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BIOLOGY LAB REPORT TITLE : TOTIPOTENCY AND PLANT TISSUE CULTURE PREPARED BY : I/C NUMBER : STUDENT ID : GROUP : LAB PARTNER : LECTURER’S NAME : PRACTICAL DATE : SUBMISSION DATE :
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Page 1: totipotency

BIOLOGY LAB REPORT

TITLE : TOTIPOTENCY AND PLANT TISSUE CULTURE

PREPARED BY :

I/C NUMBER :

STUDENT ID :

GROUP :

LAB PARTNER :

LECTURER’S NAME :

PRACTICAL DATE :

SUBMISSION DATE :

Page 2: totipotency

Abstract

Plant has the ability to divide and differentiate from a single cell into all type of cells that suppose to be in an organism. This ability is known as totipotency. In this experiment, we are to demonstrate the ability of totipotency of plant cells by extracting the explants of Brassica Rapa and growing them in the agar medium , for about 7 days. Observations were made daily for any changes in the conditions of explant and were recorded in a table.

Introduction

1. Brassica Rapa(1)

The explant used in this experiment is largely known as Pak Choy or Chinese cabbage. Scientific

name that was given to this plant is Brassica Rapa Campestris but commonly called as Brassica Rapa.

This plant is usually used in Chinese cuisine and also used in making kimchi, originating from Korea.

Pak Choy contains high amount of Vitamin A and C, thus acting as antioxidant source. Pak Choy also

said to contain glucosinolates, a compound which has been identified in preventing cancer in small

doses, but toxic for human race. It may cause hypothyroidism which probably will lead to

myxedema coma. Other milder symptoms from over-consumption of Pak Choy, such as nausea,

dizziness and indigestion in people with weaker digestive systems and sometimes this is caused by

not thoroughly cooking.

Figure 1 : Brassica Rapa (2)

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2. Totipotency (3)

Totipotency is known as the ability of a cell to divide and differentiate into all type of cells in order to

develop a complete organism. Only plant has the ability to remain totipotent for its whole live and it

means, any part of a plant can be used to give rise to a new, complete, genetically identical plant

clone. Other cells (animal cells) usually will lose its ability of totipotence in early stages of live and

continue to differentiate into more specialized and complex form. Actually, totipotency in plants

depends on the species of plant tissue and the part where we obtain the tissue.

3. Plant tissue culture (4)

Plant tissue culture involves the growth of cells in a suitable culture medium or through in vitro (in

glasses). Main purpose of this technique is to produce organism through asexual reproduction and

this technique is carried out based on application of the knowledge that plant cells are totipotent.

Micropropagation is also a commercial method used to produce identical seedlings in limited space

and time. Diagram below shows how the tissue culture technique is carried out.

Diagram 2 : Tissue Culture (5)

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Diagram 1 : How tissue culture carried out?(6)

Diagram 2 : Pros and Cons of tissue culture(7)

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4. Meristematic cells (8)

A meristem is the tissue in most plants consisting of undifferentiated meristematic cells; potential to

give rise to various part, found in zone of cell division of the plant. Both root and shoot apical

meristem divide rapidly and are considered to be indeterminate. Structure wise, meristematic cells

are packed closely enough without intercellular cavities and very thin primary cell wall present.

Apical meristems at the buds of shoots and the tips of roots contribute to primary growth in the

newest parts of plant resulting in increase of length of plant. Lateral meristems (vascular cambium

and cork cambium) develop in the older regions of the shoots and roots, contributing to secondary

growth resulting increase in the girth of the plant. The root tip contains an extra structure, a root

cap, which protects the root tip from damage as the root pushes down through the rough soil

particles. Shoot apical meristem will transform into inflorescence and later into floral meristem in

flowering plants to produce sepals, petals, stamens and carpels.

