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Preventing Germination

Updated on April 25, 2013
Tables 1-6
Tables 1-6
Figure 1
Figure 1
Tables 7-9
Tables 7-9

The effects of different concentrations of tomato extract on tomato seed germination

Abstract

In general, most seeds never germinate inside their fruit despite the ideal conditions for germination present. The ability of tomato extract, to inhibit seed germination was studied. Various concentrations of tomato extract ranging from 0-60% were given to different groups of seeds. The seeds were monitored over a period of eight days. The number of seeds germinating per day in each group of seeds was counted and the percentage germination on each day was calculated. The percentage germination decreased as concentration of the tomato extract increased because of the presence of abscisic acid (ABA), an inhibitory hormone in the extract. In addition, generally, seeds in the lower concentrations of tomato extract germinated earlier than the seeds in the higher concentrations. The factor that inhibits germination of tomato seeds is the ABA found in the pulp of tomato fruits.

Outline plan

Observations

Although surrounded by sufficient moisture and nutrients ideal for germination to take place tomato seeds fail to germinate inside their fruit. Germination occurs only after the seed leaves the fruit complex and passes through the alimentary canal of an animal or undergoes leaching by rainfall.

Hypothesis

Percentage germination of tomato seeds decreases as the concentration of tomato extract increases. The decrease in percentage germination is due to increasing concentration of an inhibitory hormone in the tomato extract known as abscisic acid, (ABA). In order for the seed to germinate, production of this inhibitory hormone has to be inactivated or its concentration reduced.

Variables

Temperature

Oxygen concentration

Light intensity

Volume of germination medium (here the medium being the tomato extract)

Viability of seeds

Concentration of medium (the tomato extract)

Percentage germination of seeds

Dependent variables

Percentage germination of seeds. This will be calculated by dividing the number of seeds germinated at the end of the experiment by the total number of seeds in the Petri-dish and the answer multiplied by 100. The percentage germination of the different experiments and at different concentrations will be compared and the average percentage per concentration will be calculated.

Independent variables

I will be varying the concentration of tomato extract in each sample- e.g. control (distilled water), 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, and 90%,

Controlled variables

The temperature must not be too low or too high as low temperatures result in a slow experiment whilst temperatures that are too high may cause denaturation of enzymes involved in the hydrolysis of food reserves. Very low temperatures cause the water to freeze and that may cause mechanical damage to the seed. In addition temperature affects enzymes for seeds in any species of plant. Extremely high temperatures can cause denaturation of enzymes within the seeds causing germination to come to a halt; and there is an optimum temperature at which germination rate is at its highest provided no other factor is limiting germination. So the seeds must and will be germinated at room temperature to ensure an experiment which will not take too long, to avoid effects of extremely high or low temperatures and to provide a constant environment for the germinating seeds in each experiment and concentration of tomato extract. This way all seeds at each concentration and in each experiment will experience the same temperatures and more accurate results can be achieved.

Oxygen concentration also varies like temperature, but so long as the seeds are all germinating in the same room and in the same area, the oxygen concentration for each seed in each concentration and experiment will be constant.

Light intensity and wave length experienced by the seeds is important because the seeds will germinate at different rates according to their exposure to optimum wavelength for germination and/or different lengths of time of heating. So the seeds will be planted at equal depths in the cotton and the Petri-dishes will be placed in an area of the room which receives illumination from all directions.

In each experiment each Petri-dish shall receive 20cm³ of the various concentrations of the tomato extract. This way the seeds have equal amounts of germination medium giving a fair test. All the extract will be prepared the same day and the same extract will be used for all the experiments so as to improve the accuracy of the experiment by reducing chances of unequal concentration of extract for every experiment.

Viability of the seeds. The seeds may be dead or may have a weak embryo, will have differing surface area to volume ratios in which case they will absorb differing amounts of water; the seeds may be of different ages or may have been harvested too early or late or may have undergone mechanical damage while they were being harvested, they will also contain differing amounts of food reserves and some may be infected by bacteria or fungi.

