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White Mangrove Development Written Proposal

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  • 1. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period Michael Gómez-Meléndez, Ana L. Velazquez-Fernández, Robert Ross (University of Puerto Rico at Cayey Introduction Abstract Mangroves are usually found in In this experiment we will identify specific environments where water is filled with high characteristics for the salt glands in young concentrations of dissolved salts. However, and old mangroves. After collect the mangroves had developed structural samples, our team preserve them and applied adaptations for this particular environment. direct techniques to identify the salt glands Salt glands are structures that allow and proceed to prepare sectioning of the mangroves to avoid the pitfalls of these samples to compare the specific harsh conditions. These glands help the morphology of the old and young mangrove secrete the excess salt that mangroves. The expected result was that receives from the water, preventing young mangroves have salt glands more dehydration. The purpose of the efficient that old mangrove because old investigation is to observe the structure of mangroves have more salt gland to liberate these glands and how the morphology of salt the salts, and the young mangrove have less glands helps the mangrove to secrete the salt glands but however it needs to eliminate excess salt. The project is also going to be the same amount of salt in less salt glands. focused on the leaf stomas and other The result was that young mangrove salt structures that can be observed on the leaf glands are bigger that mature mangrove’s surface. salt glands, because the young mangrove M. Gómez, A. Velazquez, R. Ross Page 1
  • 2. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 have less amount of salt accumulated and Find out what salt concentration is more that makes the beneficial to young and old mangroves in its habitat depending on the salt glands capacity to secrete salt. Hypothesis Materials Young mangrove salt glands are more  The materials that are going to be capable of salt secretion than older used are: ¼ L Fast Green, ¼ L of Eth mangrove salt glands (100%), ¼ L of Water, ½ liter of based on their relative size. Eth (95%), ½ L Xylene, 1 bag of Objectives ParaplastTM, 1 Garden Scissors, 1 Incubator, 1 L of TBA, 15 bottles of Compare the morphology of young 125 mL, Adhesive Solution, Coplin mangroves and old mangroves salt glands Jars, FAA (Solution of EtOH, and leaf stomas and observe how the form of Glacial Acetic Acid, Formalin and the glands benefits the function of the Water), Formaline, Hard Paper (or tissue. Paraffin boats), Heat Plate, Short term goals Microtome, Old white mangroves leafs (20), Razors blades, Slides, Observe changes in mangrove salt glands Solid Safranin (1 g), Wood Blocks, and morphology as they grow and develop. Young white mangroves leafs (20), Long term goals Light Microscope, Stereomicroscope, Coverslips twiser M. Gómez, A. Velazquez, R. Ross Page 2
  • 3. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Procedure: B. Microscopy Techniques A. Obtaining samples 1. Dissecting Microscope First, we identified in Salinas Obtain a slide and Bay the different species of put there a leaf or Mangroves and look for axillar buds and White Mangroves and obtain identified the salt leafs and axillar buds , old glands. Apply and young (extremely external light first carefully). With a knife we only. Then apply separated the axillar buds and the external and leafs. Then we cut the internal light. Turn samples in specific places as off the external light is showed on Appendix 1. and only use After that label 4 Bottles: internal light. (1) Old Mangroves A , (2) Old Mangroves B , (3) 2. Compound Microscope Young Mangroves A, (4) Young Mangroves B. Put the a. Obtain a slide and samples in the bottles with put there a leaf or FAA to preserve the tissues axillar buds with and store them until needed. water. Cover the M. Gómez, A. Velazquez, R. Ross Page 3
  • 4. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 slide(s) and identify to decant the solution and and have close-up repeat the step 1 and 2. To of the salt glands. infiltrate with paraffin pour Apply different off 1/3 volume and replace techniques to have a with an equal volume of variety of photos. melted paraffin . Then try to form a paraffin cap on top of C. Paraffin Embedding TBA and uncap the vial, As indicated in “Plant placing in the paraffin oven Microtechniques and (62o). At 4-12 h intervals Microscopy” (Ruzin, 1999) . pour out ½ volume (into a After fix the tissue paraffin waste) and bring up dehydrate , using the to volume with liquid differents solutions decanting paraffin and repeat this two the old solutions of each times. Now, pour off the sample. (Visit the Appendix paraffin and TBA mixture 2 for instructions of how to and add pure liquid paraffin prepare the solutions.) After and embed in paraffin boats. decant the solutions add a little amount (2 mL) of the solution that will be added D. Sectioning and decant again. Then add Prepare microtone, the solution and wait 8 hours sharpening and adjusting the M. Gómez, A. Velazquez, R. Ross Page 4