Apical meristems may differentiate into three kinds of primary meristem namely protoderm,

procambium and ground meristem. Under certain and suitable conditions, each shoot meristem

have potential to develop into a clone (genetical identical) through shoot cuttings that contain apical

meristem. This cloning method is known as mericloning and widely practiced in horticulture to mass-

produce plants of a desirable genotype.

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Objective

To demonstrate the totipotency of plant cells.

Problem Statement

Are plant cells are totipotent ?

Hypothesis

Plant cells are totipotent where it can differentiate into all types of cells.

Apparatus

Damp cotton wool, cling film, 100ml beaker, cling film, tissues, sterilized scissors, sterilized forceps,

pen, sticker, oven, tray

Materials

Seedlings of Brassica Rapa, molten agar

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Procedure

1. Molten agar solution and Brassica Rapa seedlings were prepared by the lab assistant.

2. Molten agar was prepared in oven at 60˚C while the Brassica Rapa seedlings were prepared in

a tray , stuffed with damp cotton wool three days before the experiment carried out.

3. 30 ml of molten agar was poured into a 100ml beaker after washing and clean the beaker

using tissues. It was then allowed to cool and solidify. Care was taken by not swirling and

moving around the beaker containing agar.

4. Next step was carried out after the agars solidify.

5. The Brassica Rapa seedling was cut below the shoot apex (growing tip) using sharp pair of

sterilized scissors while the top of the seedling was held upright by a pair of sterilized forceps.

These are known as explants. The hypocotyls (the early stem) and roots are left behind on

the damp cotton wool.

6. The explant was then carefully pushed into the solidified agar carefully. The cotyledons were

made sure not to touch the agar.

7. The beaker was then covered with cling film. Initials and the date were written on a sticker

using pen and was pasted on beaker. It is then was placed on a sunny windowsill to provide

sufficient sunlight.

8. The progress of the explants is observed daily and anything of note that develops is recorded.

9. The results are collected and recorded in the table.

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Safety precaution

In order to avoid any accident or injury during the experiment in laboratory, the

precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are

compulsory when conducting an experiment in the lab at all times to protect the skin and clothing

from spillage of any chemical substance. Washing hands thoroughly with soap and water before and

after conducting experiment is vital to avoid contamination. Furthermore, the glassware such as

beakers and boiling tubes should be handled with full care because they are fragile. The apparatus

such as forceps and scissors are also sterilized to prevent infection of microorganism. During

explants extraction, any contact with the explants should be avoided as much as possible to avoid

contamination. 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 that

may result fatal results.

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Risk Assessment

The agar solution should not take out from the oven too early to prevent it to become

solidify. Other than that, the explants are fragile that they have to be handle with care. Apart from

that, the explants were handled with sterilized apparatus such as beaker, scissors and forceps in

order to prevent infection of other microorganism. Once those apparatus was used, they were

washed and placed in different place so that they won’t be used to handle other explants without

sterilizing first. Care was taken when the seedlings were placed inside the beaker. The cotyledons

neither were make sure to not touch the agar solution nor immersed inside the agar solution as it will

affect respiration and photosynthesis of the seedlings. If the cotyledons touched by agar solution,

the whole steps were repeated again. When the explants placed into beaker, any movement is

restricted until the agar solution solidifies to prevent the seedlings from sinking into the solution.

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Results

Day Number

of Leaves

Height of

plant (cm)

Presence of

root

Other Observations

0 2 0.20 None Explant is upright.

Leaves are green in colour.

No bacterial growth.

1 2 0.20 None Explant is still upright.

Leaves are green in colour.

No bacterial growth.

2 4 0.30 Present Explant is slightly slanted.

Leaves are green in colour.

Leaves increased in size.

3 4 0.40 Present Explant becomes taller and still slanted.

Leaves are green in colour.

No significant changes in leaves size.

Roots number increased by one.

4 4 0.40 Present Leaves turn into slightly pale yellow .

Size of leaves become larger.

No change in the length of root.

Shoot turn slightly brown.

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5 4 0.40 Present Pale yellow colour proportion of leave

increases a little bit but green colour

still being dominant.

