BIOLOGY LABORATORY REPORT

A-LEVEL MEDICINE



NAME : NORHAZWANI ZUL
SID : 1311171673
GROUP: 11SC6 ( TRURO)
TITLE :TOTIPOTENCY AND PLANT TISSUE CULTURE.
DATE OF EXPERIMENT : 24
TH
JANUARY 2014
DATE OF SUBMISSION : 17
TH
FEBRUARY 2014
LECTURERS NAME: MISS FARAH ANIZA BINTI SHAHRI







Objective
1. To demonstrate the totipotency of Brassica rapa sp using plant tissue culture
technique.
2. To observe the development of the explant of Brassica rapa sp
3. To practise the process of culturing the tissue of Brassica rapa sp
Introduction
What is totipotency?
The ability of normal cell in the plant body to reproduce and to generate an entire plant is
called totipotency. This characteristic only occurs on specific types of tissue in plants called
meristematic tissue. Many somatic plant cells, including some fully differentiated types like
leaf mesophyll, provided the right information from the genetic makeup of the cell, have the
capacity to regenerate into whole plants.
[i]
Totipotent cells serve the same role in plants that
stem cells do in animals. They are found in shoot and root growing tips as meristems, and in
the cambium layer (the layer of cells between the bark and the wood) of woody plants and
trees. The activity of these meristematic regions gives rise to new roots, stems, leaves, and
flowers or fruiting structures in plants, as well as increasing the diameter of woody plant
trunks and branches. All of the structures found in a mature or growing plant are the result of
cellular material produced by meristematic tissue.
[ii]


Schwann and Schleiden in 1938 gave the theory of totipotency. They gave the concept that
cells are autonomic and are capable of regenerating to produce a complete plant.
The in vitro techniques were developed initially to demonstrate the totipotency of plant
cells predicted by Haberlandt in 1902. Basically Haberlandt was the first person done
practically totipotency by culture of isolated single palisade cells from leaves in Knop's salt
solution enriched with sucrose. The cells remained alive for up to 1 month, increased in size,
accumulated starch but failed to divide.
[iii]



Then, the modern practical was developing based from the experiment. This
totipotency sets plant cells apart from most of their animal counterparts because plant cell can
be totipotent throughout their life, and it was rst demonstrated by Steward and Reinert in the
1950s.
[iv]
Often totipotency is revealed when tissues are removed from their normal
environment and placed onto tissue culture, which act as growth medium. In plants,
totipotency is found in shoot and root of growing tips, and in the cambium layer (the layer of
cells between the bark and the wood) of woody plants and trees. We can generalize by saying
that most plants at most stages of the life cycle have some populations of cells that are
totipotent. Totipotency is of course also a property of normal undifferentiated cells, for
example in meristems.

What is a tissue culture?
Plant tissue culture also known as micro propagation, which is generally used for the aseptic
culture of cells, tissues, organs and their components
[v]
.Tissue culture practical is frankly
described which uses vegetable Brassica seedlings germinated in-vitro and cut up into root,
hypocotyl, and cotyledon explants. These are then cultured on media containing different
levels of phytohormones which induce shoot, root, and callus production in as little as four to
six weeks. The practical is simple and easy to use and does not require the use of lamina flow
cabinet.
[vi]
Tissue culture is often the fastest and most economical means to achieve this goal
as very, very small cell can develop to whole plant.
Tissue culture technique is asexual propagation was placed in sterile or aseptic culture in a
test tube, petri dish or tissue culture container containing a special culture medium. The
culture medium is the most important part of plant tissue culture. Agar medium usually
contains inorganic elements, organic compounds that are usually available from soil.
Occasionally, plant growth regulators are added to the medium to arouse cell division and
differentiation.
[vii]
Afterwards it is placed inside the agar the tissue will start to grow and form
callus.

What is a Brassica rapa sp?
Brassica rapa is also known as Brassica Campestris or brassica rapa var. chinensis.
[viii]

Brassica rapa sp are rapid-cycling Brassicas. They are members of the crucifer family of
plants,closely related to cabbage, turnips, broccoli and other cruciferous vegetables. Brassica
rapa sp require little more attention than continuous fluorescent light, water, and
fertilizer
[ix]
.Brassica rapa sp have n=10
[x]
which have a life cycle completed in just 5 weeks
and only limited space in the laboratory for growth and allow students to undertake small-
scale individual investigations within a reasonable time scale.
[xi]
Therefore, there prefer to
use it in experiment in collague.