  • 5. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 microtone blade. Prepare forward, preventing it to stick paraffin block cutting the to the microtone edge. section with the desired E. Mounting specimen. Form a truncated pyramid around the specimen Cut the paraffin ribbons in equal inside the paraffin block. sizes so they can fit the slide. Apply Make sure that the upper and a thin layer of adhesive solution to lower edges of the paraffin the slide and quickly flood the slide block are parallel. Attach the with formalin after applying the paraffin block to a small adhesive. Place the paraffin ribbons wood block. Place wood in the slide, preventing air bubbles. block with the attached Place the ribbons in section order, paraffin block in microtone from left to right. The first cut ribbon when this is ready. Align the always goes on top. Place the slide in blade to the paraffin block so a warming tray. Wait until most of the blade can cut the sections. the liquid had evaporated. Remove Start cutting the upper the excess liquid with a kimwipe paraffin. After reaching the putting the slide in vertical position specimen you’ll start getting and absorb the falling liquid with the specimen sections. Rotate the kimwipe. Place the slides vertically microtone continuously and on a test tube rack and put the rack in keep the ribbon moving the warming tray. This will dry out the remaining liquid. Visit Appendix M. Gómez, A. Velazquez, R. Ross Page 5
  • 6. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 3 for instruction of the solutions to in the young tissue. The young salt gland be prepared. has excreted less salt during their life as a consequence is less agglomeration of salt in Results their bladder. The mature salt gland has In the young mangrove leaf the salt excreted more salt and then reaches a point glands are outside the principal where the quantity start to accumulate, structure (Visit Appendix 4-A) or because the high temperatures evaporated all inside with a size that can excrete the water and only the salt is there, and the the salts almost directly outside. bladder start the inflation and the salt gland (Visit Appendix 4-B) . Also we can start reducing their size, because is see crystals of salts coming from the compressed by the salt crystals. mid vain to the salt glands in young Conclusion and mature salt glands. (Visit Appendix 5). The mature salt glands The result of this investigation was that have a bigger bladder with mangrove salt glands in petioles and leaves accumulated salts , but the structure are bigger in size in young tissue than in of the salt glands is relatively little mature tissue, with that detail we can know ( Visit Appendix 6). that the metabolic activity is bigger in the young tissue. All the data collected support Discussion our hypothesis. Analyzing the results, the young References white mangrove salt glands are bigger in size, because the metabolic activity is higher M. Gómez, A. Velazquez, R. Ross Page 6
  • 7. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 • Chanita, P. , et. al. Salt uptake and Molecular Phylogenetics and shoot water relations in mangroves. Evolution 34 (2005) 159–166. Aquatic Botany 78 (2004) 349–360 • Yong, Y. , et. al., Effects of salinity • Ruzin, et. al. , 1999.. Plant on germination, seedling growth and Microtechniquez and Microscopy. physiology of three salt-secreting University of Oxford. mangrove species, Aquatic Botany • Suhua, S. , et. al. Molecular 83 (2005) 193–205 phylogenetic analysis of mangroves: • Zouhaier, B.. , et. al. Contribution of independent evolutionary origins of NaCl excretion to salt resistance of vivipary and salt secretion. Aeluropus littoralis (Willd) ParlMolecular. Journal of Plant Physiology 164 (2007) 842—850. M. Gómez, A. Velazquez, R. Ross Page 7
  • 8. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Appendix 1 Figure 1 The boxes are the ideal places to cut off the samples to be studied. M. Gómez, A. Velazquez, R. Ross Page 8
  • 9. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Appendix 2 Solutions to be prepared. Bottle 95% EtOH 100% EtOH Water TBA Safranin A 50 mL - 40 mL 10 mL - B 50 mL - 30 mL 20 mL - C 50 mL - 15 mL 35 mL - D 50 mL - - 50 mL - E - 25 mL - 75 mL - F - 25 mL - 75 mL 0.1 g G - - - 100 mL - H - - - 100 mL - M. Gómez, A. Velazquez, R. Ross Page 9
  • 10. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Appendix 3 Bottle Bottle Contain Time for expose Coplin jar # 1 Xylene, 0.5 minutes Coplin jar # 2 Xylene 0.5 minutes Coplin jar # 3 100% alcohol 0.5 minutes Coplin jar # 4 95% alcohol 0.5 minutes Coplin jar # 5 1 % Safranin in 50% 6-12 Hours alcohol Coplin jar # 6 Wash in water 10-15 seconds. Coplin jar # 7 Wash in water 10-15 seconds Coplin jar # 8 50% alcohol 0.5 minutes Coplin jar # 9 95% alcohol 0.5 minutes Coplin jar # 10 100% alcohol 0.5 minutes Coplin jar # 11 Fast green 5-10 seconds Beaker 1 100% ethanol 1 minutes M. Gómez, A. Velazquez, R. Ross Page 10
  • 11. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Beaker 2 100% ethanol 1 minutes Coplin jar # 13 Xylene 5 minutes Coplin jar # 14 Xylene 5 minutes Coplin jar # 15 Xylene 5 minutes Appendix 4 Figure A : Young Mangrove Salt gland in a extreme outside the leaf. M. Gómez, A. Velazquez, R. Ross Page 11
  • 12. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Figure B: Young Mangrove Salt glands inside the leaf Appendix 5 Figure A: Young Mangrove Salt Crystals connected to the mid vain M. Gómez, A. Velazquez, R. Ross Page 12
  • 13. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Appendix 6 M. Gómez, A. Velazquez, R. Ross Page 13
  • 14. [Morphology changes in Laguncularia racemosa’s salt glands regarding it’s life period] April 17, 2009 Figure A: Young Mangrove Salt Gland Figure B: Mature Mangrove Salt Gland M. Gómez, A. Velazquez, R. Ross Page 14