All these factors are difficult to control and so to reduce the effect they have on the investigation, many seeds will be used in the experiment, and repeats will be carried out so that error can be minimised. Eye judgement will be used to choose seeds which look most viable and are similar in size so that the best possible results can be obtained.

Pilot

Apparatus

300 seeds

Distilled water

Tomatoes

Blender

Measuring cylinder

Beaker

Petri dishes

Cotton wool

Method

1. Lay cotton over the base of each of 10 Petri dishes.

2. Label the covers of the Petri-dishes –distilled water, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

3. Wash 10 ripe tomatoes.

4. Peel the tomatoes and grind them in a blender.

5. Prepare the various concentrations : –distilled water (control), 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

6. Pour 20cm³ of each of the different concentrations into their corresponding Petri-dishes.

7. Wait for germination, keeping the Petri-dishes covered.

8. When at least 85% of the seeds in the control have germinated, count the number of seeds that have germinated in each of the other Petri-dishes.

9. The statistical test I will be using is a goodness of fit test to find out whether there is any significant difference in the number of seeds germinating in the different concentrations of tomato extract.

Results of the pilot (Table 1)

The expected trend could be seen following the pilot experiment with one anomaly at 50% concentration. This anomaly arose because the seeds were planted deep inside the cotton and so when the extract was poured on top, it was filtered, forming a less concentrated moist that accumulated at the bottom of the Petri-dish while most of the concentrated part of the extract accumulated at the top away from the seeds. As a result, the seeds were not inhibited from germinating and the percentage of seeds germinated was an unexpected 33.3% which is higher than some percentages at lower concentrations.

Modifications

Some seeds were germinating too close to each other making it difficult for me to count them when they germinate. They will therefore be planted 15mm away from each other in 5 rows of 6. Also since these seeds have different surface area to volume ratios some will absorb more water than others so to avoid any possible competition the seeds will be planted well spaced out from each other.

To obtain fair results the seeds will be planted at the same depth of 2mm in the cotton and they will be watered using 10ml syringes. This will ensure that each seed is equally exposed to the extract in each Petri dish. This can be achieved by initially adding one drop of the extract on each seed then gently spraying each row with the rest of the required solution of tomato extract.

From the pilot experiment it was observed that seeds didn’t germinate at concentrations above 50%. This gave us insufficient results and therefore intermediate concentrations will be introduced making 60% concentration the highest concentration to show how germination above 50% concentration remains at 0%. The concentrations that will be used are- distilled water, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%.

The experiment was allowed to proceed and it was observed that the shoots grew tilted to one side (towards light) proving that the seeds were not receiving equal illumination. For this reason the Petri-dishes will be placed in an area of the room which receives light from every direction. This is important as different light intensities will cause varying rates of germination which would affect the accuracy of the results of the investigation.

Risk assessment

Wash your hands before you handle the seeds as they are prone to disease; the Petri-dishes should also be sterile before use to minimise chances of infection or build up of fungi.

Do not eat the tomatoes as pathogens and toxic chemicals present in the lab may have infected the fruit.

Care must be taken on handling the knife when peeling the tomato to prevent cuts.

Blender must always have its cover on before it is switched on to avoid extract splashing into eyes or blade of the blender flying out accidentally, and becoming a potential hazard.

Wash clothing that comes into contact with extract to prevent build up of fungi on the clothing.

Store various concentrations in a fridge to avoid accumulation of bacteria.

Be careful when handling the glass equipment such as the measuring cylinders and beakers as breakages can cause injury.

Dispose within a paper bag, any cotton used in the experiment when the experiment is done, especially any cotton that has fungi on it. This will prevent any unwanted fungal and bacterial disease and accumulation on other organisms and apparatus in the lab.

