Pollen Germination

Pollen Germination in Tradescantia, impatiens, jasmine, lily, pomegranate, California poppy, and protoplasmic streaming in hair cells of Tradescantia and leaves of Elodea These two experiments are done together with the idea of relating them in terms of mechanisms of protoplasmic movement. Ref: Rapid germination of pollen in vitro. D.J. Schimpf. The American Biology Teacher 54:168-9. 1992. Germination solution • • • • 10 g sucrose, .01 g boric acid, .03g calcium nitrate in 100 ml distilled water. This is GS. PROCEDURE Dust the pollen powder over the bottom of a tiny disposable plastic petri dish (best results occur when there is oxygen, not under a coverslip.) Replace cover, and label. Add a few drops of one of the solutions and observe under 10x objective, and when germination starts, take out a sample and place on a slide with a coverslip so you can look at it with 40x. It is important to get Impatiens flowers with colored anthers at the center of the flower, not a green solid structure. If you want to study the difference between immature and mature pollen you can use the anthers from an unopened bud, and try to see what will make it germinate. Some pollen germinates in 5-10 minutes, continues to lengthen for an hour. Others germinate after an hour. Be sure to write down the time you placed the pollen in solution. After seeing the germination in your control pollen, pick the flowers which worked the best, and continue the rest of the experiment, but also do Tradescantia since that will be used in the second half of the experiment. CAN YOU MAKE AN HYPOTHESIS ABOUT WHAT CAUSES POLLEN GERMINATION? Pollen lands many places, but the only place where it will do any good is on the stigma of the ovary. There a sugary secretion is sticky and holds it on the surface, and then after germination, the pollen grains digest their way through the tissues of the style, perhaps using the digestion products as an energy source, until reaching the egg where the sperm nuclei move from the pollen tube to fertilize it in preparation for seed formation. Prepare the control as above, in the germination solution. Also try some experimental dishes to get at the signaling mechanism and the outgrowth mechanism. The following solutions can be used: 1. 2. 3. 4. GS without sucrose GS without Calcium GS without boric acid . Chelating solution: .35M sucrose, .001 M EDTA, HEPES pH 7.2 (EDTA BINDS CALCIUM SO IT IS NOT AVAILABLE) 5. GS with colchicine 25 uM (.o1 mg/ml) 6. GS with cytochalasin 2.4 mg/ 2ml GS + 4 drops DMSO 7. .3 M KCL (cells have high K+ inside and low outside, so the gradient across the membrane which usually determines membrane potential will be changed.) 8. 1M glycerol (osmotically like sea water) 9. distilled water 10. apple peel in water (ripe apple peel has ethylene) 11. Ammonium chloride solution (alters cell pH) 10 mM (8mg/15 ml GS) 12. peroxide solution, a powerful oxidizing agent 500 uM 13. chlorox solution (dissolves some complex carbohydrates such as chitin, removing fly egg coats) use 1:1 with water, leave on only briefly, suck off or dilute with GS. The following physical factors can also be tested: cold or hot temperatures; light of different colors by wrapping in different colors of cellophane; dark; lack of oxygen; bubble oxygen through solution; bubble carbon dioxide through. Protoplasmic streaming. The same samples of pollen tubes can be used for examination of vesicle transport in white Tradescantia, pigment granule motion in purple Tradescantia. The purple ones are easier to see, but I could see movement in all tubes with GS solution, and in hair cells of Tradescantia. This takes 20x or better yet 40x magnification. Cell movements usually have an actin filament-myosin motor combo as in amoeboid movement or tubulin microtubule-dynein or tubulin-other motor combo as in pigment granule motion or vesicles in neurons. They have rather opposite optimal conditions. Actin-myosin -interaction requires Calcium release to start it and actin can be affected by pH and can be depolymerized by cytochalasin or cold. Tubulin-dynein works best at low calcium. Tubulin is depolymerized by colchicine and cold. Particles are moved toward the center of the cell by dynein, outward by kinesin. Both kinds of motion require