The document discusses pollen viability, storage, and germination. It defines viability as the ability of an organism to survive harsh conditions. Pollen viability depends on the plant's taxonomy and environment. Methods for short-term pollen storage include controlling temperature and humidity. Long-term storage uses cryopreservation techniques like freezing pollen. Factors like desiccation and biochemical changes cause pollen to lose viability over time. The document also describes classifying pollen based on longevity and methods for estimating viability, including tetrazolium and fluorescence tests.

1. Welcome to
Pollen viability, Storage & Germination
By N.Sannigrahi, Associate Professor,
Department of Botany, Nistarini College, Purulia,
723101(W.B) India
The ability of the living organism to
maintain itself or recover its
potentialities in spite of the harsh,
unhealthy & hostile conditions for the
sake of survival is defined as viability.

2. Survival of the fittest

3. POLLEN VIABILITY
• Maintenance of viability and fertilizing ability of
pollen is a serious concern of the plant breeders and
cryopreservation methods. Pollen viability is an index
of its quality and vigor of the pollen grains. The
taxonomic status of the plant and the abiotic
environmental conditions generally directs the degree
of the viability of the pollen grains-either short
duration of minutes or long duration of years. In order
to maintain the long term viability & fertilizing
capacity, special storage and preservation is required.

4. Pollen grain & Germination

5. CLASSIFICATION OF POLLEN ON THE BASIS OF
VIABILITY
• The Plant genome & external environmental conditions play
the crucial role on the pollen viability. Harrington (1970)n on
the basis of pollen longevity classified plant taxa into three
main categories-
• 1. Long lived pollen-(6 months-1 year) includes the members
of Ginkgoaceae, Pinaceae, Arecaceae, Saxifragaceae,
Rosaceae, Fabaceae, Anacardiaceae, Vitaceae, Primulaceae.
• 2. Pollen with medium life span(1-3 months) includes
members of Liliaceae, Amarylliadaceae, Salicaceae,
Ranunculaceae, Brassicaceae, Rutaceae, Scrophulariaceae and
Solanaceae.
• 3.Short lived pollens(few minutes –couple of days)
includes members of Alismataceae, Poaceae,
Cyperaceae, Commelinaceae & Juncaceae

6. FACTORS OF LOSS OF VIABILITY
• A number of causes are behind the loss of pollen viability
as below:
• 1. Deficiency of respiratory substrates
• 2. Inactivation of certain specific enzymes or growth
hormones
• 3. Different genetic factors i.e pollen cytology
• 4.Environmental conditions like humidity & temperature.
• But exception observed in cereals being short lived in
spite of their having abundant metabolites loose their
viability along with changes of amino acid composition of
stored pollen fail to explain the loss of viability.

7. CAUSES OF LOSS OF VIABILITY
• 1. Biochemical alteration of pollen-Deficiency of the
respiratory substrates due to continuous metabolic
activity. A considerable changes in the amount of
carbohydrates, amino acids and organic acids level in the
pollen of the different species have been reported as far as
the time of the pollen storage is concerned. A higher
respiratory rate in the three celled pollen leads to a
scarcity of respiratory substrate . In addition to that, the
consistent increase of aspartic acids, aminobutyric acid,
ethanolamine, isoleucine, leucine, lysine and
phenylalanine while alanine, glycine. Glutamic acid and
proline contents decrease reported in storage and viability.
The decrease of germinability due to the deficiency of
respiratory substrate followed by the inactivation of
enzymes like amylase, phosphatses associated with the
degradation of reserves stored in pollen grains.

8. CAUSES----
• Desiccation & loss of membrane integrity of pollen-
The water content is one of the regulator of viability
of the plant cells.
• Desiccation tolerant-that remain viable after
dehydration
• Desiccation sensitive-loss viability parallel to
dehydration. The water content(15-35%) ,Very high
in Poaceae pollen about 30-60% of the pollen grains
varies from species to species. Thus the pollen grains
of grasses are high sensitive to greater degree of
desiccation and normal function due to rehydration.

9. Extended Pollen viability under cryogenic temperature and relative
humidity
Taxa Storage
temperature ⁰C
RH(%) Duration of storage
Carica papya 1 10 153 days
Cocos nucifera 5 40 1.5 years
Lycopersicon
esculantum
2-4 10 252 days
Pyrus malus 2-8 10 673 days
Rhodendron sp. 2 25 124 days
Vitis vinifera 10 25 2 years

10. POLLEN STORAGE
• Pollen storage particularly in the domain of in vitro technique
is very important and it started at the end of 19th century. Large
number of crop species, forge and cereals, for fiber and fruit
crops, forge and cereals, pollen strategies are also important.
Genetic enhancement of horticultural crop needs Pollen
Cryobank from which we can draw pollen parents of choice in
the process of breeding programme.
• Storage of pollen grains retaining viability needs two kinds of
practices.
• 1.Short term storage
• 2.Long term storage

11. Short term storage
• It includes effect of temperature & humidity, pollen
storage in organic solvent
• A. Low temperature and relative humidity are
favorable for most of the taxa but a large number of
taxa can be successively stored in a limited period of
time through the manipulation of temperature and
humidity. Unsealed containers with suitable
dehydrating agents like silica gel, various conc. Of
sulphuric acid and conc. Salts are used to maintain
relative humidity.