Introduction

A seed requires oxygen, moist and warmth before it can germinate. Oxygen is required for aerobic respiration within the seed so that there is sufficient energy available for all metabolic reactions within the cells in the seed. Heat is provided for by the environment and/or metabolic activity within the seed e.g. respiration. Warmth is essential for the optimal performance of the enzymes involved in the hydrolytic break down of food reserves. The initial absorption of water is largely by imbibition- the attraction between hydrophilic colloids such as membrane-bound proteins in the seed and water molecules. The absorbed water helps in the activation of the hydrolytic enzymes (amylase), and allows hydrolysis of stored food (starch) into soluble products (glucose), capable of being transported from the storage tissues to the growing points of the embryo. With subsequent increase in number of solute molecules in embryonic tissues, water is taken up by osmosis. The swelling of the embryonic tissues rupture the seed coat thereby allowing the growing plumule and radicle to emerge.

However tomato seeds may not germinate despite the presence of favourable conditions. This is due to the existence of inhibitory hormone in the pulp of the tomato fruit preventing hydrolysis of food reserves. The inhibitory hormone in this case is known as abscisic acid ABA which binds to receptor sites on the external parts of the seed coat.

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis, i.e. enzyme activity is reduced by inhibitors. In enzyme inhibition a molecule (inhibitor) binds to an enzyme forming an un-reactive complex. Any compound which interacts with enzymes to reduce their catalytic activity can be called an inhibitor. Inhibitors often structurally resemble the inhibited enzyme’s natural substrate. Enzyme inhibitors can be used by organisms to regulate metabolism

Types of inhibitors include: Reversible inhibition, Irreversible inhibition, Competitive inhibition, and Non-competitive inhibition.

Reversible inhibitors only bind temporarily to the enzyme so that their effect is not permanent were as irreversible inhibitors bind permanently leaving the enzyme unable to carry out further catalysis. Reversible inhibitors may also be competitive or non competitive.

Some irreversible inhibitors act as enzyme inactivators. In enzyme inactivation (suicide inhibition), the inhibitor binds irreversibly to the enzyme. This has the effect of permanently reducing the total enzyme concentration and most enzyme inactivators form covalent bonds with their target enzymes. Competitive inhibition involves the inhibitor competing with substrate for enzymatic binding site and in the last type, non-competitive inhibition; the inhibitor binds to the enzyme substrate complex preventing catalysis. Mixed inhibition may also take place whereby there’s a mixture of competitive and non-competitive inhibition.

ABA negatively regulates seed germination. It inhibits precocious germination and promotes seed dormancy. This negative regulatory mechanism is carried out through the repression of transcription factors.

The purpose of the investigation is to determine the effect of varying concentrations of tomato extract on germination of tomato seeds. Tomato seeds will be planted in different concentrations of tomato extract and allowed to germinate. The conditions under which the seeds will germinate will be considered such as constant temperature, oxygen concentration, light intensity, light wavelength and volume of the various concentrations of tomato extract.

The hypothesis states that percentage germination of tomato seeds decreases with increased concentration of tomato extract. The experiments will show the gradual increase in percentage germination as concentration of the tomato extract decreases and the less concentrated solutions of will have their seeds germinating at faster rates than those in the more concentrated solutions.

Materials and their justification

2000 seeds,

Distilled water,

Tomatoes x 40,

Stirring rod,

Blender,

Measuring cylinders x2,

100ml Beakers x13, 200ml Beaker x1, 2000ml Beaker,

Petri dishes x39,

Syringe,

Apron,

Pencil, log book,

Marker pen,

Tweezers,

Cotton wool,

Wooden splint,

2000 seeds will be required. These are separated into 11 groups of 30. With this many seeds in each Petri-dish, more accurate results can be acquired as comparison of data can be made to a greater degree.

Distilled water serves as a control. When at least 85% of the seeds sown in the control have germinated, the experiment can be considered as finished and all the seeds at different concentrations of tomato extract counted, to find final number of seeds per concentration and hence calculate average percentage germination in each concentration.

Tomatoes can be used as a source of the tomato extract. This can be obtained by mashing tomato pulp in a blender.

The stirring rod can be used to stir the mixtures of distilled water and tomato extract so that each solution is uniform (i.e. there are no separate layers of water and the tomato part) before it’s poured into its corresponding Petri-dish.

The measuring cylinder can be used to measure volume of distilled water and of the tomato extract when making the various concentrations required.

