OFFICIAL BIOLOGI REPORT

TISSUE CULTURE

By:

Mariette Wahyuningsih Montgomery

(XI IA1/24)

STELLA DUCE SENIOR HIGHSCHOOL YOGYAKARTA

2010 – 2011

I. Introduction :

In the modern world, plants can rapidly grow and multiply in just a few weeks. This

technique is done with Plant Tissue Culture or Micropropagation. This technique centralizes

on the plants’ totipotency, which is the plants’ potency in multiplying its cell and

differentiate. However, not all types of cells can rapidly multiply. Tissues such as meristem

have that ability to multiply, because it is considered a young type of cell.

II. Problems :

1. What are the steps in making medium MS and to sterilize it ?

2. What are the steps that should be done in multiplying plants with tissue culture ?

3. What are the characteristics of explants that are contaminant ?

4. What are the factors that cause contiaminant explants ?

III. Objective :

1. To know how to make MS plant culture tissue medium.

2. To know the steps on how to multiply plants with tissue culture.

3. To know the characteristics of good explants and contaminant explants.

4. To know the factors that cause good explants into contaminant explants.

IV. Literature :

A. Plant Tissue Culture

Plant Tissue Culture, more technically known as micropropagation, can be broadly defined

as a collection of methods used to grow large numbers of plant cells, in vitro, in an aseptic

and closely controlled environment. This technique is effective because almost all plant cells

are totipotent – each cell possesses the genetic information and cellular machinery

necessary to generate an entire organism. Micropropagation, therefore, can be used to

produce a large number of plants that are genetically identical to a parent plant, as well as

to one another. (Raven et al., 1999)

Figure 1 - Areas of Tissue Culture Collection

(Illustration compliments of Dr. R. Daniel Lineberger, Texas A&M University)

The standard protocol for performing plant tissue culture experiments is fairly basic. First,

it is essential that a sterile environment be created. The medium used to grow the plant tissue,

the plant tissues themselves, and the environment surrounding the tissue culture, must be free

of all possible contaminants. The presence of any bacterial, fungal, algal, or viral

contaminants could potentially rob the desired plants of the nutrients provided by the culture

medium and have devastating effects upon their growth (Kyle, 2002). Once a sterile

environment has been established, tissue can be collected from the plant’s leaf, shoot, bud,

stem, or root (see figure 1). Because each of these cells is totipotent, each has the potential to

express an entire organism (see figure 2). The tissue sample can then be placed on an aseptic

(free of microorganisms), nutrient-rich medium where its cells will begin to grow and

develop into the desired plant product (Thorpe, 1981). The nature of the medium and the

nutrients that it contains is dependent upon the type of plant being grown and the properties

that the grower wishes to express. Finally, the developing tissue should be maintained in a

closely controlled chemical and physical environment, such as a greenhouse, to achieve the

best results (Thorpe, 1981).

Figure 2 - The Basic Steps of Micropropagation

(Illustration compliments of Dr. R. Daniel Lineberger, Texas A&M University)

The benefits of plant tissue culture are extensive in the agricultural world. Micropropagation

is favorable to traditional crop breeding methods in many respects, the first being that it

allows for the production of huge numbers of plants in a very short period of time. In the

Netherlands alone, over 100,000,000 plants are produced using micropropagation each year

(Hall, 1999). Plant tissue culture is also advantageous to growers because the overwhelming

number of plants can be produced using the tissue collected from a single parent plant – a

plant which itself remains unharmed in the tissue harvesting process. Crop production

through micropropagation also eliminates the possibility of any interruption in the growing

season because it can be carried out inside the carefully regulated environment of a

greenhouse. Because the chemical and physical environment inside a greenhouse can be

closely monitored, any lull in production that might typically occur as a result of seasonal

change can be avoided. (Lineberger, 2002)

Micropropagation is the art and science of plant multiplication in vitro through 'cloning’

using different parts of the plant such as the growing tip, stem, leaves, flowers and even

individual cells of a parent plant. This is also described as clonal propagation.

