TITLE AIM

The germination behaviour of Azuki bean seeds (Vigna angularis) towards microgravity -To investigate the germination behaviour of Azuki bean seeds (Vigna angularis) in simulated microgravity environment -To determine the effect of potassium nitrate, KNO3 towards germination of seed

PROBLEM What are the effects of simulated microgravity environment and potassium nitrate, KNO3 towards the STATEMENT / germination of Azuki bean seeds? RESEARCH QUESTION LITERATURE The Factors Affecting the Rate of Germination REVIEW There are four major factors affecting the germination process of seeds, which are gravity, light, temperature, air and moisture. Hideyuki Takahashi says that a microgravity environment has a great impact on plant growth and development, and it eventually affects plant yield. It is said that the shape of the living organisms from the Earth is influenced by the planet's gravitational environment. When the gravitational environment is removed (microgravity environment), some organisms will show automorphogenesis. To create a microgravity environment, a 3D clinostat can be used, which simulated microgravity by rotating the seeds. On Earth, aerial parts of the plant (shoots) grow upward while roots grow downward. However, in microgravity environment, the growth direction is unregulated, and some roots even extend in the same direction as the aerial stems. When compared, the shoots of the seeds on a normal gravitational environment grows straight, but on the other hand, the shoots of the seeds on a microgravity environment will bend. Therefore, gravitational environment do affect the germination process of seeds. Besides, light is another major factor affecting seeds germinations. Heather Jerret and Delia Gillens said that light has varied effects on germinating seeds of different plants. Some seeds need light for germination, while in some seeds germination is hindered by light. Most wild species of flowers and herbs prefer darkness for germination and should be planted deep in the soil while most modern vegetable crops prefer light or are not affected by it, and are planted shallowly to allow small amounts of light to filter through the soil. According to Victoria Lee Blackstone, some seeds, such as the sunflower and pelargoniums must have darkness for successful germination. These seeds are sown directly into the ground and covered with soils. On the other hand, some seeds, for example the impatiens and petunia seeds need to be surface-sown to allow exposure to light. If these seeds are not covered, germination will not occur. However, David Batty explained that other factors can also affect the seeds light requirement. For example, with some species (e.g. Salvia pretences and Saxifraga caespitosa) light requirement only exists immediately after harvesting whereas with Salvia verticillata and Apium graveolens (Celery) this lasts for a year and to confuse matters further other species develop a light requirement while in storage. Chemicals also, such as nitrates in the soil, can substitute for light in stimulating seeds to germinate so