100ml Beakers will be used to store the various concentrations of tomato extract. The 200ml Beaker will serve as a mixing vessel and the 2000ml will be used to store surplus tomato extract for future use.

Petri-dishes will hold the seeds in cotton while germination occurs.

The syringe is used to water the seeds individually ensuring that all the seeds are more equally exposed to the extract.

An apron will protect clothing from any spillages of tomato extract during the preparation of the various concentrations of tomato extract.

Tweezers enable better more hygienic handling and more accurate positioning of the seeds

The cotton wool acts as a base for the germination of seeds and a means of maintaining moist within the Petri-dishes.

The log book and pencils will be used in recording data

The marker pen will be used to label beakers, Petri-dishes and to mark 2mm on the wooden splint prior to seed plantation.

Method

Fifteen tomatoes were washed and pealed with a knife, then placed in a clean blender. Washing the tomatoes and cleaning blender reduce chances of bacteria and fungi forming. A blender was used in preference to manual pounding as it is cleaner, more efficient and produces a ready stirred solution of extract. To avoid any dangers of the blade in the blender flying out, or the tomato extract splashing into the eyes, the lid was kept firmly shut while the blender was switched on.

Using a marker, thirteen 100ml beakers were labelled, control, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%.

The various concentrations of tomato extract were made as follows:

The control was simply distilled water, poured into its corresponding beaker.

To prepare the 5% concentration of tomato extract, 95 cm³ of distilled water was measured in a 100ml cylinder. 5cm³ of the prepared extract was measured in a 50ml measuring cylinder. These were poured into a 200ml beaker in which the two solutions were mixed and well stirred with a stirring rod. Stirring reduces the occurrence of anomalies in the percentage germination at each concentration, such as more seeds germinating at a higher concentration than at a lower one due to the fact that the mixture poured into the Petri-dish, contained more water than it was supposed to as the dense tomato extract settled at the bottom of the beaker before the mixture was poured to its corresponding Petri-dish. From here the mixture was poured into its corresponding beaker and the 200ml beaker was rinsed with distilled water ready to mix other solutions.

Similarly the 10% concentration was made by mixing 90cm³ of distilled water with 10cm³ tomato extract, and the 15% made by mixing 85cm³ distilled water with 15cm³ extract,

the 20% by mixing 80cm³ distilled water with 20cm³ extract

the 25% by mixing 75cm³ distilled water with 25cm³ extract

the 30% by mixing 70cm³ distilled water with 30cm³ extract

the 35% by mixing 65cm³ distilled water with 35cm³ extract

the 40% by mixing 60cm³ distilled water with 40cm³ extract

the 45% by mixing 55cm³ distilled water with 45cm³ extract

the 50% by mixing 50cm³ distilled water with 50cm³ extract

the 55% by mixing 45cm³ distilled water with 55 cm³ extract

the 60% by mixing 40cm³ distilled water with 60cm³ extract.

Make sure all the solutions have been poured to their corresponding beakers.

(The actual concentration at which germination stops is at 50% concentration but this is exceeded by two concentrations to make it definite that there is no further germination after 50% concentration.)

The surplus extract is kept in the fridge in case it was required for further work to prevent build up of fungus.

The solutions were made within the same period of time (from 1pm to 2pm). Therefore the solutions were made at constant temperature. This was important because liquids expand and contract with increase and decrease in temperature thus readings on the measuring cylinders would vary if the solutions were made at different times. As a result the concentrations of solutions would not be constant relative to each other.

2000 ready dried seeds were bought from the local trading centre. A surplus of seeds was obtained so that there were a greater number of seeds to choose the healthiest looking ones from. For each experiment 390 seeds were required and these were split into thirteen groups of thirty.

Thirteen Petri-dishes were sterilised in hot water to reduce chances of fungal accumulation and disease to the seeds during the experiment. The Petri-dishes were labelled with a marker from ‘5% to 60%’, and one was labelled ‘control’. This way identification of samples is made easy and results can be obtained and recorded efficiently.

