Pollen viability, storage & germination -A basic idea

This PowerPoint offers a bird's eye view about pollen grains, its viability and storage with special reference to cryopreservation.

1. Welcome to
Pollen viability, Storage & Germination
ByN.Sannigrahi, Associate Professor,
Department of Botany, Nistarini College, Purulia,
723101(W.B) India
Theabilityofthelivingorganismto
maintainitselforrecoverits
potentialitiesinspiteoftheharsh,
unhealthy&hostileconditionsforthe
sakeofsurvivalisdefinedasviability.

2. Survival of the fittest

3. POLLEN VIABILITY
•Maintenanceofviabilityandfertilizingabilityof
pollenisaseriousconcernoftheplantbreedersand
cryopreservationmethods.Pollenviabilityisanindex
ofitsqualityandvigorofthepollengrains.The
taxonomicstatusoftheplantandtheabiotic
environmentalconditionsgenerallydirectsthedegree
oftheviabilityofthepollengrains-eithershort
durationofminutesorlongdurationofyears.Inorder
tomaintainthelongtermviability&fertilizing
capacity,specialstorageandpreservationisrequired.

4. Pollen grain & Germination

5. CLASSIFICATION OF POLLEN ON THE BASIS OF
VIABILITY
•ThePlantgenome&externalenvironmentalconditionsplay
thecrucialroleonthepollenviability.Harrington(1970)non
thebasisofpollenlongevityclassifiedplanttaxaintothree
maincategories-
•1.Longlivedpollen-(6months-1year)includesthemembers
ofGinkgoaceae,Pinaceae,Arecaceae,Saxifragaceae,
Rosaceae,Fabaceae,Anacardiaceae,Vitaceae,Primulaceae.
•2.Pollenwithmediumlifespan(1-3months)includes
membersofLiliaceae,Amarylliadaceae,Salicaceae,
Ranunculaceae,Brassicaceae,Rutaceae,Scrophulariaceaeand
Solanaceae.
•3.Shortlivedpollens(fewminutes–coupleofdays)
includesmembersofAlismataceae,Poaceae,
Cyperaceae,Commelinaceae&Juncaceae

6. FACTORS OF LOSS OF VIABILITY
•Anumberofcausesarebehindthelossofpollenviability
asbelow:
•1.Deficiencyofrespiratorysubstrates
•2.Inactivationofcertainspecificenzymesorgrowth
hormones
•3.Differentgeneticfactorsi.epollencytology
•4.Environmentalconditionslikehumidity&temperature.
•Butexceptionobservedincerealsbeingshortlivedin
spiteoftheirhavingabundantmetabolitesloosetheir
viabilityalongwithchangesofaminoacidcompositionof
storedpollenfailtoexplainthelossofviability.

7. CAUSES OF LOSS OF VIABILITY
•1.Biochemicalalterationofpollen-Deficiencyofthe
respiratorysubstratesduetocontinuousmetabolic
activity.Aconsiderablechangesintheamountof
carbohydrates,aminoacidsandorganicacidslevelinthe
pollenofthedifferentspecieshavebeenreportedasfaras
thetimeofthepollenstorageisconcerned.Ahigher
respiratoryrateinthethreecelledpollenleadstoa
scarcityofrespiratorysubstrate.Inadditiontothat,the
consistentincreaseofasparticacids,aminobutyricacid,
ethanolamine,isoleucine,leucine,lysineand
phenylalaninewhilealanine,glycine.Glutamicacidand
prolinecontentsdecreasereportedinstorageandviability.
Thedecreaseofgerminabilityduetothedeficiencyof
respiratorysubstratefollowedbytheinactivationof
enzymeslikeamylase,phosphatsesassociatedwiththe
degradationofreservesstoredinpollengrains.

8. CAUSES----
•Desiccation&lossofmembraneintegrityofpollen-
Thewatercontentisoneoftheregulatorofviability
oftheplantcells.
•Desiccationtolerant-thatremainviableafter
dehydration
•Desiccationsensitive-lossviabilityparallelto
dehydration.Thewatercontent(15-35%),Veryhigh
inPoaceaepollenabout30-60%ofthepollengrains
variesfromspeciestospecies.Thusthepollengrains
ofgrassesarehighsensitivetogreaterdegreeof
desiccationandnormalfunctionduetorehydration.