Problem statement
How does the cells in plant tissue culture able to develop into new complete plant?
Hypothesis
The explant of Brassica rapa sp will grow after 8 days to show its totipotency property.
Materials and apparatus
Seedlings of Brassica rapa sp ,agar powder, distilled water, paraffin film,100 ml beaker
,autoclaved forceps ,scissors , sticker, towel , fabrics glove
Technique
Observe the explant of Brassica rapa sp within 10 days period and record any changes
during the development of the explant into a new complete plant. The length of root , the
height of the plant and the length of the leaves are measured using ruler
Procedure
1. A beaker is used after the it has been cleaned up using towel
2. An agar solution was prepared using agar powder and distilled powder
3. 50 ml of agar was poured into the beaker.
4. The agar solution was let to cool down until it reached semi-solid state. The beaker
was covered with a piece of paper to prevent contamination to the agar solution by the
microorganism in the air
5. At the same time, three Brassica rapa sp. were cut at the tops off seedlings just below
the shoot apex.
6. When the agar reached semi-solid state, three Brassica rapa sp. were put inside the
agar solution with the bottom part of the plant did not touch the beaker.
7. The beaker was then covered with paraffin film. A sticker was adhered to mark whose
plant is.
8. The beaker was observed for 10 days.

Observation
Days Days Physical appearance Internal
environment
A B C A B C
Sat

1


-light green
-turgid
-a little bit of
air bubbles on
the inside
Sun 2

-taller leaves
-a little bit
darker
-light brown
colour found
on stem

-light
green
(uneven
colour of
green on
the leaves
)
-more air
bubbles at wall
of beaker
Mon 3

-turgid
-dark green
leaves
-healthy
-flaccid
-wilt
-stem is
brownish
-2 of the
leaves
didnt
grow (wilt
and
yellow)
-brownish
at the end
of the stem
-small of water
droplet on the
surface of
parafilm
Tue 4 -turgid
-dark green
leaves
-flaccid
-wilt
-stem thin and
-2 dark
green
leaves
-small water
droplet on the
surface of

-healthy brownish
- uneven
diameter due
pressure of
forceps apply
on explant
-small leaves
-light green



-2 yellow
green
leaves
-small
leaves
-brownish
at the end
of the stem
-healthy
on the rest
parafilm and
wall of beaker
Wed 5

-turgid
-dark green
leaves
-healthy
-flaccid
-wilt
-stem thin and
brownish
- uneven
diameter due
pressure of
forceps apply
on explant
-small leaves
-light green

-2 dark
green
leaves
-2 yellow
green
leaves
-small
leaves
-brownish
at the end
of the stem
-healthy
on the rest
-more water
droplet on the
surface of
parafilm
-very lots of
water droplets
on both
parafilm
surface and
beakers wall
Thu
-( at
balcony)
-higher
sunlight
intensity
6

-more darker
green colour
of leaves
-firm
-turgid
-medium green
leaves
-brownish
stem is more
obvious
-thin stem
-2
yellowish
small
leaves
grow in
size and
more
mixture
colour of
-more water
droplet on the
surface of Para
film
-very lots of
water droplets
on both Para
film surface
and beakers
yellow and
lust green
wall
Fri
-hot
blazing
sun
7

-move towards
sunlight
stimulus
(phototropism)
-move towards
sunlight
stimulus
(phototropism)
-not move
-not stable
as explants
fall at the
wall of
beaker
Lesser water
droplet on Para
film because
water is
condensed
Sat
(broken)
-
measured
when
uprooted
8

- Container is broken (the growth of roots can be seen
more clearly)
- Explants become more slanted
- No significant changes in height of leaves
- The leaves colour still remain dark green
Bacteria can growth after the beaker is broken as Para film is
also damaged