Fresh cotton was cut into thirteen equally sized pieces. These were placed at the bottom of each of the thirteen Petri-dishes and pressed in gently to make them stick better to the base of the dishes. Then the length of 5mm was marked using a felt-tip pen at one end of a thin wooden splint. This splint was used to make holes in the cotton in all the Petri-dishes for the seeds to be planted in. this was achieved by dipping the splint into the cotton stopping at the mark. The reason for the shallowness is too allow all the seeds total exposure to light, and the solutions of extract. Also if the seeds were planted too deep germination would be hard to spot. The holes were planted at 15mm intervals to form a 5x6 dot square in each Petri-dish. This was essential because if the seeds grew too close to each other, they were more difficult to count. The seeds were then planted with the micropyle facing upward so germination can easily and quickly be spotted.

Equal volumes of each of the solutions of the various concentrations of tomato extract was first thoroughly stirred then evenly poured into its corresponding Petri-dish. This was carried out using a 5ml syringe. The first 10cm³ of a solution was distributed drop by drop into each hole in the cotton ensuring that all the seeds get completely soaked in that particular solution. The other 10cm³ was sprayed gently across each line of the 5x6 dot squares.

The whole procedure was repeated apart from the making of solutions as the same solution was used for each experiment to ensure that the concentrations for each experiment were exactly the same.

Once planted the Petri-dishes were covered with their transparent lids. Covering the dishes helped to maintain moist conditions. The radicals begun to emerge by day 4 of the experiment; from this day on, the number of seeds germinating in each dish per day was recorded in a log book until the percentage germination was 80% or above in the control.

Results of experiment

Germination started at day 4: (Table 2)



(Table 3) Day 5

(Table 4) Day 6

(Table 5) Day7

Final results

(Table 6) Day 8

Graph of average percentage germination against concentration per day (Figure 1)





Analysis

Germination starts on day 4 (Table 2). There is no clear trend shown by the experiments themselves because at some concentrations the percentage germination is higher than the preceding concentration(s). E.g. in experiment 1, the 15% concentration has 13.3% germination while a lower concentration (10%) has only 6.7% germination. This contradicts the hypothesis. Similarly in experiment 2, 25% concentration with 10% germination, has a greater percentage germination than the preceding 20% which has 3.3% germination. This is also evident in experiment 4 at concentrations of 15% and 10%, and 30% and 25%.. This also contradicts the hypothesis which expects the lower concentration to have greater percentage germination than the other.

The average percentage also does not show any reliable trend although it vaguely shows a gradual decrease in percentage germination with increasing concentration with anomalies such as the 18.3% average germination in the 15% concentration which is supposed to be less than the 15% average germination in the 10% concentration. Other anomalies include the occurrence of a repetition of the same percentage germination in subsequent concentrations e.g. 20% and 25% concentrations which both have 9.18% average germination.

All the values for percentage germination become 0 at 50% concentrations and above.

On day 5 (Table 3), a trend becomes clearer in each experiment as well as in their average. Anomalies still exist at concentrations such as the greater percentage germination values in a subsequent concentration (At 30% and 35% concentrations in experiment 1, at 20% and 25% concentrations in experiment 2 and in the average at 20% and 25%) and again the occurrence of a repetition of percentage germination (at 10% and 15% in experiment 3, and at 20% and 25% in experiment 4).

The average percentage germination now shows an obvious trend and only has a minor anomaly at 25% where the percentage germination of 13.4 is only slightly different to the 13.3% of the preceding 20% concentration. This is contradicts the hypothesis but to a very small extent.

The percentage germination values at 50% concentrations remain as 0%.

Generally more seeds in the lower concentrations of tomato extract germinated first before any seeds in the higher concentrations.

In some experiments, the percentage germination remains the same as the previous two or three day’s e.g. from day 7 to 8 in experiment 1, at 35% concentration the percentage germination remained constant at 13.3%. Similarly from day 5 to 8 in experiment 3, at 45% concentration, the percentage germination remained 3.3%.

