Palynology and Pollen Tube Formation

1. Palynology and Pollen Tube
Formation
Espino, Fermin, Paculan, Pajinag, Quesada

2. Introduction

3. Palynology
• Greek palynos (dust)
• Deals with the morphology and ultrastructural
features of pollen
• Establish evolutionary relationships, identify
members of a taxon, and determine plant
community structures in a place (Simpson,2010).
• Pollen unit, aperture type, aperture number,
sculpturing type

4. Objectives
• describe the features of pollen grains
• observe the germination of pollen grains
• determine the factors that affect the
germination of pollen grains

5. Methodology

6. A. Examining pollen grains

7. Methods
• Collect anthers of Hibiscus rosa-sinensis, Carmona sp.,
Dianella javanica, and Ixora coccinea
• Fix in FAA
• Pollen grains were then collected from the anthers by
dissecting the anthers
• Pollen were mounted on a glass slide
• Stained with toluidine blue
Noted: pollen unit, aperture type, aperture number,
culpturing type

8. Results & Discussion

9. Pollen characteristics of selected
species
Species Pollen unit Pollen aperture Sculpturing Pollen shape
type
Hibiscus Monad Pantoporate Echinate Globose
rosa-sinensis
Dianella Monad Tricolpate Psilate 3-sided
javanica convex
Ixora Monad Zonocolporate Verrucate Globose
coccinea
Carmona sp. Monad Monoporate Psilate Globose

10. Discussion: Palynology
• The pollen grain
– Immature male gametophyte of seed plants
– Protected from desiccation and mechanical damage
by a wall composed of two layers: intine and exine
• Exine: outermost, resistant to desiccation,
decay,and other mechanical damage, with
sporopollenin
• Sporopollenin: polymer of carotenoids, fatty
acids,phenolics, and phenylpropanoids
• Intine: inner layer, composed of cellulose and
pectin (Simpson, 2010).

11. Pollen Unit
• number of pollen grains united together at the time of release
(Simpson, 2010).
• usually monads (unfused), in majority of the angiosperms
(Simpson, 2010), rarely dyads
• tetrads (microspores remain fused together)
Tetrads: based on arrangement of the pollen grains:
• tetrahedron (Ericaceae, cranberries, blueberries, manzanitas),
linear
(Typha sp.), rhomboidal, tetragonal, decussate. The grains form a
Pollen may also be polyads if the grains are connate in units more
than four (Mimosoideae and Fabaceae)
• If fusion is in irregular numbers (less than a theca), massulae
• if fusion involves the entire theca, pollinia, as in Apocynaceae and
Orchidaceae.

12. • Simpson, 2010

13. Pollen Shape
• Three-dimensional: boat-shaped, globose,
ellipsoid, or fusiform
• Two-dimensional: triangular, rhombic,
rectangular, etc.
• Ratio of the polar to the equatorial diameter, or
P/E ratio
• If equal to 1, pollen are spheroidal. If greater than
1.2, they are prolate. If less than .8, they are
called oblate (Simpson, 2010).

14. Simpson, 2010

15. Aperture
• Aperture
• Exit of pollen tube
• Harmomegathy: apertures contract in low water conditions, by
using the exine to seal the aperture (Simpson, 2010), resist
desiccation
• Porus (circular)
• Colpus (elongate)
• Zonoaperturate (equator), pantoporate (globally on surface)
• Sulcate and ulcerate: parallel to equator but similar in shape to
colpus and porus respectively
• Attach prefixes to indicate number; poly- if more than six
• Inaperturate
• Syncolpate, spiraperturate, trichotomosulcate

16. Simpson, 2010

17. Sculpturing type
• Baculate: rods, or bacula
• Clavate: club-shaped structures, clavae
• Echinate: spiny elements longer than one micrometer,
echinae
• Fossulate: longitudinal grooves
• Foveolate: pits on the surface
• Gemmate: globular structures, gemmae
• Psilate: smooth
• Reticulate: netlike muri; the spaces are called lumina
• Rugulate or rugulose: “brainlike” structures
• Spinulose or scabrate: spines shorter than one micrometer
• Striate: thin, cylindrical structures
• Verrucate: short, wart-like verrucae

18. • Simpson, 2010

19. Introduction

20. Bourne, Geoffrey.(1987)

21. • Pollen tube travels towards the ovule via
chemotropism, wherein various chemical
signals are released by the ovule and the
pollen tube is attracted towards it.