ATP. If the outgrowth is due to osmotic factors, such as uptake of water, using different concentrations of sugar, glycerol, salt can test for those effects. Using the solutions listed on the previous page, do experiments to test which mechanism might be used here. So all you need to do is take samples from the petri dishes and make slides to look for streaming in all the experimentals. Always look at your control first, so you know what to look for in the experimentals. Discuss the results with your partners. The object of this exercise is not to have fun and see pollen grow, though we do allow it. The object is to study protoplasmic motion mechanisms. Gather careful data. Figure out how best to display it. Can you draw any conclusions? Write them in your summary report. Pollen from the anther (the male part of the flower) germinates on the stigma or female part of the flower. The result is a long tube which grows down to the ovary where the eggs are. The pollen tube takes the sperm nucleii down to the egg to fertilize it and the result is a seed. There are many processes which we could investigate using pollen. Pollen is thought to produce extracellular enzymes which will hydrolyse the stigma tissue to allow the tube to grow between the cells, and to use the digestion products for energy. Factors influencing growth of roots, stems, seeds. 1. Effect of gravity on corn seed rootlets. Culture of seeds:Ref:Plant Physiology Laboratory Manual. C.W. Ross Wadsworth Publishing Co. Belmont CA 1985. Draw an arrow on the dish. Place 8 corn seeds, point down in the same direction as the arrow, across a line in a petri dish. Tape them down. Cover with 6 layers of filter paper. Add water or other medium to dish and then drain. Tape lid closed. Stand on edge so arrow points down so all roots will grow downward. Allow to grow for 2 days at 23 C so that primary roots are 2 cm long. Take out 4 of the seedlings and cut off 2mm from the tip. Place them in a fresh dish aligning with an arrow, place new paper and moisten as before, pour off. Now repeat this for 8 more seedlings. Measure the length of each root before and after experiment. Number the seeds on the bottom of the dish or in a drawing so you know which one is which. Place one set (4 cut and 4 uncut) with the arrows pointing down, and one set with the arrow pointing sideways. Observe after 2 hours. Carefully record your data and then after discussion with your group come to some conclusions. CAN YOU MAKE AN HYPOTHESIS ABOUT WHAT CAUSES ROOT DIRECTIONAL GROWTH? NEXT: TEST YOUR HYPOTHESIS. Prepare the control as above, in water. Also try some experimental dishes to get at the signaling mechanism and the outgrowth mechanism. The following solutions can be used: 1. Chelating solution: .35M sucrose, .001 M EDTA, HEPES pH 7.2 (EDTA BINDS CALCIUM SO IT IS NOT AVAILABLE) 2. calcium ionophore A23187 makes channels in the membrane so calcium can enter or leave the cell. 3. GS with colchicine 4. GS with cytochalasin 5. .3 M KCL (cells have high K+ inside and low outside, so the gradient across the membrane which usually determines membrane potential will be changed.) 6. 1M glycerol (osmotically like sea water) 7. ammonium chloride solution (alters cell pH) 8. peroxide solution, a powerful oxidizing agent 9. chlorox solution (dissolves some complex carbohydrates such as chitin) The following physical factors can also be tested: cold or hot temperatures; light of different colors by wrapping in different colors of cellophane; dark; lack of oxygen; bubble oxygen through solution; bubble carbon dioxide through. Cell movements usually have an actin filament-myosin motor combo as in amoeboid movement or tubulin microtubule-dynein or tubulin-other motor combo as in pigment granule motion or vesicles in neurons. They have rather opposite optimal conditions. Actin-myosin -interaction requires Calcium release to start it and actin can be affected by pH. Using the solutions listed on the previous page, do experiments to test which mechanism might be used here. So all you need to do is take samples from the petri dishes and look for bending of the primary root. Always look at your control first so you know what to look for in the experimentals. Gather careful data. Figure out how best to display it.