that some light requiring seeds will still germinate if covered with fertile soil. According to Wikipedia (en.wikipedia.org), Seeds from different species and even seeds from the same plant germinate over a wide range of temperatures. Seeds often have a temperature range within which they will germinate, and they will not do so above or below this range. Many seeds germinate at temperatures slightly above 60-75 F (16-24 C) [room-temperature if you live in a centrally heated house], while others germinate just above freezing and others germinate only in response to alternations in temperature between warm and cool. Some seeds germinate when the soil is cool 28-40 F (-2 - 4 C), and some when the soil is warm 76-90 F (24-32 C). Heather Jerret as well as Delia Gillens also thought that germination can take place over a wide range of temperature and is specific to individual crop types, and can be specific to varieties. They say, the optimum for most crops is between 65-75F, but exceptions do apply. Jacob J. Wright, on the other hand tells that at standard, the temperature for general bean seeds is at 70 degree Fahrenheit (21 degree Celsius), but each bean species may need different soil temperatures for germination, based on adaptations to where they are native. As for moisture, Heather Jerret and Delia Gillens said that a dormant seed only contains 10-15% of water and is essentially dehydrated. In order to become active, the seed has to absorb water. It is imbibed by the seed coat and enzymes within the seed become active and functional, metabolizing stored food reserves. The embryo then begins to swell. The softened seed coat ruptures as the seed grows too big for its encasement and germination has commenced. This shows that without water, seeds cannot germinate. Jacob J. Wright says that even though a bean seed feels and looks dry, minute amounts of moisture within the seed keep the dormant embryo alive. For the embryo to grow, some external water source causes the hard testa to soften and split and trigger metabolism in the dormant embryo. Water enters the seed in a tiny hole in the testa called the micropyle. He again tells that a delicate balance exists in the appropriate amount of water necessary for bean seed germination. Too little moisture and the seed begins to sprout but then dries up and dies; too much water and the embryo dies and rots. During germination, air is another factor that could affect the whole germination process. Heather says in the dormant condition the seeds respiratory rate is very low and so oxygen is required in very small quantities. But for germination, oxygen is needed in large quantities. The seeds obtain oxygen that is dissolved in water and from the air contained in the soil. If soil conditions are too wet, an anaerobic condition persists, and seeds may not be able to germinate. Furthermore, Jacob J. Wright tells that plants absorb carbon dioxide from the atmosphere, but plants also need oxygen to metabolize food and to grow. During germination, air must reach embryo and that is why a moist but crumbly, loose soil environment promotes best germination. Last but not least, germination process can be affected when the seeds are soaked in different solution as a pre-treatment for the seeds. By soaking the seeds, this treatment can stimulate the rate of germination of seeds, which then make them to germinate faster. According to rhodielady_47, Salt Petre, also known as potassium nitrate, (KNO3) is often used to stimulate germination of dormant or irregular seeds. The seeds are soaked in a 1000 to 3000ppm solution (1 - 3 grams per litre), or are germinated on pads soaked in this solution. Seeds remain dormant to ensure the best conditions for germination. Basically, potassium nitrate helps to break the seeds

dormancy, and therefore, encouraging the seeds to germinate faster. HYPOTHESIS -The Azuki bean seedlings show automorphogenesis. - Pre-soaking seeds in potassium nitrate, KNO3 solution increases the rate of germination of Azuki bean seeds (Vigna angularis). VARIABLES MANIPULATED RESPONDING -Simulated microgravity -The length of radicle and plumule -The suppleness of the stem CONSTANT -Type of beans -Room temperature -Volume of water -Duration of germination APPARATUS 250ml beaker, forceps, 10ml measuring cylinder, container (21cm 15cm 4cm), opaque box, thermometer, cutter knife, ruler, volumetric flask. MATERIALS Distilled water, Azuki bean seeds (Vigna angularis), 0.1% potassium nitrate, KNO3 solution, rockwool blocks (3cm 3cm 3cm), graph paper, plasticine clay, gloves, thread, and label paper.

PROCEDURE

Figure 1, the apparatus set-up of the experiment

1. On Day 1, 18 Azuki beans are pre-soaked in 100ml of 0.1% potassium nitrate, KNO3 solution in a beaker. 2. After 30 minutes, the seeds are brought out and rinsed with distilled water. 3. Three holes are made on each rock wool block with a knife. 4. The rinsed seeds are placed on the rock wools securely with the hilum facing upwards. 5. All the seeded rockwool blocks are placed into the plastic container and fitted with the plasticine. The plasticine is used to avoid the rockwool blocks from falling off the container

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

when tilted. Each rockwool block is labelled 1 to 6 and watered with 15 ml of distilled water. The container is tilted to discard any excess water that is not absorbed by the rockwool. The container is closed with the lid and kept in an opaque box to keep it in total darkness. Every 6 hours, the container is tilted 90 clockwise to simulate microgravity. After 24 hours, on Day 2, three seedlings from Block 1 are gently removed and placed on a graph paper. The photographs of seedlings that are placed on the graph paper are taken. The length of radicle and plumule for each seedling is measured from one end to another using a thread and ruler. The mean length of radicle and plumule is calculated and recorded. After that, the rockwool blocks are watered with the fixed amount of distilled water which is 15ml. Steps 10 to 14 are repeated for Blocks 2 - 5 on Days 3 6. On Day 7, steps 10 13 are repeated for Block 6. The suppleness of the stems of the seedlings on Block 6 is measured with spring balance The suppleness of the stems of the seedlings is recorded in a table.

