Phosphate ions, PO43-, have a variety of functions in plants; these include ATP and nucleic acids.
ATP, adenosine triphosphate, consists of adenine linked to a pentose sugar, ribose, and a string of three phosphates; it powers the majority of energy-requiring reaction that occur. As mitosis is a reaction that requires excessive amounts of ATP, a solution lacking in phosphate ions would leave the plant without enough ATP, and the mitotic cycle would suffer.
Nucleic acids are found in living cells in two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They are composed of long string of nucleotides, which can be hydrolysed to give a nitrogenous base, a pentose sugar and phosphoric acid. If a plant were to be cultured in a solution without sufficient phosphate ions, DNA replication and protein synthesis could not occur because there would be enough free nucleotides. This would negatively affect the mitotic cycle.
With this understanding of the role of phosphate ions in a plant, I predict that any seedling cultured in a solution that has a low concentration of phosphate ions will show very little or no mitotic activity when examined under a microscope. When the concentration is higher, there will be more available nucleotides to allow for DNA replication and protein synthesis to occur, and there will be sufficient ATP to provide the energy required.
Plants also require more than just phosphates in the solution, needing the nutrients nitrates and sulphates, calcium, magnesium, iron and potassium ions. Therefore, to fully ensure that the number of cells dividing by mitosis is accurate, the plants should be cultured in otherwise perfect conditions, at the optimum temperature, with all the required nutrients in the culture solution. It’s then easier and more accurate, to define that a change in mitotic activity is due to a change in phosphate concentration. This will maintain a fair test.
To carry out this experiment, I will need the following materials and apparatus:
Test Tube
Garlic Cloves Beaker
Acetic Orcein Stain Watch Glass
Hydrochloric Acid Cotton Wool
Warm Water Needle
Culture Solutions Microscope Slide and Coverslip
Microscope
The culture solution will have a constant volume of the other desired macronutrients, enabling a fair test – to change the concentration of phosphate ions, I will merely replace it will distilled water. For example, in a culture solution the correct concentration* of phosphates is 41 mg dm-3, and I will take that to be 100% concentration and this will be my control. The ranges that I will use are concentrations of 120%, 80%, 60%, 40%, 20%.
This will offer me the ability to examine a range of different concentrations to see what the impact is on the number of cells dividing by mitosis – I believe the figure quoted is the optimum concentration, and that any less will cause a negative impact. I have included a higher concentration just to ensure that there is no significant impact, and that 100% is the optimum concentration.
Method
1 – Half fill a test tube with the 100% concentration culture solution, and insert garlic clove, wrapped in cotton wool, allowing roots to be submersed in solution.
2 – Culture in a warm, bright environment for 4-5 days and then cut of the apical meristem 5mm from the tip of a growing lateral root.
3 – Submerse in watch glass, containing 10 parts stain to 1 one part acid, and warm over beaker filled with hot water for 5 minutes.
4 – Cut tip in half transversely, place on slide and add 2 drops of stain.
5 – Using a needle, break the root tip to spread it thinly as possible. Cover with coverslip, and examine under microscope for stages of mitosis.
6 – Repeat steps 1-5 twice more for concentration 100%.
7 – Repeat steps 1-6 for each concentration.
By having three root tips from each concentration, I am more likely to overcome any anomalous results and prevent errors, by averaging the Mitotic index***, which is the number of cells undergoing mitosis divided by the number of cells visible. The average can be recorded in the table shown below, and I can then fully determine what the effect phosphate ions have on cell division in root tips.
References
* Water Culture definition withdrawn from the textbook Advanced Biology: Principles and Applications by C.Clegg and D.Mackean. (Page 1, Paragraph 2)
Data regarding water culture solution extracted from textbook. (Page 2, Paragraph 2)
** Allium Mitosis Labels, diagram taken from website (Page 1, Paragraph 3)
*** Mitotic Index definition extracted from (Page 2, Final Paragraph)