Results
Days Days length of roots/ cm Height of explant/
cm
Size of leaves/
cm
No of leaves
a b C a b c a b c a B c
Sat 1 0.00 0.00 0.00 0.7 0.5 0.6 0.2 0.2 0.2 4 4 4
Sun 2 0.00 0.00 0.00 0.9 0.5 0.7 0.3 0.2 0.3 4 4 4
Mon 3 0.02 0.02 0.02 1.0 0.5 0.7 0.3 0.2 0.3 4 4 4
Tue 4 0.05 0.05 0.05 1.0 0.5 0.7 0.3 0.2 0.3 4 4 4
Wed 5 0.07 0.07 0.07 1.0 0.5 0.7 0.3 0.3 0.3 4 4 4
Thu 6 0.10 0.10 0.10 1.0 0.5 0.7 0.3 0.3 0.3 4 4 4
Fri 7 0.20 0.10 0.40 1.0 0.6 0.7 0.3 0.3 0.3 4 4 4
Sat 8 0.50 0.00 0.60 1.0 1.0 0.7 - - - 4 4 4



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Interpretation data
This experiment was carried out to investigate the totipotency in plant which is Brassica
Rapa sp using the tissue culture technique. The Brassica Rapa sp was taken and the stem was
cut out just below the shoot apex as this was where the meristematic cell lies. The seedling
were now called the explants, and was inserted in the agar solution which is the tissue culture
medium. The growth and development of the explants are observed in 8 days and the number
of leaves, the length of root, the height of the explants, the size of the leaves, any physical
increment and changes to the internal environment of the agar solution were recorded.
The graph portrays, the height of Brassica rapa sp increases over the 8 day of
observation. First day the height of explant A, B and C were only 0.7 cm,0.5 cm and 0.6 cm
respectively. After 8 days of observation, the height of the explants had amplified about 0.3
cm. There was no increment in the height of the explants within third days and sixth day due
to the explants have to focus more in growth of root . Therefore, more food was supply to
root in order to help the growth of the root of explant. Nevertheless, the rate of growth of
plant increases afterward and stays constant until the ends of the observation as the food
supply have finished up. The explant have achieved the optimum height of plant as the beaker
was closed so the further production food cannot synthesizes by leaves.
The number of leaves and the size of the leaves were also observed and recorded. The
number of leaves does not increase at 4 while the size of leaves of all explants show an
augmentation about 0.1 cm over the period of observation. The colour of the healthy explant
A show a dark green while light green for explant B and mixture of dark green and yellow
colour of explant C. Green colour of the leaves show that the explant have more and more
green pigment which is chlorophyll . The relationship between chlorophyll synthesis,
chloroplast structure and the development of photosynthesis , as measured by carbon dioxide
uptake , oxygen evolution or by partial reactions of isolated chloroplast
[xii]
. It is well known
are the photosynthetic activity lags behinds chlorophyll formation.
[xiii]
Therefore , the
photosynthetic activity lags of explant A is at the maximum as the leaves has dark green
colour. The explants C have the yellow colour of leaves display that the photosynthetic
activity lags at the explant C is at minimum rate. The stem colour in explant B is reddish
brown and have damaged stem due to excessive applied pressure applied when forceps are
used to insert the explant into the agar solution. That is why the explant B flaccid and wilt on
fifth day.
The explant A and B bend toward the strongest light on the seventh day. They do this
by elongating the cells of the stem on the side that is farthest from the light. This type of
light-oriented growth is called phototropism
[xiv][xv]
. The shoot explant move towards sunlight
to perform photosynthesis in order to make its own food so that it can endure. The increment
in the height and size of leaves shows that the nutrients obtained via photosynthesis is used
for growth by the explants. Moreover, the cotyledons itself are photosynthetic and they have
their own storage of food for the plant to survive for a few days. Consequently, it enables the
explants to maintain its healthy state. The raise in height also shows that the shoot growing
against as the root is negative phototropism
[xvi]
. The root growth downward as the root is
positive gravitropism or geotropism, dictates upward shoot growth to ensure a proper
positioning of the leaves for efficient photosynthesis and gas exchange. It also directs roots to
grow downward in soil, where they can reach out to take up the water and mineral ions
required for plant growth and development
[xvii]
. The growth of roots shoots and leaves from
the explants proves that the plant cells are totipotent.

Discussion
Agar solution supporting structure to the explant as it is resistant to force for the developing
explant. In this experiment, student the recommended to use small beaker to prevent wasting
of agar and shorter time taken to solidify the agar. Nutrient agar is not suitable to be used for
this experiment as it can stimulate the growth of bacteria. The other function of agar is to
supply water to the explant for photosynthesis process. The seedlings were used as explants
when they have just started to unfold their cotyledons before the totipotent cells at the apex
start to differentiate. This is because the cotyledons act as leaves in seedlings and are vital for
the development of the explant. If there is no supply of food from cotyledons, the explant will
do photosynthesis to make their own food.
The short-necked test tube was recommended because easier to put the explants into
the agar so that the forceps used does not touch the agar that will lead to contamination. If the
long necked test tube was used, a greater depth should be required. If you need use the long-
necked test tube is used due some limitation, put more agars into the test tube until more than
three quarter of the beaker so that the explants will be put at the opening of the test tube. Else
you will need to use a longer tweezers to put the explants.