Although for each experiment there are still some vivid anomalies (experiment 1, percentage germination at 10% and 15% concentration both had 46.7% germination, and at 25% concentration was 33.3% which was a higher percentage germination than the percentage germination at the preceding concentration (20%), which was 30.0%) by the end of the experiment a clear general trend is evident and the results support the hypothesis. The average percentage of seeds that have germinated at the end of the experiment decreases with increased concentration of the tomato extract.

Figure 1 illustrates that average percentage germination for everyday decreases as the concentration of extract increases and that more germination occurs each new day.

Fungi were observed accumulating on the cotton. As the concentration of extract increases so does the density of fungus accumulating.

The χ² test shows that percentage germination is affected by variations in the concentration of ABA. Degree of freedom=13-1=12, χ²=107.3097

Value in the table is 21.026.

The Χ² value is greater than value in the table (p<0.05). Therefore the null hypothesis is rejected and the alternative is accepted stating that the concentration of ABA in tomato extract has some effect on the number of seeds germinating.












Discussion and Evaluation

The results support the hypothesis. There was a decrease in average percentage germination as concentration of extract increased (Figure 1). E.g. at 5% concentration, on day eight, average percentage germination was 71% according to Figure 1, then at a higher concentration-20%, it was 35%, then at an even higher concentration-40%, where percentage germination was 10%, evidently approaching the 0 percent germination at 50% concentration. Although the hypothesis is supported by the results, it still needs to be modified to account the fact that at the end of the experiment, some groups of seeds may not have reached maximum germination so can end up having the same or smaller percentage germination compared to the preceding concentration(s). It should rather state that ‘provided all viable seeds are allowed to germinate, the percentage germination will decrease with increased concentration of tomato extract’.

In natural cases, the ABA is either hydrolysed by acids in the alimentary canal of animals or the seed undergoes leaching by rainfall. After this and a period of dormancy, the cell can germinate. The increase in tomato extract concentration increases the ABA concentration. Generally ABA works at the transcription process to decrease mRNA synthesis for amylase (i.e. preventing the expression of the amylase gene), therefore reducing the total amylase concentration within the seed. ά-amylase catalyzes conversion of starch to sugar which is used as an energy source for the embryo in the germinating seed. It is also possible that ABA suppresses GA-responsive genes essential for tomato seed germination, including the GA-responsive amylase gene. In the presence of appropriate ABA concentrations, the biosynthesis of hydrolytic enzymes, notably α-amylase, will be prevented resulting in the seed’s inability to breakdown the stored nutrients within the endosperm. ABA also inhibits water uptake by preventing cell wall loosening and enhancing the effects of embryo dormancy.

Percentage germination of 80% or greater was considered as maximum percentage germination and this marked the end of the experiment. This was decided because there was no time to wait for 100% germination. The percentage germination of the control determined the maximum concentration because many more seeds germinated in the control than in any other concentration. As the distilled water contained no tomato extract it was labelled a suitable medium to serve as a control. Less seeds germinate as concentration of the tomato extract increases because the tomato extract contains abscisic acid (ABA) thus increased concentration of tomato extract means increased concentration of ABA.

Initially there was no clear trend and the results didn’t seem to support the hypothesis. I.e. some higher concentrations showed greater percentage germination than their preceding concentration e.g. Table 2, exp 1, percentage germination at 35% concentration was greater than percentage germination at 30%, 25% and 20% concentration. This was because percentage germination in those particular Petri-dishes had not yet reached its maximum, and the seeds that germinated in the 35% concentration were more viable, than the seeds that had not yet germinated in preceding concentration as well as other seeds within the same Petri-dish and had not absorbed any ABA prior to germination. ABA plays a role in reducing the viability of the seeds by its inhibitory effects already explained previously. Gradually the trends start to become clear as inhibition of germination by ABA took place to different extents at each concentration. For example (Table 4), experiment 4, the percentage germination in day 6 was greater at 20% (26.7 % germination), than at 15% (23.3% germination) but in day 7 became less at 20% (26.7% germination) than at 15% (36.7% germination) because the seeds were approaching maximum germination. The ABA concentration in the 20% is higher than in the 15% therefore maximum percentage germination would be lower in the 20%. Germination for the seeds in the 20% will thus come to a halt at this concentration before the preceding concentration because the action of the ABA is to a greater extent in a larger number of seeds. If percentage germination remains the same for over three days for a particular concentration, until the experiment is over, then the seeds in that concentration have undergone maximum germination. I.e. no more seeds can germinate as ABA has fully accumulated in them. As concentration of ABA increases, more seeds are completely affected by the ABA as greater amounts of it are acting in each seed leading to less food breakdowns in the seeds and hence percentage germination decreases. Similarly in experiments with repeated percentage germination, there are seeds in the lower concentration of ABA, that have not yet germinated and are less viable than the seeds (here factors other than ABA are causing low viability) in the higher concentration but eventually the number of seeds that have germinated in the lower concentration will exceed the number of those which have already germinated in the higher concentration due to the action of the greater amounts of ABA in the higher concentration acting to deprive the seeds of food reserves required for germination to take place.