Size of leaves and root retain the same.

No change in length of shoot.

Shoot become light brown.

6 4 0.40 Present Leaves turn a bit more yellow but still

green being the dominant.

Number and size of leaves and roots

were the same.

No change in length of shoot.

Shoot become browner.

7 PLANT DIED The explants collapsed and touch the

surface of agar.

The leaves become bright yellowish.

Shoot are in brown colour.

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Data Interpretation

In this experiment, we have decided to demonstrate the totipotency of Brassica Rapa seedlings by

measuring the growth and developments of the explants. This was done by measuring and recording

the length of shoot, number and size of leaves and number and length of roots. Other qualitative

observations such as colour of leaves were also noted as side notes. However, since the explants is

so small and the beaker was covered with the cling film, it was difficult to measure its length. Thus,

the length was calculated by placing the ruler outside the beaker and the length of the shoot of

explants was measured. So, it may lead to inaccuracy of length reading if good care is not taken

during the activity.

Table 1 shows the observation made at daily bases. Every single change was noted to obtain

as much as accurate result of the explants. At day 0, no noticeable changes in growth were recorded

since the explants were just planted into the agar. Since the explant was cut by leaving the

hypocotyls (the early stem) and roots are left behind the tray, the explants doesn’t possess roots yet.

The explant was still freshly germinated, thus it have green coloured fresh little sized leaves and

there is no sign of bacterial growth on the molten agar.

The next day, the condition of explants stays the same. On the following day, the explants

slightly slant to the source of the light (towards the window). This shows that the shoot is growing

towards sunlight to perform photosynthesis in order to make food so that it can survive. The sizes of

the leaves also noted to increase. This shows that the nutrients obtained via photosynthesis is used

for growth of leaves by the explants. Furthermore the cotyledons itself are photosynthetic and they

have their own storage of food for the plant to survive for a few days. Thus, it enables the explant to

maintain its healthy state without much problem and give arise to two more leaves.

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On the next day, it was observed that the shoot become taller than the day before. This

shows that the shoot growing towards sunlight in order to obtain more sunlight so that it can

perform photosynthesis. The leaves were still in green colour, showing that the explants still full of

nutrient and healthy. Number of root of the explants increased by one showing that the explant has

started to look for water source as for medium to transport obtained nutrients to all parts of the

explants. The growth of roots shoots and leaves from an explants proves that the plant cells are

totipotent. The root grows downwards because of gravity influence.

On the fourth day, the leaves started to turn into slightly pale yellow and shoot become

slightly brown. This shows that the nutrients that stored in the explants start to wear out and mark

the start of demand for nutrient. But still, the leaves become larger as the light source still present.

No significant changes were noted for the shoot and root length. On the very next day, it was noted

that the proportion of pale yellow colour increase a little bit, but the green colour still remain being

dominant while brown colour intensity increases a little. This indicates, despite the photosynthesis,

the demand for needed nutrient increase. Size of the leaves, number of roots and the length of

shoot noted to be same.

On the sixth day, the leaves continue to turn yellow; same goes to the shoot colour and as

before, the number and size of leaves, number of roots and the shoot length stay the same. On the

next day particularly at late in the evening, it was noted that the explants collapsed and touch the

surface of agar. The leaves also turned into brighter yellow than the day before and the shoot was

noted to turn into brown colour, darker than before. It was then concluded that the explants died

because of insufficient nutrients especially mineral ions, oxygen and water.

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Water shortage happen because the water content in the agar is used by two explants and

the volume of agar used also only 30ml, which is not much. Since sugar is absent in the molten agar,

thus no bacterial or microorganism growth was noted. Humidity inside the beaker was maintained

by the cling film which was used to cover the mouth of beaker. Other than prevent water

evaporation, this also prevent bacteria and microorganism contamination which may affect the

condition of explant. The cling film is also transparent, thus allows entry of sunlight and enables

process of photosynthesis.