Micropropagation will be crucial to the agricuture industry in the future because it is used to

produce plants which have been genetically modified and selected for their ability to resist

certain indigenous environmental stresses. Currently, scientists and members of the

agricultural community have joined forces to investigate the possibility of creating lines of

tomatoes that possess increased salt tolerance (to be grown in areas in which the soil is high

in salinity), plants that are completely resistant to various viral, bacterial, algal, and fungal

infections, tobacco plants whose leaves can withstand freezing temperatures, and crops that

are entirely resistant to harmful and destructive insects. (Lineberger, 2002)

Figure 1: Micropropagation from different plant parts

Stages of Micropropogation

There are four major stages of micropropagation. Murashige proposed three (I to III) stages,

Debergh and Maene added stage ‘0′. Currently we follow five stages procedure (0 to IV).

Stage 0 – Selection and preparation of the mother plant

Stage I - Initiation of culture

Stage II - Multiplication

Stage III - Rooting

Stage IV - Transfer to soil 

Stage 0

Selection and maintenance of stock plants for tissue culture initiation - In this stage stock

plants are grown under more hygienic conditions to reduce the risk of contamination. 

Stage I

Initiation and establishment of aseptic culture - Vegetative parts or reproductive parts are

used for the propagation and shoot tip and auxiliary buds are often used for this. In this

process explants are surface sterilized by treating it with disinfectant solution such as Ethyl

alcohol, bromine water, mercuric chloride etc.

Establishment of explant on appropriate medium – There is not any universal culture

medium; however modifications of Murashige and Skoog basal medium (Murashige and

Skoog, 1962) are most frequently used.

Stage II

Multiplication of shoots - Often media are not changed between stage I and stage II, but

cytokinin proportion is increased for stage II to produce numerous shoots.  

Stage III

Rooting of regenerated shoots - In this stage, shoots are transferred on a rooting medium

containing an auxin. Elongation of shoots prior to rooting, rooting of shoots (individual or

clumps), and prehardening cultures are carried out to increase the survival rate.   

Stage IV

Hardening - This stage is carried out under greenhouse environment. The plantlets are

placed in sterilized soil in a high humidity environment to harden off the plant.

Figure 2: Stages of Micropropagation

Limitations of Micropropagation

1. Sophisticated facilities are required.

2. Production cost is high.

3. Requirement of skill in handling and maintenance.

4. Somaclonal variations may arise during in vitro culture.

5. Vitrification can be a problem in some species.

V. Hypothesis :

VI. Tools and Materials :

A. Tools :

1. Autoclave2. “Lamp in air Flow”3. Analytic balance4. Encase5. Skalpel6. Long Tweezers7. Short tweezers

8. Petridisc9. Erlenmeyer10. Oven11. “Aluminium Foil”12. Bottle13. Umbrella paper

B. Materials :

1. The plants parts like a root, stem, leaf, and seed from soft and hard plants.

2. The chemicals materials for sterilization and materials for media.

VII. Working Procedure :

A. Procedures on making MS plant culture tissue medium :

1. Prepare one Erlenmeyer of 150 mL.

2. Add 1.5 mL/L of Macronutrient I stock.

3. Add 1.5 mL/L of Macronutrient II stock.

4. Add 1.5 mL/L of FeNa2EDTA.

5. Add 0.075mL/L of Micronutrient stock.

6. Add 1.5mL/L of Myoinositol

7. Add 0.015mL/L vitamin stock.

8. Add 4.5 gram/100 mL of sucrose.

9. Add sytocinin hormone and auxin hormone with a fraction of 5:1 (sytocinin:auxin)

10. Measure the pH until 5.8

11. Add 1.8 gram/L of Agar-agar powder

12. Heat it until it boils.

13. Pour it into the inoculation.

14. Sterilize it in the autoclave for 15 minutes with the temperature of 121° C .

B. Sterilization of Plants Physically / Flambir :

1. Wash the carrots with Rinso then rinse thoroughly with clean water.

2. After it is clean, about 10 cm from the tip to the middle part and cut 3 cm of the carrot.

3. Cut then sterilize with the flambir as much as 3 times.

4. In the petridish, the outer part of the carrot are to be cut, then chop cross-sectional as thick as 0.5 cm.

5. Cut tiny pieces (0.5 x 0.5 cm), then separate it from the cambium, empulur, and the phloem.

6. Then add the solid medium.

7. Repeat step 1 – 6 for the potato and garlic explants.

C. Sterilization of Plant Chemically :

1. Wash the leaves with Rinso until it is cleaned

2. All of the tools to scatter, benlate, Clorox, tweezers, and explants are to be inserted into the (laminair air flow) that was sterilize beforehand.