Generally, the seeds have different sizes (surface area to volume ratios), amounts of food reserves etc and so their viability will vary. Because of this the seeds will germinate at different rates but in favourable conditions (very low ABA concentrations) average germination rates are equal.

Another hormone contributing to germination is giberellic acid (GA), the primary hormone involved in germination, which induces the transcription of hydrolytic enzymes that break down endosperm food reserves. In order to germinate, a seed’s radicle must break through the seed coat. GA allows the radicle to breakthrough more easily by activating and increasing the activity of cell-wall degrading enzymes to weaken the endosperm. GA enhances α-amylase mRNA accumulation while ABA inhibits the accumulation.

It is possible that the lost germination response is due to the acidity of the tomato extract. The seeds may grow better at certain pH values than others as enzymes are sensitive to change in pH (the particular enzyme here being amylase). But this does not affect the proposed investigation as the enzymes would have caused more seeds to germinate at a concentration other than the control in response to an optimum pH.

Another possible explanation for these results could be the varying osmolarity of the tomato extract.  The water potential outside the seed is higher than inside the seed at low tomato extract concentrations and at high tomato extract concentrations, the water potential outside the seed is lower than inside.  So water gradients are set up and water moves into the seed by osmosis at low concentrations of the extract and out of the seed by osmosis at high concentrations of the extract.  This means the seed does not rupture and the radicle and plumule does not emerge so percentage germination is very low at high concentrations of the extract.  Also the presence of water allows breakdown of starch to occur and so if there is less water available, then there will be less starch hydrolysed, and less glucose as a substrate for respiration to occur and produce energy required for germination to take place. Although water potential does get lower outside the cell as concentration of extract increases, this theory cannot be true as the germination is dependent on availability of amylase and not water.  Although water activates amylase, ABA has a prevailing effect of preventing its synthesis.

The seed in a tomato has warmth, moist, and oxygen which can be absorbed through the surface of the fruit or leaves of the plant. But the seeds don’t seem to have direct exposure to the different wavelengths of light making ‘wavelength’ appear to be the limiting factor for germination; but tomato seeds can germinate in the dark so this is not a limiting factor for germination of tomato seeds within their fruit.

No seed was observed in the 50% concentration because ABA accumulates in the whole of the seed and so germination is completely inhibited.

The experimental procedure of counting the seeds was very erroneous and I made a map to show which seeds had been counted in order to prevent these errors. This can be very time consuming and so a better method of counting would be to pull out any seed that has germinated as I count therefore preventing the error of counting one seed twice. Although the seeds were placed in an area were they experience the same temperatures, oxygen concentration and light intensity and wavelength, there is a possibility still that any of these variables had increased temporarily over half of the Petri-dishes causing inconsistency of the germination of the seeds. To eliminate this source of error the seeds could instead be incubated in a vessel with constant controlled temperature, light and oxygen.

The fungus that was observed appeared because of the extract which in high concentrations provides the ideal environment for fungal population.

The Χ² test proved that the concentration of ABA in tomato extract has some effect on the number of seeds germinating. The value of Χ² (107.3097) is greater than the value in the table (21.026) for a 5% significance level so the null hypothesis which assumes that there is no significant difference in the number of seeds germinating in the different concentrations of tomato extract, is rejected.

