Mangrove Development Research Proposal

[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

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


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


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


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


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


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.


April 17, 2009

Appendix 1

Figure 1

The boxes are the ideal places to cut off the samples to be studied.


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 -


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 # 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 # 11 Fast green 5-10 seconds

Beaker 1 100% ethanol 1 minutes


April 17, 2009

Beaker 2 100% ethanol 1 minutes

Coplin jar # 13 Xylene 5 minutes



Appendix 4

Figure A : Young Mangrove Salt gland in a extreme outside the leaf.


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


April 17, 2009

Appendix 6


April 17, 2009

Figure A: Young Mangrove Salt Gland

Figure B: Mature Mangrove Salt Gland


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