3. Put in the explants in the sterilize Erlenmeyer, then add the benlate.

4. Shake it slowly and softly for 5 minutes (adjust it with the how thick/thin the leaf)

5. After 5 minutes, the benlate are to be thrown in a Beaker glass.

6. Then clean with a sterilize aquadest, afterwards shake it for 1 minute twice.

7. Sterilize the explants with Clorox 1% and tween as much as 2 drops.

8. Throw away the Clorox, explants is washed with sterilize aquadest by shaking it slowly and softly for 1 minute. Repeat it three times.

9. Cut the explants with a sterilize scapel. Each of the chopped explant should be about 1 cm

10. Differ the tip of the leaf part, stem of the leaf, and the carcass of the leaf.

11. Each of the cut explants are to be put in the medium inside the bottle. Each bottle is to be filled with 1 leaf.

12. After it is put inside the medium, the bottle should be closed tightly. Give a label for each bottle and the explant.

VII. Observation Data :

No Name of

Explants

Number of OberservationI II III IV

Fungi

Bacteria

Browning

Fungi Bacteria Browning

Fungi Bacteria Browning

Fungi Bacteria Browning

1 Leaf 1 - - - - - - - - -2 Leaf 2 - - - - - - - - - -3 Root 1 - - - - - - - -4 Root 2 - - - - - - - - - - - -5 Stem 1 - - - - - - - - -6 Stem 2 - - - - - - - -7 Carrot 1 - - - - - - - -8 Carrot 2 - - - - - - - -9 Garlic 1 - - - - - - - - -

10 Garlic 2 - - - - - - - - -11 Potato

1- - -

12 Potato 2

- -

Explanation() : There’s fungi / browning / bacteria

(-) : There’s not contamination or browning.

IX. Discussion :

In this experiment, we learned how to multiply plants with the tissue culture

technique. Tissue culture technique or micropropagation is a modern way of rapidly

multiplying plant stock using tissue cultures. This technique uses the plant’s cell potency to

differentiate cell types or also called totipotency. Totipotency is the ability of a single cell to

divide and produce all the differentiated cells in an organism, including extraembryonic

tissues and meristem tissue. These types of tissues are still young and undifferentiated, thus

can rapidly experience cell growth. In some organisms, cells can dedifferentiate and regain

totipotency. For example, a plant cutting or callus can be used to grow an entire plant.

Plant tissue culture is not like planting with soil; however it uses medium MS that’s

filled with hormones and nutrients for a plant to grow well. Tissues such as meristem need

just the right amount of nutrients and hormones for growth. There are important

substances that are needed in its medium. They are: macronutrient, micronutrient, glucose,

vitamin, and hormones.

Group Type Concentration mg/liter

Macronutrient NH4NO3

KNO3

CaCl.2H2O

MgSO4.7H2O

KH2PO4

Na2-EDTA

FeSO4.7H2O

H3BO3

MnSO4.H2O

ZnSO4.7H2O

1.650,00

1.900,00

440,00

370,00

170,00

37,30

27,80

6,20

16,90

8,60

Micronutrient KJ

Na2MOO4.2H2O

CuSO4.5H2O

0,83

0,25

0,025

CoCl2.6H2O 0,025

Vitamin Thiamin-HCl

Nicotinic acid

Pyriddoxin-HCl

Myo-Inositol

Glysin

0,50

0,05

0,50

100,00

0,02

Sugar Sacharosa 4.500,00

Hormones Cytochinin

Auxin

750,00

150,00

PH 5,8

Medium MS is one of the most important parts of the plant tissue culture. This is

because it is the essential material in which the plant will grow. As said before, there are five

components in making medium MS (macronutrient, micronutrient, glucose, vitamin, and

hormones). The main component of the medium is macronutrient (essential nutrient that

are big in size) and micronutrient (essential nutrient that are small in size). This is the

substance in which the plant will need to grow efficiently. Glucose is also an important

substance. Glucose works as an energy supply for the plants. Vitamins work as a

strengthener for the plants to grow strong. There is also a substance called Myo-inositol or

inositol is a chemical compound with formula C6H12O6 or (-CHOH-)6, a sixfold alcohol (polyol)

of cyclohexane. Myo-inositol plays an important role as the structural basis for a number of

secondary messengers in eukaryotic cells . Then there are hormones, in which the plants

need for rapid growth. These hormones include cytochinin and auxin.

