Biology Laboratory Report Totipotency

BIOLOGY LABORATORY REPORT

A-LEVEL MEDICINE

NAME NORHAZWANI ZUL

SID 1311171673

GROUP 11SC6 ( TRURO)

TITLE TOTIPOTENCY AND PLANT TISSUE CULTURE

DATE OF EXPERIMENT 24TH JANUARY 2014

DATE OF SUBMISSION 17TH FEBRUARY 2014

LECTURERrsquoS 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 Knops 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 first 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

plantsclosely 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 film100 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

didnrsquot

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

beakerrsquos 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 beakerrsquos

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 1 000 000 000 07 05 06 02 02 02 4 4 4

Sun 2 000 000 000 09 05 07 03 02 03 4 4 4

Mon 3 002 002 002 10 05 07 03 02 03 4 4 4

Tue 4 005 005 005 10 05 07 03 02 03 4 4 4

Wed 5 007 007 007 10 05 07 03 03 03 4 4 4

Thu 6 010 010 010 10 05 07 03 03 03 4 4 4

Fri 7 020 010 040 10 06 07 03 03 03 4 4 4

Sat 8 050 000 060 10 10 07 - - - 4 4 4

The h

eight

of pl

ant a

gains

t day

s

A B C

051

152

253

354

455

556

657

758

020406081

Days

Heigh

t of e

xplan

tcm

The l

ength

of ro

ots ag

ainst

days

A B C

051

152

253

354

455

556

657

758

01020304050607

Days

Leng

th of

roots

cm

The s

ize of

leav

es ag

ainst

days

A B C

051

152

253

354

455

556

657

758

005010150202503035

Days

Size o

f leav

escm

The n

o of l

eave

s aga

inst

days

A Serie

s 1

Serie

s 2

051

152

253

354

455

556

657

758

1234

Days

No of

leav

es

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 07 cm05 cm and 06 cm

respectively After 8 days of observation the height of the explants had amplified about 03

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 01 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 totipotencyThe hypothesis is accepted

References

1 Genetic control of totipotency of plant cells

Ezhova TA Ontogenez 2003 Jul-Aug34(4)245-52

Biological Faculty Moscow State University Vorobevy gory Moscow 119899

Russia

2 httpdavesgardencomguidesarticlesview1777

3 httpplantissueculturecomp=417

4 httpedugreenteriresinexplorebiotissuehtm

i Dr Brian J Atwell School of Biological Sciences Macquarie University Dr Paul E Kriedemann Research School of Biological Sciences Australian National University Dr Colin GN Turnbull Department of Botany University of Queensland Plants in Action Adaptation in Nature Performance in Cultivation httpplantsinactionscienceuqeduauedition1q=content10-2-1-concept-totipotency ii httpdavesgardencomguidesarticlesview1777iii ibidiv Dr Brian J Atwell School of Biological Sciences Macquarie University Dr Paul E Kriedemann Research School of Biological Sciences Australian National University Dr Colin GN Turnbull Department of Botany University of Queensland Plants in Action Adaptation in Nature Performance in Cultivation httpplantsinactionscienceuqeduauedition1q=content10-2-1-concept-totipotencyv httpamritavlabcoinsub=3ampbrch=187ampsim=1100ampcnt=1 vi Michael P Fuller and Frances M Fuller 13 Dec 2010 pages 53-59 Plant tissue culture using Brassico seedlings Journal of Biological Education Volume 29 Issue 1 1995 DOI1010800021926619959655419viihttppasselunledupagesinformationmodulephp idinformationmodule=956783940amptopicorder=5ampmaxto=10ampminto=1viii John Adds Erica Larkcom Ruth Miller Robin Sutton Tools Techniques and Assessment in Biology 2001ix Dr Williams Department of Plant Pathology at the University of Wisconsin-Madison since 1962THE STORY OF FAST PLANT page 1 httpwwwfastplantsorgaboutthe_story_of_fast_plantsphp 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 2001xii Kenneth D Nadler2 Helen A Herron and S Granick Plant Physiol 1972 March 49(3) 388ndash392PMCID PMC365971 Development of Chlorophyll and Hill Activity 1httpwwwncbinlmnihgovpmcarticlesPMC365971 xiii R J Dowdell A D Dodge Planta 2 VI 1971 Volume 98 Issue 1 pp 11-19 Chlorophyll formation and the development of photosynthesis in illuminated etiolated pea leavesxiv Prof Claus Schwechheimer from the Chair of Plant Systems Biology at the Technische Universitaumlt Muumlnchen (TUM) How do plants grow toward the light Scientists explain mechanism behind phototropism May 28 2013 page 1httpwwwsciencedailycomreleases201305130528105946htm xv Craig W Whippo Phototropism Bending towards Enlightenment Department of Biology Indiana University Bloomington IN 47405-7107 doi httpdxdoiorg101105tpc105039669The Plant Cell May 2006 vol 18 no 5 1110-1119 page 1 httpwwwplantcellorgcontent1851110full xvi Unknown httpusersrcncomjkimballmaultranetBiologyPagesTTropismshtmlxvii Rujin Chen Elizabeth Rosen and Patrick H Masson Gravitropism in Higher Plants1 1 Laboratory of Genetics 445 Henry Mall University of Wisconsin Madison Wisconsin 53706doi http dx doi org 10 1104 pp 120 2 343 Plant Physiology June 1999 vol 120 no 2 343-350 page 1 httpwwwplantphysiolorgcontent1202343fullfn-2 xviii Unknown httpenwikipediaorgwikiParafilm xix LXu UNajeeb GXTang HHGuGQZhang YHe WJZhou Advances in Botanical Research 2007

Biological Faculty Moscow State University Vorobevy gory Moscow 119899 Russia

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