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

Mangroves are usually found in

environments where water is filled with high

concentrations of dissolved salts. However,

mangroves had developed structural

adaptations for this particular environment.

Salt glands are structures that allow

mangroves to avoid the pitfalls of these

harsh conditions. These glands help the

mangrove secrete the excess salt that

receives from the water, preventing

dehydration. The purpose of the

investigation is to observe the structure of

these glands and how the morphology of salt

glands helps the mangrove to secrete the

excess salt. The project is also going to be

focused on the leaf stomas and other

structures that can be observed on the leaf

surface.

Abstract

In this experiment we will identify specific

characteristics for the salt glands in young

and old mangroves. After collect the

samples, our team preserve them and applied

direct techniques to identify the salt glands

and proceed to prepare sectioning of the

samples to compare the specific

morphology of the old and young

mangroves. The expected result was that

young mangroves have salt glands more

efficient that old mangrove because old

mangroves have more salt gland to liberate

the salts, and the young mangrove have less

salt glands but however it needs to eliminate

the same amount of salt in less salt glands.

M. Gómez, A. Velazquez, R. Ross Page 1


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April 17, 2009

The result was that young mangrove salt

glands are bigger that mature mangrove’s

salt glands, because the young mangrove

have less amount of salt accumulated and

that makes the

Hypothesis

Young mangrove salt glands are more

capable of salt secretion than older

mangrove salt glands

based on their relative size.

Objectives

Compare the morphology of young

mangroves and old mangroves salt glands

and leaf stomas and observe how the form of

the glands benefits the function of the

tissue.

Short term goals

Observe changes in mangrove salt glands

and morphology as they grow and develop.

Long term goals

Find out what salt concentration is more

beneficial to young and old mangroves in its

habitat depending on the salt glands capacity

to secrete salt.

Materials

The materials that are going to be

used are: ¼ L Fast Green, ¼ L of Eth

(100%), ¼ L of Water, ½ liter of

Eth (95%), ½ L Xylene, 1 bag of

ParaplastTM, 1 Garden Scissors, 1

Incubator, 1 L of TBA, 15 bottles of

125 mL, Adhesive Solution, Coplin

Jars, FAA (Solution of EtOH,

Glacial Acetic Acid, Formalin and

Water), Formaline, Hard Paper (or

Paraffin boats), Heat Plate,

Microtome, Old white mangroves



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April 17, 2009

leafs (20), Razors blades, Slides,

Solid Safranin (1 g), Wood Blocks,

Young white mangroves leafs (20),

Light Microscope,

Stereomicroscope, Coverslips twiser

Procedure:

A. Obtaining samples

First, we identified in Salinas

Bay the different species of

Mangroves and look for

White Mangroves and obtain

leafs and axillar buds , old

and young (extremely

carefully). With a knife we

separated the axillar buds and

leafs. Then we cut the

samples in specific places as

is showed on Appendix 1.

After that label 4 Bottles:

(1) Old Mangroves A , (2)

Old Mangroves B , (3)

Young Mangroves A, (4)

Young Mangroves B. Put the

samples in the bottles with

FAA to preserve the tissues

and store them until needed.

B. Microscopy Techniques

1. Dissecting Microscope

Obtain a slide and

put there a leaf or

axillar buds and

identified the salt

glands. Apply

external light first

only. Then apply

the external and

internal light. Turn

off the external light

and only use

internal light.



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April 17, 2009

2. Compound Microscope

a. Obtain a slide and

put there a leaf or

axillar buds with

water. Cover the

slide(s) and identify

and have close-up

of the salt glands.

Apply different

techniques to have a

variety of photos.

C. Paraffin Embedding

As indicated in “Plant

Microtechniques and

Microscopy” (Ruzin, 1999) .

After fix the tissue

dehydrate , using the

differents solutions decanting

the old solutions of each

sample. (Visit the Appendix

2 for instructions of how to

prepare the solutions.) After

decant the solutions add a

little amount (2 mL) of the

solution that will be added

and decant again. Then add

the solution and wait 8 hours

to decant the solution and

repeat the step 1 and 2. To

infiltrate with paraffin pour

off 1/3 volume and replace

with an equal volume of

melted paraffin . Then try to

form a paraffin cap on top of

TBA and uncap the vial,

placing in the paraffin oven

(62o). At 4-12 h intervals

pour out ½ volume (into a

paraffin waste) and bring up

to volume with liquid

paraffin and repeat this two

times. Now, pour off the

paraffin and TBA mixture



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April 17, 2009

and add pure liquid paraffin

and embed in paraffin boats.

