单倍体细胞培养技术在禾草育种中的应用

单倍体细胞培养技术

在禾草育种中的应用

单倍体细胞培养主要用花药/小孢子/未受精子房在人工培养基上进行培养。可以直接发育成胚状体，然后长成单倍体植株；或者通过愈伤组织诱导分化出芽和根，最终长成植株。单倍体细胞培养在植物育种中已取得了很大成就。

1964年，Guha和

Maheshwari

将曼陀罗的成熟花粉培养在适当的培养基上，发现花粉能转变成活跃的细胞分裂状态，从药室中长出胚状体，最终得到胚状体植株。使细胞全能性理论在生殖细胞水平上得到验证。microsporemothercellMeiosismicrospores(n)firstmitosissimilarnucleinuclearfusiondiploidembryogenerativenucleustubenucleusnormalgerminationtubenucleusdividesgenerativenucleusdisintegratestubenucleusdisintegratesgenerativenucleusdivideshaploidembryoMicrosporeshavetheremarkablecapacitytodevelopintohaploidsviaembryogenesisinvitro.Stresstreatmentactsasatrigger.Gametophytictosporophyticpathway（配子体---孢子体）PollenDevelopmentABtalldefDiseaseresistanceabtallDefDiseasesusceptibility

Geneset1Geneset2ABtalldefDiseaseresistanceABtalldefDiseaseresistance

Geneset1Geneset2

Eachcellcontains2setsofgeneticinformationwhicharenotidentical.

Adoubledhaploidplantshascellscontaining2genesetswhichareexactlyidentical.Whatisadoubledhaploidplant?ABtalldefDiseaseresistanceABtalldefDiseaseresistanceABtalldefDiseaseresistance

Howaredoubledhaploidsproduced?Pollenhasonlyonesetofgeneticinformation(haploid)Thisgeneticinformationisduplicatedtoproducea“doubledhaploid”DiseaseResistantHighYieldDiseaseResistantHighYieldDiseaseResistantPoorYieldDiseaseSusceptibleHighYieldDiseaseSusceptiblePoorYieldDiseaseResistantHighYieldDiseaseResistantHighYieldDiseaseResistantHighYieldDiseaseResistantHighYieldHowdodoubledhaploidsacceleratebreeding?

PlantsselectedfromaconventionalbreedingpopulationdonotbreedtrueSelectedplantProgeny:(Discard)(Discard)(Discard)(Select)Progeny:(Select)(Discard)(Discard)(Discard)Continuefor10-12generationsuntilalloffspringbreedtruetotypePlantselectedfromadoubledhaploidbreedingpopulationalwaysbreedtrueSelectedPlantProgeny:Thisattributeofdoubledhaploidsallowsnewcultivarstobereleased3-5yearsearlierthanthosewithconventionalbreeding.DiseaseSusceptibleHighYieldDiseaseSusceptibleHighYieldDiseaseSusceptiblePoorYieldAdvantagesofDoubledHaploidBreeding

Timesavingbreedingmethod

-uniformityreachedimmediately

-yieldtrialswithinseveralyears

-seedmultiplicationsafeandfast

Increasedselectionefficiency

-facilitateselectionofmajorgene

-selectionofqualitativemajorgenecharacters

-especiallyselectionofquantitativecharacters

Targetvaluebreeding

-efficientuseofeliteparents

Invitromethods:Antherculture(androgenesis)-productionofhaploidplantsfrommicrosporesAnthercultureforproductionofhaploidsreportedinabout250speciesSolanaceae,Cruciferae,Gramineae,mostcommonOvuleculture(gynogenesis)productionofhaploidplantsfromunfertilizedeggcellHaploidPlantFormationFactorsinfluencingandrogenesisGenotypeofdonorplantsAntherwallfactorsCulturemediumandculturedensityStageofmicrosporeorpollendevelopmentEffectoftemperatureand/orlightPhysiologicalstatusofdonorplantWheatanthercultureFestuloliumanthercultureTimothyanthercultureProductionofembryos(calli)andregeneratedplantsfromanthercultureofdifferenttimothygenotypes.

____________________________________________________________________________________GenotypeEm/100anthers+SD*

Albinoplants

Greenplants Source

(Total)

(Total)

____________________________________________________________________________________Adda 870

+121

>600 150 IcelandKämpeII

610

+207

many 11 SwedenSaga 420

+55

26 0

Sweden57A29 390

+65

36 0 FinlandVåti7701 250

+40

110 4 NorwayVåti7702 200

+45

56 0 NorwaySvÅ0918 170

+41

90 4 SwedenGrinstadt 140

+39

many 29

NorwayAlma

86

+29

20 86

FinlandIki

75

+26

24 8 FinlandBodin

26

+11

45 0 NorwaySvÅ0896

17

+12

12 0

SwedenTopas

5

+3

27 0 HollandHJA80102

21

+9

many 3 FinlandHJA1341

2

+2

many 1

Finland120B8

15

+6

many 10

Finland____________________________________________________________________________________*SD:standarddeviation.Ploidyleveloftheregeneratedtimothyplantscv.Adda.____________________________________________________________Samples Numberofdihaploids

Numberofhaploids

(2n=6X=42)

(n=3X=21)______________________________________________________41

24(58.5%)

14(41.5%)______________________________________________________Effectofmicrosporedevelopmentalstageonmicrosporecellvital,microsporedivisionandembryogenesisinmicrosporecultureintimothycv.Grinstadt.

