Student Sheet 4 www.saps.org.uk Pollen Tube Growth Technical and teaching notes Safety Care should always be taken when handling and mixing chemicals. Wear eye protection and consult CLEAPSS HAZCARDS for full information about different chemicals. Preparation of materials Recipe for Brassica Pollen Germination Medium Mineral Salt Solution to make 1 litre: 0.417g calcium nitrate (Ca(NO3)2); 0.2g boric acid (H3BO3); 0.101g Potassium nitrate (KNO3); 0.217g magnesium sulphate (MgSO4.7H2O); 3.5 cm3 of 1.0M ammonium hydroxide (NH4OH). Sugar solution 1.2M sucrose (41g sugar in 100 cm 3 of distilled water) Equal volumes of these two solutions should have a pH of 8.8. Vary the NH 4OH in the first solution to adjust the pH. Note Using this method with pollen from other species, Butler published a list of the best flowers and the best concentrations to use throughout the spring and summer. You will find this in Butler K.G. "Pollen germination across the seasons" School Science Review 2000 82 (298) p93f. You can read a copy of this article on the SAPS website. Student Sheet 4 www.saps.org.uk Background Information When a pollen grain is released from an anther, it is partially dehydrated. If it lands on the surface of a stigma of the same flower (self-compatible) or of a different flower of the same species (self-incompatible), it rehydrates. Some species have ‘dry’ stigmas, and it is thought that lipids in the surface coat of the pollen grain bring about a concentration gradient of water that directs the growth of the pollen tube. If the humidity is very high, the pollen will rehydrate anyway. Other plants have ‘wet’ stigmas and the stigma exudate hydrates the pollen grain itself, so surface lipids in the pollen are not so important. The growth of pollen tubes is easily observed under medium power of a microscope (see below), but the ‘in vivo’ growth is often much faster because of several factors that are known to enhance the growth. Some of these are calcium, boron, and sucrose: this last compound acts as a source of carbon as well as exerting an osmotic effect. Some species produce flavinols in both the pollen and anther, which is necessary for growth. Mutant maize plants that are deficient in the enzyme that synthesises flavinols are self sterile. Addition of flavinols restores the ability of the pollen to grow and fertilize the ovules of the female plant. As pollen tubes grow, callose plugs are produced close to the aperture of the pollen grain, so the cytoplasm becomes concentrated towards the growing tip. Pollen tube pictures are available on the SAPS website. Pollen allergens Many people suffer from hay fever and other respiratory illnesses brought on by inhaling pollen. The cause of this is certain proteins present in the pollen grain. The pollen specific genes for these are very similar in a wide range of plants, and their base sequences are highly conserved, suggesting they play an important role in pollen development. The proteins coded by these genes are show similarities to pectate lyases, and it is thought that they enable the pollen to partially dissolve tissue in the style of the female flower, so the pollen tube can successfully travel down to the ovary. Other allergens found include profilin (in birch pollen), which binds actin and is therefore important in developing the architecture of the pollen tube (cytoskeleton and microfilaments). Grass pollen contains expansin like proteins. These are involved in cell wall expansion. The presence of proteins in pollen can be seen under the microscope by staining with Coomassie Blue or similar. Put a drop of water containing pollen on a microscope slide and place a cover slip on top. Place a few drops of stain, containing some sucrose or glycerol, at one side of the cover slip (these compounds have an osmotic effect on the pollen grain causing the cytoplasm to ooze through the apertures. Place some tissue paper at the opposite side and allow the stain to percolate through the pollen. The grains within the emerging cytoplasm of the pollen will be stained blue. These are the proteins. Observing pollen tube growth This has a reputation of being unreliable, being dependent on the age of the pollen, the species being used, and the constituents of the growth medium. The SAPS protocol given here can be unsuccessful with some flowers. A simplified method, using just sucrose (0.6 mol dm-3), works very well with a number of plants commonly found in school grounds. These are in flower in the different seasons, so the experiment can be undertaken at various times in the school year. The table below indicates plants that have proved successful. Month March/April Successful Birch Not successful Student Sheet 4 www.saps.org.uk April Cotoneaster Crab Apple April/May Ceanothus Fraxinus Cherry Viburnum May/June/July Chestnut Mountain Ash July/August Lime Indian Bean Chestnut pollen will produce rudimentary pollen tubes even when incubated in water alone, so one suspects the actual concentration of sucrose used is not critical, though it may influence the speed of growth. We have produced images of the growth over a period of one hour: These are available in the zipped file available on the SAPS website so students can analyse the data to work out the rate of growth of the pollen tubes. This lends itself to investigative work at GCSE and post-16, with variables such as sucrose concentration, temperature, pH, and the addition of mineral ions.