PRESENTATIO The length of plumule and radicle N OF DATA Day/length(average) Pictures Day 1

Radicle (mm) 0

Plumule (mm) 0

Observation No changes or increase in size

Day 2

1% bigger than actual size

Day 3

1.67

2 radicles emerge out of 3 seeds

Day 4

8.67

All radicles emerge from the seeds

Day 5

21.33

7.33

All radicles emerge from the seeds and one out 3 plumules emerge from the seeds

Day 6

10.00

5.33

All radicles emerge from the seeds and 2 out of 3 plumules emerge from the seeds All radicles and plumules emerge from the seeds

Day 7

39.67

23.67

Suppleness of stem Seed 1 Seed 2 DISCUSSION 2.5 N 2.7 N

In this experiment, the Azuki bean seedlings (Vigna angularis) show automorphogenesis and the rate of germination of the seeds increased when pre-soaked in potassium nitrate, KNO3 solution. The Azuki bean seedlings (Vigna angularis) are observed and watered for seven days. They were tilted clockwise for 90 every 6 hours. Plasticine clay is used to avoid the rockwool blocks from falling off the container when tilted. Every 24 hours, the radicle and the plumule of the seedlings are measured and their photographs are taken. On the 7th day, the growth direction of the radicles is unregulated while the plumules bend instead of growing straight upwards. The experiment focused on two factors affecting the germination process which are microgravity and pre-soaking treatment. Normally, a plumule grows straight upwards and this is what occurred on the seedlings in the previous experiment. However, most of the plumules bend in this experiment. This phenomenon is called automorphogenesis. This happens due to the simulated microgravity that has been applied towards the seedlings. The simulated microgravity causes the seedlings to show automorphogenesis. Automorphogenesis is a response of a plant when gravity is removed or in microgravity. Automorphogenesis shows curvatures or unregulated growth of plumule and radicle of the plant. There are many other effects of microgravity towards the germination of seeds. They are automorphogenesis and Diageo tropism. Diageo tropism is the tendency of plant to grow in a direction

horizontal to the surface of the earth. Basically, microgravity causes automorphogenesis and growth direction of plumule and radicle to be unregulated or others call it curvatures. Another factor which is pre-soaking treatment is widely used in the agriculture industry. In this experiment, the Azuki bean seeds are pre-soaked in potassium nitrate, KNO3 solution before it is planted into the rock wools to stimulate the growth of the radicle. Potassium nitrate is one of several nitrogen-containing compounds which majorly used as a fertilizer. Usually, a seed takes 3-5 days to germinate after it is planted. However, this experiment is conducted for 7 days only. Therefore, the germination of the radicles and plumules need to be stimulated by soaking it in potassium nitrate, KNO3 solution which is widely used in germinating seeds. When the growth of radicle is stimulated, the seeds will sprout quickly and the effects of microgravity towards the radicle and plumule can be seen more apparently. CONCLUSION The Azuki bean seedlings (Vigna angularis) show automorphogenesis and the rate of germination of the seeds increased when pre-soaked in potassium nitrate, KNO3 solution. Hypothesis are accepted. REFERENCES http://www.ncbi.nlm.nih.gov/pubmed/11538807, http://link.springer.com/article/10.1007%2FBF00008074, http://www.kiwispace.org.nz/display/SSAF/Hypothesis+1+-+Automorphogenesis http://www.jaxa.jp/article/special/kibo/takahashi_e.html http://www.researchgate.net/publication/11803400_Autotropism_automorphogenesis_and_gra vity http://www.highmowingseeds.com/sb-factors-affecting-germination-of-organic-seeds.html http://forums.gardenweb.com/forums/load/tomato/msg0317254629885.html http://www.thompson-morgan.com/effect-of-light http://www.iss.jaxa.jp