9. Extended Pollen viability under cryogenic temperature and relative
humidity
Taxa Storage
temperature ⁰C
RH(%) Duration of storage
Caricapapya 1 10 153 days
Cocosnucifera 5 40 1.5 years
Lycopersicon
esculantum
2-4 10 252 days
Pyrusmalus 2-8 10 673 days
Rhodendronsp. 2 25 124 days
Vitisvinifera 10 25 2 years

10. POLLEN STORAGE
•Pollenstorageparticularlyinthedomainofinvitrotechnique
isveryimportantanditstartedattheendof19
th
century.Large
numberofcropspecies,forgeandcereals,forfiberandfruit
crops,forgeandcereals,pollenstrategiesarealsoimportant.
GeneticenhancementofhorticulturalcropneedsPollen
Cryobankfromwhichwecandrawpollenparentsofchoicein
theprocessofbreedingprogramme.
•Storageofpollengrainsretainingviabilityneedstwokindsof
practices.
•1.Shorttermstorage
•2.Longtermstorage

11. Short term storage
•Itincludeseffectoftemperature&humidity,pollen
storageinorganicsolvent
•A.Lowtemperatureandrelativehumidityare
favorableformostofthetaxabutalargenumberof
taxacanbesuccessivelystoredinalimitedperiodof
timethroughthemanipulationoftemperatureand
humidity.Unsealedcontainerswithsuitable
dehydratingagentslikesilicagel,variousconc.Of
sulphuricacidandconc.Saltsareusedtomaintain
relativehumidity.

12. Conditions of pollen storage
•Pollenstorageinorganicsolvent
•Organicsolventisveryimportantforpollenstorage.Iswanami
&Nakamura(1972)firstdemonstratedtheuseoforganic
solventinthisregard-benzene,petroleum,diethylether,
acetone,chloroform,cyclohexaneandotherorganicsolvents
areveryusefulinthisregard.
•1.Citruspollenmaintainedviabilityindifferentsolventsfor
threemonths
•2.Chrysanthimumpollenlosesviabilityindryconditionsbut
iftreatedwithdiethylether,itincreasesviability.
•Storageinorganicsolvents,thereisnolossofviability
referredasabsolutedormancy(Iwanami,1984).
•Plantsolventhasprovedtobebetterforstorageofanypollen
thanlowtemperature&humidity.

13. Effect of organic solvents on pollen tube growth
TreatmentLilium
auratum
Lilium
auratumvar
platyphyla
L.longifloru
m
Camellia
sasanqua
Impatiens
balsamina
Fresh pollen11.8(mm) 14.2(mm) 10.4(mm) 2.1(mm) 2.1(mm)
Solvents ----Average
tube length
--after 24h
at 28⁰C
Acetone 14.4 21.6 16.4 6.8 2.2
Benzene 15.2 16.1 12.4 6.1 2.3
Petroleum
benzene
16.2 15.9 16.0 6.3 2.2
Petroleum
ether
16.3 15.2 15.1 6.3 2.2
Chloroform15.1 16.0 12.4 5.0 2.1
Benzyl
alcohol(Inhi
bit growth)
0.0 0,0 0.0 0.0 0.0
Ethanol 4.0 1.9 5.9 4.2 0.1
Methanol 0.6 0.7 0,3 0.7 0.1

14. LONG TERM STORAGE
•Storageofpollengrainsabove0⁰Cslowsdownmetabolic
activityofpollen,resultingthegradualdecreaseandfinally
totallossofpollenviability.Thuslongtermpollenstorage
requiresCryogenictechniquesfortheeffectiveandlongterm
pollenpreservation.
•1.usingthestoragetemperatureof-10⁰cand34⁰c,the
longevityofthebi-cellularpollenandpollenwithoriginallow
contentofthemoisturehasbeensuccessivelyextended
between1-3years.
•2.Lyophilization-Freezedryingorvacuumdryinginvolves
rapidfreezingofpollentosubzerotemperaturesof-60⁰Cto-
80⁰Cusinginertheliumornitrogenandthegradualremovalof
waterundervacuumsublimation.Innumberoftaxa,freeze
dryingwithcombinedlyophilisationpromisesbetterpollen
storageandpreservation.

