Exercise 3

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Palynology and Pollen Tube Formation

Espino, Fermin, Paculan, Pajinag, Quesada

Introduction

Palynology

• Greek palynos (dust)• Deals with the morphology and ultrastructural

features of pollen• Establish evolutionary relationships, identify

members of a taxon, and determine plantcommunity structures in a place (Simpson,2010).

• Pollen unit, aperture type, aperture number,sculpturing type

Objectives

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

germination of pollen grains

Methodology

A. Examining pollen grains

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

Results & Discussion

Pollen characteristics of selected species

Species Pollen unit Pollen aperture Sculpturing type

Pollen shape

Hibiscusrosa-sinensis

Monad Pantoporate Echinate Globose

Dianella javanica

Monad Tricolpate Psilate 3-sided convex

Ixora coccinea

Monad Zonocolporate Verrucate Globose

Carmona sp. Monad Monoporate Psilate Globose

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).

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 aPollen 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.

• Simpson, 2010

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 than1.2, they are prolate. If less than .8, they arecalled oblate (Simpson, 2010).

Simpson, 2010

Aperture• Aperture• Exit of pollen tube• Harmomegathy: apertures contract in low water conditions, by

using the exine to seal the aperture (Simpson, 2010), resistdesiccation

• 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

Simpson, 2010

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

• Simpson, 2010

Introduction

Bourne, Geoffrey.(1987)

• 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.

B. Germinating Pollen GrainsModified from Zung and Catron, 1997

Methodology

Medium A 10% sucrose

Medium B

10% sucrose

100mg/L boric acid

300mg/L calcium nitrate

Medium C

10% sucrose

100mg/L boric acid

200mg/L magnesium sulfate

100mg/L potassium nitrate

Medium D

10% sucrose

100mg/L boric acid

1% agar

Growth Chamber

Results & Discussion

Hibiscus rosa-sinensisSucrose Concentration (%)

Initial 15 mins 30 mins 45 mins 1 hour

10 (Paculan, et al)

0 0 0 0 0

15 (Se, et al)

0 0 0 0 0

20(Gonzaga, et al)

0 0 0 0 0

30(Talana, et al)

0 5 µm(only in Medium C)

5 µm(only in Medium C)

Ixora coccineaSucrose Concentration (%)

Initial 15 mins 30 mins 45 mins 1 hour

10 (Paculan, et al)

0 0 0 0 0

15 (Se, et al)

0 0 0 0 0

20(Gonzaga, et al)

0 0 0 0 0

30(Talana, et al)

0 0 0 0 0

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

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

Pollen grain

• Packed with biochemicals like sugar, starch, lipids and phytic acid

• With protein exudates for pollen-stigma interaction

Pollen Tube

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

Four stages of pollen germination

1. Adhesion2. Rehydration from stigma3. Germination proper4. Penetration and tube elongation

Bourne, Geoffrey.(1987)

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.

Jain et al.(2008)

http://www.brown.edu/Departments/Molecular_Biology/pgl/KH%27s%20animation/Animation/Basic%20Animation/PollenAnimationBasic.html

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

Nutrients Needed for Pollen Germination

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).

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

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

According to Baloch et al. (2008)

• 20% sucrose concentration gained the most pollen grain germination in Hibiscus escuelentus.

• 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.

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

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

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)

Calcium

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

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)

• 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).

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

• 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).

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.

• 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.

Recommendation

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

References• Baker, H. B. and Baker, I. (1979). Starch in angiosperm pollen grains and its evolutionary significance. Amer J.Bot. 66 (5): 591-600.• 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.• Bourne, Geoffrey.(1987).Pollen: cytology and development.Orlando, Florida:Academic Press, Inc.• Brewbaker J.L. and Kwack B.H.,(1963). The essential role of calcium ion in pollen germination and pollen tube growth. Amer J. Bot. 50: 859-865• Dabgar, Y. B. and. Jain, B. K. (2001). Effect of sucrose, boron, calcium and magnesium during in vitro pollen germination and tube growth in

Abelmoschus esculentus Moench. J. Swamy Bot Club. 8: 25-29.• Delph- Lynda, F., Weining, C. and Suttivan, K. (1998).Why fast growing pollen tubes given rise to vigorous progeny. Proc. Royal Soc. London

series 13 Biol. Sci.265: 935-939.• Dickinson, D.B. (1967). Permeability and respiratory properties of germinating pollen. Physiol. Plant 20:118-127.• Fan, Liu-Min., Wang, Yong-Fei., Wu, Wei-Hua.(2001).In vitro Arabidopsis pollen germination and characterization of the inward potassium

currents in Arabidopsis pollen grain protoplasts.Department of Plant Sciences, College of Biological Sciences, Key Research Laboratory in Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, China

• Franklin, Martin.(1972).In Vitro Measurement of Pollen Tube Growth Inhibition. Plant Physiol. (1972) 49, 924-925• Jain B.K. and Shah C.K.,(1991). Histochemical study of in vitro pollen germination and tube growth of pollen in najas marina L. Biovigyan 17: 30-

37.• Jain et al.(2008)Textt Book of Botany: Angiosperms.India:Capital Offset Press• Kevan, P.G., Eisikowitet D., and Rathwell B.,(1989). The role of nectar in the germination of pollen in Asclepias syriace L. Bot. Gaz. 150: 266-270• Nair, P.K.K.(1985). Essentials of palynology, plant breeding. Monogr. Theor. Appl. Genet.Vol. II: Berlin, Germany.• Raghavan, V.(1997).Molecular embryology of flowering plants.USA:Cambridge University Press• Shivanna, K.R. (1979). Recognition and rejection phenomena during pollen pistil interaction. Proc. Ind.Acad. Sci. 88 (B): 115-141• Simpson, Michael.(2010). Plant Systematics. Academic Press, California.• Soni et al.(2010).Effect of Different Concentration Of Sucrose During In Vitro Pollen Germination and Pollen Tube Growth in Cleome Gynandra L.

Dist: Sabarkantha, North Gujarat

Exercise 3

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