Exercise 3


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Palynology and Pollen Tube Formation

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Exercise 3

  1. 1. Palynology and Pollen Tube FormationEspino, Fermin, Paculan, Pajinag, Quesada
  2. 2. Introduction
  3. 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. 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. 5. Methodology
  6. 6. A. Examining pollen grains
  7. 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 blueNoted: pollen unit, aperture type, aperture number,culpturing type
  8. 8. Results & Discussion
  9. 9. Pollen characteristics of selectedspeciesSpecies Pollen unit Pollen aperture Sculpturing Pollen shape typeHibiscus Monad Pantoporate Echinate Globoserosa-sinensisDianella Monad Tricolpate Psilate 3-sidedjavanica convexIxora Monad Zonocolporate Verrucate GlobosecoccineaCarmona sp. Monad Monoporate Psilate Globose
  10. 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. 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 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.
  12. 12. • Simpson, 2010
  13. 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. 14. Simpson, 2010
  15. 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. 16. Simpson, 2010
  17. 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. 18. • Simpson, 2010
  19. 19. Introduction
  20. 20. Bourne, Geoffrey.(1987)
  21. 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. 22. B. Germinating Pollen Grains Modified from Zung and Catron, 1997
  23. 23. Methodology Medium A Medium B Medium C Medium D10% 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. 24. Growth Chamber
  25. 25. Results & Discussion
  26. 26. Hibiscus rosa-sinensisSucrose Initial 15 mins 30 mins 45 mins 1 hourConcentration (%)10 0 0 0 0 0(Paculan,et al)15 (Se, et 0 0 0 0 0al)20(Gonza 0 0 0 0 0ga, et al)30(Talana, 0 5 µm 5 µm 5 µm 5 µmet al) (only in (only in (only in (only in Medium C) Medium C) Medium C) Medium C)
  27. 27. Ixora coccineaSucrose Initial 15 mins 30 mins 45 mins 1 hourConcentration (%)10 0 0 0 0 0(Paculan,et al)15 (Se, et 0 0 0 0 0al)20(Gonza 0 0 0 0 0ga, et al)30(Talana, 0 0 0 0 0et al)
  28. 28. Bhojwani S.S. and Bhatnagar S.P. (1994)
  29. 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. 30. Pollen grain• Packed with biochemicals like sugar, starch, lipids and phytic acid• With protein exudates for pollen-stigma interaction
  31. 31. Pollen Tube• grows chemotropically and intercellularly into the style due to a concentration gradient of calcium-boron-inositol sugar complex (Pandey, 2006
  32. 32. Four stages of pollen germination1. Adhesion2. Rehydration from stigma3. Germination proper4. Penetration and tube elongation
  33. 33. Bourne, Geoffrey.(1987)
  34. 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. 35. Jain et al.(2008)
  36. 36. Jain et al.(2008)
  37. 37. Jain et al.(2008)
  38. 38. http://www.brown.edu/Departments/Molecular _Biology/pgl/KH%27s%20animation/Animatio n/Basic%20Animation/PollenAnimationBasic.h tml
  39. 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. 40. Nutrients Needed for Pollen Germination
  41. 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. 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. 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. 44. According to Baloch et al. (2008)• 20% sucrose concentration gained the most pollen grain germination in Hibiscus escuelentus.
  45. 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. 46. Boron• Facilitates the uptake of sugar from the medium and that it is involved in the biosynthesis of cell wall precursors.
  47. 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. 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. 49. Calcium• Calcium is an important cation involved in many key metabolic reactions, especially signal transduction, in plants and animals (Raghavan, 1997 ).
  50. 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. 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. 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. 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. 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. 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. 56. Recommendation• For future studies, it is recommended that pollen from other species aside from those already mentioned be studied.
  57. 57. 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