15. LONG TERM PRESERVATION
•Cryopreservation
•Areductioninthepollenwatercontentbelowathresholdlevel
belowbeforelowtemperatureexposureseemstobeimportant
forachievingviability.Thuspartiallydehydratedpollen
possesslessfreezablewaterandcansurvivedeepfreezing
Lowtemperaturebelow-70to-196Cisveryeffectiveinthis
regardthatcanincreasethepollenstorageandviability.A
reductionofthepollenwatercontentbelowlowtemperature
exposureseemstobeimportantforachievingviability.Thus
partiallydehydratedpollenpossesseslessfreezablewaterand
cansurvivedeepfreezing.Anumberofpollengrainshave
beenpreservedsuccessfullybycryopreservationlikeBeta
vulgaris,Brassicaoleracea,Caricapapaya,Glycinemax,
Prunusperica,Viciafaba,Zeamays,Helianthusannus,Pyrus
malusetc.

16. Taxa Storage temperature Storageduration
Beta vulgaris -196⁰C 1 year
Brassica olracea -196 16 months
Capsicum annum -196 42 months
Carica papaya -196 485 days
Glycine max -196 21 days
Helianthus annus -196 4 years
Lycopersiconesculentum-196 1062 days
Prunus persica -196 1 year
Pyrus communis -196 7 months
Pyrus malus -196 673 days
Solanumtuberosum -196 2 years
Vicia faba -196 1 month
Zea mays -196 10 years

17. Estimation of Pollen viability
•Pollenviabilityisanimperativefactorinthestudyofreproductive
biologybeforeadoptionanybiologicalprogrammefordifferent
attributes.Theveryquick&dependablemethodtodeterminethe
viabilityofthepollengrainisveryimportantforanykindof
successfulbreedingprogramme.Anumberofmethodsareemployed
forthesameasfollows-
•Invitrogerminationtest-Observationofpollentubeafter
germinationundermicroscope.
•Pollengerminationonstigma-Growthofthepollentubethrough
style.
•Enzymesassaymethod-Activityofcertainenzymeslike
dehydrogenasebythetriphenyltetrazoliumchloride(TTC)
•Fruit&seedtest-Degreeofsettingfruitbyeffectivefertilization
•Tetrazoliumtest-by2,3,5-triphenyltetrazoliumchloridetest
•Fluorochromaticreactiontest(FCR)orFluoresceindiacetate
test(FDA)

18. Continuation----
•Itisbasedonthereductionofsolublecolourlesstetrazolium
salttoreddishinsolubleformazaninthepresenceof
dehydrogenase.Ifthepollengrainsareincubatedin
tetrazoliumsolution(0.1-1%)for30-60minutesat30-37⁰C,
viablepollengrainstakeareddishcolourduetotheformation
offormazen.Since,thetetrazoliumsaltscanbeeasilyreduced
bylight,itisnecessarytokeepthesolutionandthepollen
grainsindarkness.
•Inadditiontothat,anothertestnon-polar,non-fluorescent
Fluoresceindi-O-acetatecanbereadilyhydrolysedbyacetyl
esteraseactivitytofluorescein.Thisisalsousedforthetesting
oftheviabilityofthepollengrains.

19. POLLEN GERMINATION
•Pollen germination is a systematic pathway that involves the
following steps-
•Pollen –pistil interaction
•Pollen attachment & Hydration
•Pollen germination & tube growth
•Recognition of the Pollen by the stigma
•Pollen stigma interactions
•The stigmatic surfaces provides essential prerequisites for a
successful germination. In wet stigma, the role of the stigmatic
exudates in pollen germination is highly variable. In dry
stigma, the pellicle plays a vital role in germination. A number
of biophysical pathways are involved in this regard.

20. Thanks a lot for your journey with me.