____________________________________________________________________________Stage

AverageSwollencell%Divisioncells/dishEmbryos/dish____________________________________________________________________________Earlyuninucleate 14.8µm

7+3* 64+15

11+5Middleuninucleate16.3µm 11+3 112+29

49+16Lateuninucleate 18.1µm 34+11 395+27 124+38Earlybinucleate 19.0µm 59+14 511+52 294+45Middlebinucleate 19.0µm 29+8 170+33

65+24____________________________________________________________________________*Standarddeviation

Effectofcarbohydrateonmicrosporeswollen,divisionandembryogenesisoftimothymicrosporeculturecv.Grinstadt._______________________________________________________________________________Carbohydrate Microsporeswollen%

Divisioncells/dish Embryos/dish_____________________________________________________________________Maltose 65+14%* 490+66 230+51Sucrose 31+11% 160+29

90+18Glucose 37+9% 180+35

49+9_____________________________________________________________________*Standarddeviation

Effectoflightintensityonplantregenerationintimothyantherculture._______________________________________________________________Lightintensity Culturedcallus

Albinoplants

Greenplants(

molm-2s-1)

number

(%) (%)_______________________________________________________________High(94.7)

1620

520(32.10%) 23(1.42%)Low(5.5)

1740 710(40.80%) 86(4.94%)_______________________________________________________________GenotypeAddawasused.Dataistheaverageof4experiments.CompositionofPG-96basalmedium_______________________________________________________ Components mg

l-1_______________________________________________________KNO3 1500(NH4)2SO4 150KH2PO4 125MgSO4.7H2O 200Ca(NO3)2.4H2O 200KCl 50 FeSO47H2O 27.8Na2EDTA.2H2O 37.3 MnSO4.4H2O 10ZnSO4.7H2O 3H3BO3 5KI 0.83NaMoO4.2H2O 0.25CuSO4.5H2O 0.025CoCl2.6H2O 0.025 Ascorbicacid(VitaminC) 10Proline 20Asparticacid 10Citricacid 10Biotin 0.05 Glycine 2Inositol 100Thiamine-HCl 10Pyridoxine-HCl 1L-Glutamine 500Nicotinicacid 0.5Glutathione 10L-Serine 20Caseinhydrolysate 200L-Alanine 10 2,4-D 1.5Kinetin 0.5 Maltose 90,000-130,000pH 5.7_______________________________________________________KM8PMediaForProtoplastCultureEffectofcoldpretreatmentatdifferentlengthsofexposureat4degreesonembryoyieldoftimothycv.AddaandKämpell180878106504302403801906103501606900100200300400500600700800900123456Coldpretreatmenttime(weeks)Embryoyield/100budsAddaKämpellGuoetal.,PlantCellReports19:761-767Guoetal.,PlantCell,TissueandOrganCulture57:85-93

RyeisolatedmicrosporecultureMorphologicalanalysisofDHplants

inmicrosporecultureofrye_____________________________________________________________________________Plant

Height

Tillernumber

DHplants

Height

Tillernumber_____________________________________________________________________________

1

74.1(cm)

9

1 48.0(cm) 16

2

86.5 11

2 47.5 21

3

79.0

8

3 49.3 18

4

77.3

8

4 57.6 15

5

78.3

9

5 49.1 18

6

75.2 10

6 55.6 17

7

72.6 10

7 50.6 20

8

80.7

6

8 43.9 16

9

81.5 10

9 49.3 19

76.5 11 10 47.5 17Average 78.2

9.2 Average 49.8 17.7_____________________________________________________________________________________GenotypeAmilowasusedinthistest.EffectofosmoticpressureinPG-96Minductionmediumonmicrosporesurvivalpercentageinryemicrosporeculture.70607441554301020304050607080901000.1090.1740.2210.2820.3440.3942%4%6%8%10%12%Maltosepercentageandosmolalityvalueofinductionmedium.MicrosporesurvivalpercentageaftertwodaysProductionofembryos/calliandregeneratedplantletsfrom11respondinggenotypesinryemicrosporeculture(PG-96M)._____________________________________________________________________________Genotype

Androgenicembryogenesis Greenplants Source (embryos/calliperdisha) (total)______________________________________________________________________