Evaluation

Based on the obtained results, we can conclude that the Brassica Rapa explant shows

totipotency by differentiate into different cells such as roots and leaves. But, since there is

insufficient nutrients and suitable conditions thus explant cannot continue survive and maintains its

growth rate.

Page 15: totipotency

Limitation

Several factors in this experiment may limit the result of this experiment. Following shows

the limitation that encountered in this experiment and how they were handled to avoid fluctuation

of result.

Firstly, there are some possibilities for microorganism to invade the seedlings which may alter

the growth of the explants, resulting in stunted growth or death of the plant in short period.

This was avoided by treating the seedlings using aseptic techniques and this was carried out

by laboratory assistant before the experiment was handled by us, the students. We were told

that the seedlings were treated with chlorox (10% -20% sodium hypochloride). They were

soaked in the chlorox for 10 to 20 minutes and then was rinsed with distilled water several

batches. Then, they were dried using filter paper and germinated on cotton wool that was

soaked with water. They left for 3 days for germinate before the next step was carried by

use. Treatment with chlorox was carried out for getting rid of contaminants. Chlorox also can

be replaced by 1% of potassium permanganate.

Other than that, since agar (made of seaweed) can easily turn into solid state when exposed

to room temperature, thus the agar was kept into oven at 60˚C to retain its molten state.

Agar was handled with extra care and it was poured into beaker as fast as can until required

volume reached. This is to avoid those extra agar from solidify and prevent wastage. The

molten agar also left to be solidify before placing the explants into the agar to prevent sinking

of explants that may lead to stunted growth of explants.

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Source of Error

Some errors that may occur during this experiment should be taken good care so that it won’t

alter the condition of the Brassica Rapa explant and indirectly contribute to unreliable result. One of

the errors that I may encountered during this experiment is leakage of surrounding air into the

beaker. The cling film which was used to cover the mouth of beaker is ultra thin and may exposed if

handled rough or come into contact with sharp objects accidently. This may cause surrounding air

which is full of contaminated particles and microorganism to enter the territory of the explants, thus

alter humidity of explants surrounding causing the explants to wilt. Other than that, microorganism

and pollutants also may enter molten agar when they were transferred into beaker. Since molten

agar in conical flask was used by different group members at different period of time, the molten

agar may be invaded by microorganism in the air during the interval when the conical flask was

passed to the others. This may also alter the growth of the explant, affecting the result.

Conclusion

From this experiment, it can be concluded that plant cells are totipotent, which has potential to

differentiate into all types of cells. Explant will continue growing and forming new parts if the

conditions are suitable or favorable and free from contamination. The hypothesis is accepted.

Further Investigation

Presence or absence of cotyledon also influences condition of explants. Thus, another experiment

can be carried out to find the relationship between presence or absence of cotyledon on explants

condition.

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References

1. Wikipedia Foundation. 2009. Brassica Rapa. Available from http://en.wikipedia.org/wiki/Chinese_cabbage . Accessed on 10th January 2012.

2. http://www.google.com.my/search?q=brassica&hl . Accessed on 16th January 2012.

3. Wikipedia Foundation. 2009. Totipotency. Available from http://en.wikipedia.org/wiki/Cell_potency. Accessed on 10th January 2012.

4. Wikipedia Foundation. 2009. Tissue Culture. Available from http://en.wikipedia.org/wiki/Tissue Culture. Accessed on 10th January 2012.

5. http://www.google.com.my/imgres?q=plant+tissue+culture+process. Accessed on 16th January 2012

6. Gan W.Y . 2007. Biology SPM Success. Edition 4. 122.p.Shah Alam : Oxford Fajar Sdn.Bhd.

7. Gan W.Y . 2007. Biology SPM Success. Edition 4. 123.p.Shah Alam : Oxford Fajar Sdn.Bhd.

8. Wikipedia Foundation. 2009. Meristem. Available from http://en.wikipedia.org/wiki/meristem. Accessed on 14th January 2012.