Limitations

Fungal build up was a limitation as the fungi may infect the seeds and prevent/slow their germination thereby disrupting the results. Also the fungi make it difficult to see and count the seeds so if not careful seeds at higher concentration may have germinated but left unaccounted for.

Size of the seeds varied and so I couldn’t get exact values of percentage germination as some seeds may have been viable but due to their large surface area to volume ratio may have taken in insufficient water in order to germinate on the day I chose to count the number of seeds that germinated. This day was when I counted over 85% germination in the control.

Time was a limitation as I could have obtained more accurate results by using more seeds and a wider range of concentrations of tomato extract. Counting a larger number of seeds preparing more concentrations, sowing the seeds at a fixed depth and spacing would have been very time consuming.

The amount of space available in the lab was insufficient for the number of students carrying out experimental work, as a result it was difficult to find areas with constant variables such as light intensities and wavelengths as the spot was already taken occasionally; and if left outside, the experiment stood high chances of being tampered with either by children that pass by the lab, or by pests such as rats, when the samples are left unattended.

Conclusion

Tomato pulp contains an inhibitory hormone called abscisic acid (ABA), which prevents the tomato seeds from germinating within their fruit. The rate of germination of tomato seeds decreases with increasing concentrations of ABA. The time taken for a seed to germinate depends upon its viability.

Further work

I would increase the number of seeds (70+), using a larger dish to contain them and also monitoring them over a longer period of time to find the exact days at which germination comes to a halt in each sample.

Investigating how different species of tomato react to different concentrations of tomato extract and/or abscisic acid.

Investigating the effect of red light far red light and darkness on (scientific name for my tomatoes)

Investigating whether filtering the extract before use will produce better results.

Investigating whether the distance between the planted seeds affects germination.

Carry out experiment in a greenhouse where temperature, lighting, oxygen and carbon dioxide levels are more easily regulated. For example, windows are electronically controlled so when the temperature is too high, the windows open and if the temperature is lower than required, the windows close. Regulation mechanisms ensure that the seeds germinate under constant conditions.

Biological significance

None of the environments in which the seeds are germinated are pure tomato extract so the experiment does not show exactly what happens to the seeds in the tomato fruit itself. But nevertheless, the experiments still show the effect of reducing the concentration of the environment that the seeds would normally experience and they illustrate the idea that as long as the tomato extract is present, there will be some inhibition of germination.

Naturally the seeds would enter the gut of an animal in which certain chemicals neutralise the ABA coating the seed and once it is passed out of the gut, provided conditions are suitable and that the seed has undergone its period of dormancy, germination can occur.


Bibliography

1. T. Kolusheva & A. Marinova

‘A study of the optimal conditions for starch hydrolysis through thermostable amylase’, Journal of the University of Chemical Technology and Metallurgy, 42, 1, 2007, 93-96

2. Hegarty, T.W., ‘The influence of environment on seed germination’, Aspects of Applied Biology pages 7, 13-31., year 1987

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Acknowledgements:

Mr Chimonjo

Mr P.R Harwood

Mr Chimenya

Mr G. MacKay

The Kamuzu Academy Library

Statistical test (Table 7)

Null hypothesis

The concentration of ABA in tomato extract has no effect on the number of seeds germinating.

Alternative hypothesis

The concentration of ABA in tomato extract has some effect on the number of seeds germinating.

Expected frequencies (E) =Sum of number of seeds that germinated/ number of different concentrations

Average sum of seeds germinated=113 (sum of all seeds in the 4 experiments/4)

Number of concentrations=13

Therefore expected frequencies: 113/13=8.6923 in each concentration. (Table 8)

Χ²=107.3097

Degree of freedom=13-1=12

Therefore p < 0.05 and the null hypothesis is rejected.

(Table 9)Chi-Square Probabilities

The areas given across the top are the areas to the right of the critical value. To look up an area on the left, subtract it from one, and then look it up (i.e.: 0.05 on the left is 0.95 on the right)

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