22. B. Germinating Pollen Grains
Modified from Zung and Catron, 1997

23. Methodology
Medium A Medium B Medium C Medium D
10% sucrose 10% sucrose 10% sucrose 10% sucrose
100mg/L boric acid 100mg/L boric acid 100mg/L boric acid
300mg/L calcium nitrate 300mg/L calcium nitrate 300mg/L calcium nitrate
200mg/L magnesium sulfate 1% agar
100mg/L potassium nitrate

24. Growth Chamber

25. Results & Discussion

26. Hibiscus rosa-sinensis
Sucrose Initial 15 mins 30 mins 45 mins 1 hour
Concentr
ation (%)
10 0 0 0 0 0
(Paculan,
et al)
15 (Se, et 0 0 0 0 0
al)
20(Gonza 0 0 0 0 0
ga, et al)
30(Talana, 0 5 µm 5 µm 5 µm 5 µm
et al) (only in (only in (only in (only in
Medium C) Medium C) Medium C) Medium C)

27. Ixora coccinea
Sucrose Initial 15 mins 30 mins 45 mins 1 hour
Concentr
ation (%)
10 0 0 0 0 0
(Paculan,
et al)
15 (Se, et 0 0 0 0 0
al)
20(Gonza 0 0 0 0 0
ga, et al)
30(Talana, 0 0 0 0 0
et al)

28. Bhojwani S.S. and Bhatnagar S.P. (1994)

29. Four media
• Medium A
– Sucrose (10%, 15%, 20%, 30%)
• Medium B
– Sucrose (10%, 15%, 20%, 30%)
– 100 mg/L boric acid
– 300 mg/L calcium nitrate/ calcium chloride
• Medium C
– Sucrose (10%, 15%, 20%, 30%)
– 100 mg/L boric acid
– 300 mg/L calcium nitrate/ calcium chloride
– 200 mg/L magnesium sulfate
– 100 mg/L potassium nitrate
• Medium D
– Sucrose (10%, 15%, 20%, 30%)
– 100 mg/L boric acid
– 300 mg/L calcium nitrate/ calcium chloride
– 1% agar

30. Pollen grain
• Packed with biochemicals like sugar, starch,
lipids and phytic acid
• With protein exudates for pollen-stigma
interaction

31. Pollen Tube
• grows chemotropically and intercellularly into
the style due to a concentration gradient of
calcium-boron-inositol sugar complex (Pandey,
2006

32. Four stages of pollen germination
1. Adhesion
2. Rehydration from stigma
3. Germination proper
4. Penetration and tube elongation

33. Bourne, Geoffrey.(1987)

34. According to Jain et al. (2008)
• Stigmatic fluid with lipids, resins, sugar, etc.,
and thus provides a suitable medium for the
germination of the pollen grains
• Pollen grain with hydrolytic enzymes such as
acid and alkaline phosphatase, ribonuclease,
esterase and amylase in the intine, principally
below the aperture region of the pollen grain.
These enzymes play a significant role in the
process of pollen germination.

35. Jain et al.(2008)

36. Jain et al.(2008)

37. Jain et al.(2008)

38. http://www.brown.edu/Departments/Molecular
_Biology/pgl/KH%27s%20animation/Animatio
n/Basic%20Animation/PollenAnimationBasic.h
tml

39. Growth Chamber
• Humid environment is needed
– Dehydrated pollen grain sown in a growth
medium should be adjusted to a favourable pH
and osmolarity under appropriate conditions of
temperature and humidity (Raghavan, 1997).
– Moist environment is required because the pollen
tube absorbs water for it to grow longer

40. Nutrients Needed for Pollen
Germination

41. Sucrose
• Provides a carbon energy source that can initiate
the metabolic processes that trigger germination
and support pollen tube growth
• A secondary function, that of an osmoticum, has
evolved for sucrose and other carbohydrates in
pollen germination, because a high osmotic
environment of the medium prevents the
bursting and collapse of pollen grains immersed
in a hypotonic medium (Raghavan, 1997).