D. Sectioning

Prepare microtone,

sharpening and adjusting the

microtone blade. Prepare

paraffin block cutting the

section with the desired

specimen. Form a truncated

pyramid around the specimen

inside the paraffin block.

Make sure that the upper and

lower edges of the paraffin

block are parallel. Attach the

paraffin block to a small

wood block. Place wood

block with the attached

paraffin block in microtone

when this is ready. Align the

blade to the paraffin block so

the blade can cut the sections.

Start cutting the upper

paraffin. After reaching the

specimen you’ll start getting

specimen sections. Rotate the

microtone continuously and

keep the ribbon moving

forward, preventing it to stick

to the microtone edge.

E. Mounting

Cut the paraffin ribbons in equal

sizes so they can fit the slide. Apply

a thin layer of adhesive solution to

the slide and quickly flood the slide

with formalin after applying the

adhesive. Place the paraffin ribbons

in the slide, preventing air bubbles.

Place the ribbons in section order,

from left to right. The first cut ribbon

always goes on top. Place the slide in

a warming tray. Wait until most of



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April 17, 2009

the liquid had evaporated. Remove

the excess liquid with a kimwipe

putting the slide in vertical position

and absorb the falling liquid with the

kimwipe. Place the slides vertically

on a test tube rack and put the rack in

the warming tray. This will dry out

the remaining liquid. Visit Appendix

3 for instruction of the solutions to

be prepared.

Results

In the young mangrove leaf the salt

glands are outside the principal

structure (Visit Appendix 4-A) or

inside with a size that can excrete

the salts almost directly outside.

(Visit Appendix 4-B) . Also we can

see crystals of salts coming from the

mid vain to the salt glands in young

and mature salt glands. (Visit

Appendix 5). The mature salt glands

have a bigger bladder with

accumulated salts , but the structure

of the salt glands is relatively little

( Visit Appendix 6).

Discussion

Analyzing the results, the young

white mangrove salt glands are bigger in

size, because the metabolic activity is higher

in the young tissue. The young salt gland

has excreted less salt during their life as a

consequence is less agglomeration of salt in

their bladder. The mature salt gland has

excreted more salt and then reaches a point

where the quantity start to accumulate,

because the high temperatures evaporated all

the water and only the salt is there, and the

bladder start the inflation and the salt gland

start reducing their size, because is

compressed by the salt crystals.

Conclusion



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April 17, 2009

The result of this investigation was that

mangrove salt glands in petioles and leaves

are bigger in size in young tissue than in

mature tissue, with that detail we can know

that the metabolic activity is bigger in the

young tissue. All the data collected support

our hypothesis.

References

Chanita, P. , et. al. Salt uptake and

shoot water relations in mangroves.

Aquatic Botany 78 (2004) 349–360

Ruzin, et. al. , 1999.. Plant

Microtechniquez and Microscopy.

University of Oxford.

Suhua, S. , et. al. Molecular

phylogenetic analysis of mangroves:

independent evolutionary origins of

vivipary and salt secretion.

Molecular Phylogenetics and

Evolution 34 (2005) 159–166.

Yong, Y. , et. al., Effects of salinity

on germination, seedling growth and

physiology of three salt-secreting

mangrove species, Aquatic Botany

83 (2005) 193–205

Zouhaier, B.. , et. al. Contribution of

NaCl excretion to salt resistance of

Aeluropus littoralis (Willd)

ParlMolecular. Journal of Plant

Physiology 164 (2007) 842—850.



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April 17, 2009

Appendix 1

Figure 1

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



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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 -



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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% alcohol

6-12 Hours

Coplin jar # 6 Wash in water 10-15 seconds.

Coplin jar # 7 Wash in water 10-15 seconds





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April 17, 2009


Coplin jar # 11 Fast green 5-10 seconds

Beaker 1 100% ethanol 1 minutes

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.



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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



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April 17, 2009

Appendix 6

Figure A: Young Mangrove Salt Gland



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April 17, 2009

Figure B: Mature Mangrove Salt Gland


Technology

Mangrove Development Research Proposal