The test tube must be covered by the parafilm to prevent the growth of bacteria and
fungi and maintains the humidity inside the beaker as parafilm have special characteristics as
ductile, malleable, waterproof, odourless, thermoplastic, semi-transparent and cohesive.
[xviii]

Besides it also act to prevent the moisture loss and enabling the penetration of sunlight inside
so the explants can make photosynthesis. The beaker was covered by parafilm to avoid
contamination to the agar solution from bacteria or fungi and microorganism present in the
air. The parafilm can also maintain humidity by preventing moisture loss and allows light in
so that the explant can perform photosynthesise for its food production. Once the experiment
is completely set up, the parafilm would not be opened again within the 8 days of experiment
to provide a fixed internal situation to the explants which were developing and dividing
actively. The tubes should not be opened again so that they will not be further polluted with a
microorganism.

We should not open the test tube again once we set them up to prevent
contamination of dangerous microorganisms which might compete the same need as the
explants.

The measurement that should be made as the explants grow is the height of the
explants, length and number of roots, size of leaves and the intensity colour of leaves present
in each explant throughout the experiment. . The time taken for each physical development
on the explant can be recorded.

This experiment proved that plant particularly Brassica Rapa apex as the explants
can grow into a new complete plants which have new leaves, stems and roots which more
known as totipotent . The cotyledons can also photosynthesise and also contain their own
store of nutrients, so they can grow and develop even though no additional nutrients are
added to the agar during the days of experiment
The futher information that can be gain by extending this experiment by just
growing the shoot apex or only isolated cotyledons and make comparison are some indication
of the extent to which development of the explants is dependent on the presence of apex,
hypocotyls or cotyledons.


Validity and reliability
The result should be considered as valid because the experiment was done in controlled
condition. The growth of plant Brassica rapa sp was observed once in days throughout the
experimental was done. The height of the plant was measured using ruler, the colour of the
leaves and the presence of root were observed

Source of errors
The apparatus used would be some sources of errors. This is because the content of
microorganisms that might contaminate the agar nutrient. This would be a reason for
experiments which had some black spots or even maggot on its agar nutrient. The forceps and
scissors used are also could be contaminated by microorganisms before the experiment was
begin. Mouth of agar bottle also could contain microorganisms as a product of previous used
of the bottle by pouring it into any container.
Besides that, the state of agar nutrient used during the insertion of Brassica rapacould be
too liquid or too solid. This would cause instability to the plant, thus may cause the plant to
fall against the agar. This may also cause the bottom part of the plant to move too downward
until it reaches the surface of the beaker. If the agar is too hard, it is impossible to put the
plant into the agar. Other than that, other microorganism in the air would enter the beaker via
the mouth of the beaker. This might alter the result of the microorganism in the air are
succeeded to get into the beaker.
Limitations
It is impossible to make sure none of the microorganisms is in the beaker during the
experiment. Despite autoclaving and lighting the surface of the beaker and the mouth of the
agar bottle, the microorganism would still enter the beaker by exhalation of human during the
experiment was carried out. Water droplets from mouth which would came out during
conversations during experiment would contribute to the presence of microorganism in the
beaker.
Besides that, the state of agar actually cannot be justified by swirling it. As touching the
agar could transmit microorganisms to the agar, swirling the beaker is seems to be the best
alternative to make sure the solid is in semi-solid state. However, the concentration of the
agar could be spoiled due to swirling of the beaker, thus the agar may not become hard after a
long period of cooling.

Modifications and further works
In the following experiment, the person in charges in the experiment should wear face
mask to limitation the transmission of microorganism via exhalation or conversation into the
beaker. This should give better and more reliable result.

