Observing the growth of pollen tubes
Students often complain that plants are boring, and they don’t do anything quickly. Pollen tube growth is a surprisingly rapid process in certain conditions, and the pace of growth is measurable under a microscope during the course of a (double) lesson. Simple observations of the diversity of pollen grains can lead to discussions about allergic reactions to different pollens, and to mechanisms ensuring fertilisation of ovules by pollen from the correct species.
This will depend on whether your intended learning outcomes relate to handling the pollen and observing the pollen tubes, or whether you want to try to assess investigative skills as well.
Although some pollen grains (Note 1) will begin to germinate quickly, you will need over an hour of working time to measure rates of growth and to compare different kinds of pollen or different growth media. If different groups of students use different concentrations of culture medium (with and without crushed stigma) and different types of pollen, this will increase your chances of success in the time available.
If you have access to a video microscope, it would be possible to display any fast-growing tubes to the whole class and record the sequence for future lessons.
Apparatus and Chemicals
For each group of students:
Microscope, monocular x 100 and x 400 with eyepiece graticule, 1 per group
Stage graticule, 1 per 3 or 4 groups
Plants in flower (for example, Pelargonium sp., Impatiens sp., Narcissus sp., Liliaceae – Note 3)
Petri dishes, 4 per group
Marker pens, to label dishes and slides
Measuring cylinders or syringes to measure 1 cm3 and 10 cm3
Stop clock/watch, 1
Access to a balance
Access to scissors
Forceps, 1 per group
Mounted needle, 1 per group
Dropping pipette, 1 per group
For the class – set up by technician/ teacher:
Sucrose solutions (Note 1)
Pollen germination medium (Note 2)
Health & Safety and Technical notes
There are no hazards associated with this procedure if students are behaving in a manner appropriate to a laboratory practical.
1 Sucrose concentration: various sources list concentrations from 1 per cent to 20 per cent w/v. The SAPS protocols recommend a starting point of 1.2 M sucrose, which is about 40% w/v, diluted by half on mixing with mineral salts culture medium. The optimum sucrose concentration varies with species – so it may be necessary to use trial and error to find a reliable concentration. 10-20% final sucrose concentration seems to be ideal for most. This solution will keep for some months in a refrigerator.
2 Mineral salts pollen culture medium:
This solution needs to be made up fresh – immediately before use. To make one litre, add 0.417 g calcium nitrate (Ca(NO3)2) (Hazcard 19A – oxidising and irritant at concentrations of greater than 0.8 mol dm–3), 0.200 g boric acid (H3BO3 – Hazcard 14 – low hazard), 0.101 g potassium nitrate (KNO3 – Hazcard 82 – oxidising, explosive with some metals and dangerous with some combustible materials), 0.217 g magnesium sulphate (MgSO4.7H2O – Hazcard 59B – low hazard), and 3.5 cm3 of 1.0M ammonium hydroxide (NH4OH – Hazcard 6 – corrosive at 6 mol dm–3 or above). Mixed with the sucrose solution the mineral salts medium should give a pH of 8.8. Add the ammonium hydroxide last to adjust the pH to the desired value.
In some cases, pollen germination is speeded by adding a little crushed mature stigma directly to the slide from the tip of a mounted needle.
CLEAPSS Laboratory guide, section 15.5, recommends the ‘hanging drop technique’ for setting up the microscope slides and suggests this simpler recipe for culture medium: For 40 cm3 of 0.25M medium, mix in a clean 100 cm3 beaker, 2 g domestic sugar (sucrose), 1.4 g thick flower honey, 2-3 small crystals of boric acid, a thin smear of Marmite (to cover about 1 cm2 of beaker surface), and 40 cm3 of deionised water.
3 Impatiens should begin to germinate in 15 minutes or so. Pelargonium will begin to germinate after 45 minutes or so. Many practitioners report success with Liliaceae (such as florist lilies in any season) and with Narcissus and other spring-flowering bulbs. SAPS report reliable results with rapid-cycling ‘fast’ brassicas, and with other garden brassicas such as rape or salad rocket. One trick seems to be to use flowers of a range of ages, although it seems that often more mature flowers give more reliable results. If results are poor, try different concentrations of sucrose in the pollen culture medium or sucrose alone.