12. Conditions of pollen storage
• Pollen storage in organic solvent
• Organic solvent is very important for pollen storage. Iswanami
& Nakamura( 1972) first demonstrated the use of organic
solvent in this regard-benzene, petroleum, diethyl ether,
acetone, chloroform , cyclohexane and other organic solvents
are very useful in this regard.
• 1. Citrus pollen maintained viability in different solvents for
three months
• 2.Chrysanthimum pollen loses viability in dry conditions but
if treated with diethyl ether, it increases viability.
• Storage in organic solvents, there is no loss of viability
referred as absolute dormancy(Iwanami, 1984).
• Plant solvent has proved to be better for storage of any pollen
than low temperature & humidity.

13. Effect of organic solvents on pollen tube growth
Treatment Lilium
auratum
Lilium
auratum var
platyphyla
L.longifloru
m
Camellia
sasanqua
Impatiens
balsamina
Fresh pollen 11.8(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
Chloroform 15.1 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
• Storage of pollen grains above 0⁰C slows down metabolic
activity of pollen , resulting the gradual decrease and finally
total loss of pollen viability. Thus long term pollen storage
requires Cryogenic techniques for the effective and long term
pollen preservation.
• 1. using the storage temperature of -10⁰c and 34⁰c , the
longevity of the bi-cellular pollen and pollen with original low
content of the moisture has been successively extended
between 1-3 years.
• 2.Lyophilization-Freeze drying or vacuum drying involves
rapid freezing of pollen to sub zero temperatures of -60⁰C to -
80⁰C using inert helium or nitrogen and the gradual removal of
water under vacuum sublimation. In number of taxa, freeze
drying with combined lyophilisation promises better pollen
storage and preservation.

15. LONG TERM PRESERVATION
• Cryopreservation
• A reduction in the pollen water content below a threshold level
below before low temperature exposure seems to be important
for achieving viability. Thus partially dehydrated pollen
possess less freezable water and can survive deep freezing
Low temperature below -70 to -196C is very effective in this
regard that can increase the pollen storage and viability. A
reduction of the pollen water content below low temperature
exposure seems to be important for achieving viability. Thus
partially dehydrated pollen possesses less freezable water and
can survive deep freezing. A number of pollen grains have
been preserved successfully by cryopreservation like Beta
vulgaris, Brassica oleracea, Carica papaya, Glycine max,
Prunus perica, Vicia faba, Zea mays, Helianthus annus, Pyrus
malus etc.

16. Taxa Storage temperature Storage duration
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
Lycopersicon esculentum -196 1062 days
Prunus persica -196 1 year
Pyrus communis -196 7 months
Pyrus malus -196 673 days
Solanum tuberosum -196 2 years
Vicia faba -196 1 month
Zea mays -196 10 years

17. Estimation of Pollen viability
• Pollen viability is an imperative factor in the study of reproductive
biology before adoption any biological programme for different
attributes. The very quick & dependable method to determine the
viability of the pollen grain is very important for any kind of
successful breeding programme. A number of methods are employed
for the same as follows-
• In vitro germination test-Observation of pollen tube after
germination under microscope.
• Pollen germination on stigma-Growth of the pollen tube through
style.
• Enzymes assay method-Activity of certain enzymes like
dehydrogenase by the triphenyl tetrazolium chloride (TTC)
• Fruit & seed test-Degree of setting fruit by effective fertilization
• Tetrazolium test-by 2,3,5-triphenyltetrazolium chloride test
• Fluorochromatic reaction test(FCR) or Fluorescein diacetate
test(FDA)

18. Continuation----
• It is based on the reduction of soluble colourless tetrazolium
salt to reddish insoluble formazan in the presence of
dehydrogenase. If the pollen grains are incubated in
tetrazolium solution(0.1-1%) for 30-60 minutes at 30-37⁰C,
viable pollen grains take a reddish colour due to the formation
of formazen.Since, the tetrazolium salts can be easily reduced
by light, it is necessary to keep the solution and the pollen
grains in darkness.
• In addition to that, another test non-polar, non-fluorescent
Fluorescein di-O-acetate can be readily hydrolysed by acetyl
esterase activity to fluorescein .This is also used for the testing
of the viability of the pollen grains.

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.