997

[19] S L Stone E M Anderson R T Mullen and D R GoringldquoARC1 is an E3 ubiquitin ligase and promotes the ubiquitination


of proteins during the rejection of self-incompatible Brassicapollenrdquo Plant Cell vol 15 no 4 pp 885ndash898 2003

[20] H Qiao H Wang L Zhao et al ldquoThe F-box protein AhSLF-S2 physically interacts with S-RNases that may be inhibited bythe ubiquitin26S proteasome pathway of protein degradationduring compatible pollination in Antirrhinumrdquo Plant Cell vol16 no 3 pp 582ndash595 2004

[21] B A McClure J E Gray M A Anderson and A E ClarkeldquoSelf-incompatibility inNicotiana alata involves degradation ofpollen rRNArdquo Nature vol 347 no 6295 pp 757ndash760 1990

[22] S Y Hu Reproductive Biology of Angiosperms Higher Educa-tion Press Beijing China 2005

[23] C Wilcock and R Neiland ldquoPollination failure in plants why ithappens and when it mattersrdquo Trends in Plant Science vol 7 no6 pp 270ndash277 2002

[24] T B Batygina Embryology of Flowering Plants Terminologyand Concepts Volume 1 Generative Organs of Flower SciencePublishers Enfield NH USA 2002

[25] Y L Wang Z Y Guan F D Chen W M Fang and N J TengldquoPollen viability pistil receptivity and embryo development inhybridization ofNelumbo nuciferaGaertnrdquoThe Scientific WorldJournal vol 2012 Article ID 678706 8 pages 2012

[26] M A Samuel Y T Chong K E Haasen M G Aldea-BrydgesS L Stone and D R Goring ldquoCellular pathways regulatingresponses to compatible and self-incompatible pollen in Bras-sica and Arabidopsis stigmas intersect at exo70a1 a putativecomponent of the exocyst complexrdquo Plant Cell vol 21 no 9pp 2655ndash2671 2009

[27] B Jin L Wang J Wang et al ldquoThe structure and roles of sterileflowers in Viburnum macrocephalum f keteleeri (Adoxaceae)rdquoPlant Biology vol 12 no 6 pp 853ndash862 2010

[28] B Jin N Li N Jia W Z Zhou L Wang and C B ShangldquoObservations on the anatomy of reproductive organs and thepollinators of Viburnum macrocephalum f keteleeri (Caprifoli-aceae)rdquoActa Phytotaxonomica Sinica vol 45 no 6 pp 753ndash7682007

[29] J L Meng Genetics of Plant Reproduction Science PressBeijing China 1997

[30] P P Wang T Shi Z H Gao Z Zhang and W B ZhuangldquoInsertion mutation of pollen SFB gene in self-compatibility ofJapanese apricot cultivars native to Chinardquo Acta HorticulturaeSinica vol 39 pp 413ndash460 2012

[31] G G Xu H Q Wu J Wu C Wang K J Qi and S L ZhangldquoMolecular mechanism and genetic characterization of self-compatibility in peachrdquo Acta Horticulturae Sinica vol 39 pp1035ndash1044 2012

[32] Z Y Fang YM Liu LM Yang et al ldquoCabbage self-compatibleline lsquoZhonggan 87-534rsquordquo Acta Horticulturae Sinica vol 39 pp2535ndash2536 2012

[33] D Tang ldquoAn inquiry into the lower fruiting and germinationrate of Chrysanthemum cvsrdquo Journal of Southwest ForestryCollege vol 20 pp 200ndash204 2000

[34] S Fukai T Nagira and M Goi ldquoCross compatibility betweenchrysanthemum (Dendranthema grandiflorum) and Dendran-thema species native to Japanrdquo Acta Horticulturae vol 508 pp337ndash340 2000

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Food ScienceInternational Journal of

Agronomy


International Journal of



Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of


Plant GenomicsInternational Journal of


Biotechnology Research International


Forestry ResearchInternational Journal of


Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of


Veterinary Medicine International


Cell BiologyInternational Journal of


Evolutionary BiologyInternational Journal of



petals) of all peripheral ray florets should be entirely cut offby scissors until the stigmas are visibleThen the emasculatedinflorescences are bagged [3]Thus the emasculation of chry-santhemum inflorescences not only is very laborious butalso sometimes causes injuries to the inflorescences as aconsequence resulting in low breeding efficiency In additionthe emasculation process significantly reduces the numberof florets that can be used for production of hybrid seeds[9 10] For example if chrysanthemum is self-incompatiblethe small central disk florets can be also used for productionof hybrid seeds because it is unnecessary to remove themTherefore it is very urgent and necessary to reveal if chrysan-themum is self-incompatibility or not

According to our knowledge there have been no studiessystematically investigating self-incompatibility of chrysan-themum so far We therefore carried out a systematicalinvestigation on pistil receptivity seed set and compatibleindex of 24 chrysanthemum cultivars with high ornamentalvalues in this study In addition we also examined pistilreceptivity seed set and compatible index of some progenyof one chrysanthemum cultivar The purpose of this studyis to solve the above-mentioned argument among chrysan-themum breeders through identifying chrysanthemum self-incompatibility and related cellular mechanisms In additionwe also hope that the expected results will provide valu-able information for increasing breeding efficiency duringchrysanthemum hybridization breeding in the future