SAFETY: Technicians should handle certain of the solid chemicals in the mineral salts medium with care (Note 2), but at the dilutions used the hazard to students is low.
a Collect a range of flowers.
b Make up the solutions of sucrose and mineral salts pollen culture medium.
c Place a filter paper in each of four Petri dishes, moisten the paper with water and replace the lids.
d Take 5 cm3 of pollen culture solution and make it up to 10 cm3 with 40% sucrose solution. This now contains 20% sucrose.
e Take four absolutely clean microscope slides and place a drop of medium in the centre of each slide. Label the slides with the names of the flowers you are investigating, such as Pelargonium, Impatiens, Narcissus or Lilium.
f Use a low power stereo binocular microscope to help you to select two or three flowers of each type that are mature and shedding pollen. The ripeness of the pollen seems to be critical to success, so sampling from more than one flower increases the chance of successful germination.
g Place a microscope slide under the field of view of the microscope and gently rub the point of a mounted needle over the anthers so that pollen falls onto the medium. Transfer any pollen from the needle by tapping the needle against a pair of forceps placed next to the slide.
h Clean the needle thoroughly and repeat step g so that you have two slides with pollen for each of the types of flower you are investigating. Do not add coverslips.
i Note the time of adding pollen to the medium and place the slides in the Petri dishes. Handle the slides with great care so that the drops of pollen medium remain in the centre of the slides.
j Use a microscope, with x100 magnification and an eyepiece graticule, to observe the slides over the next 15 minutes for signs of pollen germination. Between quick observations, turn the microscope lamp off and return the slide to the Petri dish.
k Once the pollen tubes have started to grow, measure the length of four or five tubes at three minute intervals for about 30 minutes. If the pollen tubes do not grow, try again with a different concentration of pollen culture medium or of sucrose, or add a little crushed mature stigma to one of the slides and note any effect.
l Collate the group results and work out the average growth with time for each type of pollen, in each medium. Draw graphs to show the growth of pollen tubes with time, compared with a graph of group results, for each solution. Are the units of measurement eyepiece units or micrometres (µm)?
The concentration of sucrose solution is only one environmental factor that could be varied. If the students can suggest others during planning, you could investigate those also. Temperature, age of pollen, composition and concentration of growth medium could all affect the growth rate.
Further samples of growing pollen tubes could be stained with, for example, lactopropionic orcein, to show up the nuclei. If time permits, students should observe prepared microslides showing pollen tube growth down a style.
If you calibrate the microscopes, the units of growth can be converted into µm (micrometres).
Pollen tubes of Impatiens tend to grow straight with only an occasional change in direction. In contrast, those of Pelargonium grow extremely contorted under these conditions. This may be the result of unsuitable osmotic conditions in the medium, insufficient sucrose, or lack of an ‘organizer’ substance produced by the carpel.
The pollen grain and tube represent the male gametophyte. The pollen grain nucleus divides mitotically to form first the vegetative and generative nuclei and then the latter divides again to form two male nuclei (gametes). Thus the pollen grain is much more than just a gamete.
Between germination of the pollen grain and formation of the seed, there are several steps. These include: growth of the pollen tube down the style, release of two gametic nuclei, double internal fertilisation, formation of the embryo, endosperm and related structures.
Advantages of retaining the ovule and embryo inside the tissues of the adult are:
- decreased dependence upon environmental water
- the provision of nutrients for early development
- dispersal of offspring with a food reserve increasing the probability of successful germination
Disadvantages of retaining the ovule and embryo are:
- problems of gametes meeting; the need for pollen tube formation for gamete movement, and a mechanism to ‘direct’ the growth of the tube
- the complex nature of the seed, and increased use of energy for the food store – an energy-expensive process compared with other means of reproduction
A websearch for scanning electronmicrographs + pollen + grain will reveal several high resolution images showing differences between grains of pollen.
Health & Safety checked, September 2009
Download the student sheet Observing the growth of pollen tubes (70 KB) with questions and answers.
A link to the SAPS worksheet on pollen tube growth. (Website accessed October 2011)