Further experiment can be done by varies the treatment of cotyledon excision of the
embryo in order to stimulate the embryo germination and development of plants from
embryos. This can determined whether which way to germinate seed is better .
[xix]

Moreover ,we also can further experiment by taking different part of Brassica rapa to
show how effect the growth of plant. For instance, if the shoot apex is grown without its
cotyledon, the growth and development of the explant in the tissue culture will be slower
compared to a growing shoot apex together with its cotyledon. This is because the cotyledons
provide food and nutrients for the explant as they are growing in the agar solution which also
provides nutrients for the explant. The explant gets the nutrients both from cotyledon and
agar solution before they can make the food on their own in the process of photosynthesis.
Therefore, without cotyledon, the explant require more nutrient from the agar solution thus
their development would become harder and slower compared to the growing shoot apex
with its cotyledon attached to themselves.

Advance experiment on totipotency can be done in varies temperature of agar solution.
This might be held in the variety of agar solution temperature. Hence, we could see at which
optimum temperature the totipotency of plant stem cell could be the best. Besides, the
Brassica rapa could be planted in different type of nutrients. From the result obtained, we
could determine which nutrient is the most important for the plant for totipotency.

Safety precautions
Throughout the experiments, there are precautions that were taken to avoid the conditions
that might lead to the inaccuracy of the results obtained.
Individual
1. Laboratory coat is worn when conducting the experiment to avoid spillage of molten
agar solution which can stain clothes
2. Suitable shoes must be worn during conducting the experiment.
3. Goggles also must be worn to avoid the eyes become irritate due to chemical
substances .
4. Wear gloves substance may cause irreversible eye injury
5. The sharp apparatus such as scissors was handled carefully
6. Washing hands thoroughly with soap and water before and after conducting
experiment is vital to avoid contamination.
7. Laboratory apparatus should be handled carefully and properly such as measuring
cylinder to prevent any injuries or accident while conducting the experiment since
they can break easily
8. The apparatus such as forceps and scissors are also sterilized to prevent infection of
microorganism. After using all samples and apparatus at the end of experiment, they
should be discarded properly and returned back to their places to avoid injuries and
unnecessary accidents.
Experiment
1. The beaker was covered with a piece of paper while waiting for the agar solution to
cool and solidify to prevent contamination
2. The agar is ensured to be in semi-solid state ,so easier Brassica rapa sp is pushed into
agar. The surface of the agar must not be touched when pushing the explants into the
agar by forceps to avoid contamination of bacteria and microorganism which can
affect the internal environment for the explants to grow and divide.
3. The beaker is covered with a piece of parafilm to avoid the entering of microorganism
and bacteria therefore minimise the contamination in the beaker which can affect the
growth of Brassica Rapa.
4. The parafilm will not be opened during 10 days period of observation once the beaker
has been sealed safely with it.


Conclusion
The explant of Brassica rapa sp grow its root and size of leaves in 8 days. Therefore, the
explant shows the totipotency.The hypothesis is accepted.

References
1. Genetic control of totipotency of plant cells
Ezhova TA. Ontogenez. 2003 Jul-Aug;34(4):245-52.
Biological Faculty, Moscow State University, Vorob'evy gory, Moscow, 119899
Russia.

2. http://davesgarden.com/guides/articles/view/1777/
3. http://plantissueculture.com/?p=417
4. http://edugreen.teri.res.in/explore/bio/tissue.htm
5. http://edugreen.teri.res.in/explore/bio/tissue.htm


i
Dr Brian J. Atwell, School of Biological Sciences, Macquarie University ,Dr Paul E. Kriedemann, Research
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University of Queensland, Plants in Action ,Adaptation in Nature, Performance in Cultivation
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ii
http://davesgarden.com/guides/articles/view/1777/
iii
ibid
iv
Dr Brian J. Atwell, School of Biological Sciences, Macquarie University ,Dr Paul E. Kriedemann, Research
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v
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vi
Michael P. Fuller and Frances M. Fuller , 13 Dec 2010 , pages 53-59, Plant tissue culture using
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vii
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viii
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ix
Dr. Williams ,Department of Plant Pathology at the University of Wisconsin-Madison since 1962.THE STORY
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x
Geoffrey R. Dixon

,Vegetable Brassicas and Related Crucifers, 2006, CABI, 2007 - Gardening page 4
xi
John Adds, Erica Larkcom, Ruth Miller, Robin Sutton; Tools, Techniques and Assessment in Biology, 2001
xii
Kenneth D. Nadler,2 Helen A. Herron, and S. Granick Plant Physiol. 1972 March; 49(3): 388392.
PMCID: PMC365971, Development of Chlorophyll and Hill Activity 1
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