2 Materials and Methods

21 Experimental Materials Twenty-four chrysanthemumcultivars with high ornamental values were used as materialsin this study They were grown in the ChrysanthemumGermplasm Resource Preserving Center Nanjing Agricul-tural University China (32∘051015840N 118∘901015840E)

22 Determination of Selfing Seed Set and Compatible Indexthrough Selfing Experiment In order to get some informationon seed set and compatible index of the 24 chrysanthemumcultivars we first carried out selfing experiment The selfingexperiment was performed in early November 2011 Twentyto thirty inflorescences were randomly selected from eachcultivar and then bagged with waterproof and breathablepaper bags at around 3 days before anthesis Cultivarsrsquonames date and the names of researchers were markedon the vegetable parchment tied with each inflorescenceAlthough the paper bags were waterproof and breathablerelative humidity sometimes was much higher inside thanoutside bags Therefore a small hole was made from eachbag to prevent mildew After two weeks all the bags wereremoved from the inflorescences because they had lost pistilreceptivity Around two months later (in the middle ofJanuary 2012) the inflorescences were cut off from plantsfor seed collection After that seed set (the average seednumber per inflorescencethe average floret number perinflorescence) and compatible index (the seed number ofall inflorescencesthe number of all inflorescence) were thencounted

23 Artificial Self-Pollinated Experiment Because it was dif-ficult to know the exact dates of anther dehiscence andpollen dispersal in the selfing experiment it was very difficultto determine pollen-stigma interaction or pistil receptivityWe therefore did artificial self-pollinated experiment as thepollination time can be controlled In the artificial self-pollinated experiment five inflorescences were randomlyselected from each cultivar at around 3 days before anthesisthen emasculated and bagged Around three days later whenthe shape of pistils became Y it meant that the pistils hadstrong receptivity [3 5] At this time artificial self-pollinatedexperiment was performed with fresh mature pollen grainsthat were collected from mature inflorescences on the sameplant or other plants of the same cultivar After artificialpollination the inflorescences were bagged again

24 Determination of Pistil Receptivity Because our previ-ous study indicated that the number of germinated pollengrains on chrysanthemum stigmas reached the peak at 24 hafter artificial pollination we thus here collected the self-pollinated inflorescences at 24 h after pollination in theartificial self-pollinated experimentThe self-pollinated inflo-rescences were cut off from each cultivar and immediatelyfixed and stored in FAA solution The pistils were strippedout softened in 1molL NaOH solution for 8 h at roomtemperature then washed with distilled water one time andimmersed in aniline blue staining fluid (01molL K

3PO4+

18 glycerol) for one to two hours in the dark [1 11] Afterthat the pistils were placed on the glass slides and gentlypressed with coverslips Then the germination behaviors ofpollen grains on stigmas and the growth of pollen tubes wereobserved under a fluorescence microscope (Zeiss Axioskop40) Good representative images were captured with anAxiocam MRC camera For each cultivar 30 pistils wereobserved and the numbers of germinated pollen grains oneach stigma were recorded

25 Examination of Embryo Development after Chrysanthe-mum Selfing Because low seed set during selfing experi-ments may be caused by two factors that is self-incom-patibility (pistil receptivity or pollen-stigma interaction) andembryo abortion we examined embryo development afterchrysanthemum selfing for the purpose of ruling out thepossibilities that low seed set was caused by embryo abortionAt two weeks after the inflorescences were bagged in theselfing experiment five inflorescences were sampled fromeach of 24 cultivars and immediately fixed in FAA solution(95 ethanol distilled water formalin glacial acetic acid =63 27 5 5) until use Ovaries were collected form the floretsof each inflorescence and subjected to dehydration througha graded series of ethanol solutions and then immersed inparaffin wax After that sections were cut to a thicknessof 8ndash10 120583m and stained with Heidenhainrsquos hematoxylin andthen observed and photographed under an Olympus BX41microscope [1 3] Because the cultivar ldquoQX-118rdquo did notproduce any selfing seeds and there were a very high numberof pollen grains germinated on its stigmas at 24 h after
















3 Results




(a) (b) (c)

(d) (e) (f)

Pg Pt

St

Sty

St

(g) (h) (i)






Cc

Ec

Ds

(a)

Des

In

(b) (c)

Co

(d) (e) (f)


























Pg Pt

St

Sty

St

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)




4 Discussion













Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014







































3 Results




(a) (b) (c)

(d) (e) (f)

Pg Pt

St

Sty

St

(g) (h) (i)






Cc

Ec

Ds

(a)

Des

In

(b) (c)

Co

(d) (e) (f)


























Pg Pt

St

Sty

St

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)




4 Discussion













Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014

























(a) (b) (c)

(d) (e) (f)

Pg Pt

St

Sty

St

(g) (h) (i)






Cc

Ec

Ds

(a)

Des

In

(b) (c)

Co

(d) (e) (f)


























Pg Pt

St

Sty

St

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)




4 Discussion













Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014

























Cc

Ec

Ds

(a)

Des

In

(b) (c)

Co

(d) (e) (f)


























Pg Pt

St

Sty

St

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)




4 Discussion













Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014





































Pg Pt

St

Sty

St

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)




4 Discussion













Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014


























4 Discussion













Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014































Acknowledgments


References







































Journal of




Agronomy





Microbiology




Volume 2014











































Journal of




Agronomy





Microbiology




Volume 2014
























Research Article Identification of Chrysanthemum...

Documents

Transcript of Research Article Identification of Chrysanthemum...