42. According to Soni et al. (2010)
• The concentration of sucrose varies from the
species to species
• In Cleome gynandra L., the maximum % of
germination and tube growth is seen in 10%
sucrose
• Najas marina (Jain & Shah, 1991), Datura
metal (Patel, 2002), Tradescantia paladosa
(Tanaka, 1981)and showed highest
germination in 10% sucrose solution

43. According to Soni et al. (2010)
• 11 to 15% sucrose concentration is best for
Asclepias syriace (Kevan et.al,1989 )
• 25% in Trapa bispinosa (Hoque and Arima,
2000)
• 15% sucrose medium in Abelmoschus
esculents (L.) Moench (Dabgar & Jain, 2002)
• 30% in Bambusa vulgaris (Koshy and Jee, 2001

44. According to Baloch et al. (2008)
• 20% sucrose concentration gained the most
pollen grain germination in Hibiscus
escuelentus.

45. Boron
• For pollen tube growth in higher plants
• Reduces bursting of pollen tubes and
enhances percentage of germination
(Bhojwani and Bhatnagar, 2005)
– Frequent bursting of pollen grains and pollen
tubes is major difficulty in the work of pollen
culture. This is due to uptake of large quantities
of water thus can be controlled by adjusting
osmotic concentration of the medium.

46. Boron
• Facilitates the uptake of sugar from the
medium and that it is involved in the
biosynthesis of cell wall precursors.

47. Calcium
• Ca2+ is an essential requirement of pollen
tube growth (Bendnarska, 1989)
• Controls the permeability of pollen tube
membrane (Dickinson, 1967)
• Absence of calcium in the medium results in
an increase in the membrane permeability
leading to the loss of internal metabolites

48. Calcium
• Higher concentration of calcium in the
medium prevents diffusion of the calcium
from the pollen. Thus supplementation of
calcium in the medium lead to development
of straight and rigid pollen tube with vigorous
growth. A positive correlation between speed
of pollen tube growth and quality of the
resulting progeny is also explained (Delph-
Lynda et al., 1998)

49. Calcium
• Calcium is an important cation involved in
many key metabolic reactions, especially
signal transduction, in plants and animals
(Raghavan, 1997 ).

50. Magnesium and Potassium
• According to Brewbaker and Kwack (1963)
magnesium ions enhance the effect of calcium
ions resulting in vigorous growth of pollen
tube
• effect of many of these ions could be due to
induced changes in the pH of the medium, or
their effect on the uptake, binding, and
activity of calcium ions (Branscheidt, 1930)

51. Agar
• provides stability so that the growth of
individual pollen tubes can often be
monitored (Martin, 1972).
• the solidified surface of the germination
medium could mimic the micro-milieu
conditions for pollen germination in vivo (Fen
et al., 2000).

52. Agar
• The higher density of agar medium causes the
pollen tube to grow slower because of the
resistance of motility compared to liquid
medium.

53. • The requirements of pollen grain germination
of various plants are different. However,
sugars in various concentrations are of
primary need and besides the carbohydrates,
Calcium and Boron is usually required for
achieving optimum germination (Nair, 1985).

54. Conclusion
• To conclude, palynology is the study of pollen,
as well as their features like pollen unit,
aperture type, aperture number, sculpturing
type, and presence or absence of starch.
These details are important in establishing
evolutionary relationships among plants,
identifying members of a taxon, and in
determining plant community structures in a
place.

55. • The four different media only differ in
composition, either addition or subtraction of
one or more components. Medium C has the
complete set of nutrients needed for pollen
germination, thus, this could give the most
appropriate medium in vitro. In accordance to
different sucrose concentration, this will
depend per species.

56. Recommendation
• For future studies, it is recommended that
pollen from other species aside from those
already mentioned be studied.

57. References
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600.
• Bhojwani S.S. and Bhatnagar S.P. (1994). The embryology of angiosperms. Vikas publ. Hou.PVT Ltd. New Delhi, India.
• Bendnarska, K. (1989). The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth –investigation
with the use of 45Ca2+, verapamil, La3+ andruthenium red Plant Reprod. 2: 53-58.
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J. Bot. 50: 859-865
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tube growth in Abelmoschus esculentus Moench. J. Swamy Bot Club. 8: 25-29.
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Cleome Gynandra L. Dist: Sabarkantha, North Gujarat