Hormon cytochinin and auxin has different function for the plant’s growth and

development. Hormon cytochinin’s main function is to multiply the plant’s shoot. And the

auxin is to develop the plant’s root and strengthen it. The figure below will give a deeper

explanation :

Auxin promotes growth by stimulating cell elongation.

It triggers enzyme activities that loosen the cell wall fibers (normally tightly woven).

To make Medium MS (Murashigie and skoog) we need an Erlenmeyer glass (150

mL). Add the macronutrient I, macronutrient II, FeNa2EDTA, Micronutrient, Myo-inositol,

vitamin, sucrose, and hormones. However, we need just the right amount of these

substances.

Adding the 1.5 mL / L Macronutrient I stock, Macronutrient II stock,

FeNa2EDTA, and Myo-inositol :

(1.5

1000) ÷ 10 = 0.15100

So, for every 100 mL medium solution 1 mL of Macronutrient I stock, 1 ml

Macronutrient II stock, and 1 ml FeNe2EDTA stock, and also 1 ml the Myo-

inositol Stock is added.

Adding the 0.075 mL / L of micronutrient stock :

(0.0751000 ) ÷ 10 =

0.0075100

So, for every 100 mL medium solution 0.0075 mL of micronutrient stock is

added.

Adding 0.015 mL / L of vitamin stock :

(0.0151000 ) ÷ 10 =

0.0015100

So, for every 100 mL medium solution 0.0015 mL of vitamin stock is added.

Adding 4.5 grams / L of sucrose :

(4.5

1000) ÷ 10 = 0.45100

So, for every 100 mL medium solution 0.45 grams of sucrose is added.

Adding the hormone cytochinin and the auxin :

o These two hormones work together to rapidly develop the explant’s

organs and for its growth. Thus, the proportion of these hormones

are 5 : 1 (cytochinin : auxin) to create a medium with a

proportionate ingredients.

o M1 . V1 = M2 . V2

o M1 = 500 ppm

o M2 = 100 ppm

o V2 = 0.75

o 500 . V1 = 100 . 0.75

o V1 = 0.15

Thus the right proportion of cytochinin is 0.15 mL : 0.75 mL.

After adding all the materials in the exact proportion, we measured the pH level of

all the nutrients and substance combined. The targeted pH level is 5.8. This is to make the

medium similar to soil (pH level of soil is the same – 5.8). Thus, the medium will be an

optimal place for the explant to grow and develop.

In this experiment, we used a digital pH meter to measure its pH level. If it is lower

then 5.8, then we add HCL solution until the pH level equivalent or about 5.8.

Afterwards, the substance that’s been added will be boiled with agar-agar powder

and water. While we wait for it to boil, we stir it often so the substance mixes with the agar-

agar. After it boils, we turn off the heater, and slowly pour it into small jars and close it with

a tin foil and brown paper with a tight rubber band around it so it is free of bacteria and

fungi.

Nutrient substance (pictured when the pH level

was measured)Medium MS as it is boiled with sucrose and agar-

agar powder and water

If the entire medium is filled in the jar and closed tightly, we put each of them inside

a machine called an autoclave. An autoclave is a machine that gives heat and pressure to

sterilize the medium so it does not get contaminated.

(A Figure of an autoclave)

The medium is put inside the autoclave for an estimation of 15 minutes at

121° C temperature.

As said before, micropropagation only uses young plant that still has

meristem tissue. This is to increase the chance for the plant to grow, because

meristem tissues are still young tissues and cells, thus it will experience cell changes

and will rapidly experience develop and grow.

In the initiation process, we choose the right – young explant to be put into

the medium MS. However, before we do that we need to sterilize the explant for it

to grow well (uncontaminated). There are two types of sterilization. They are

sterilization physically and chemically. Sterilization physically is for explants that do

not get burnt easily, such as carrots, garlic, etc. However, sterilization chemically

sterilize explant that does get burnt easily, such as leaf, stem, root, etc.

Sterilization Physically:

Physic Sterilization is done with burning the explant (garlic, potato, carrot) as

its first step. This is to sterilize the explant, so it doesn’t get contaminated. Flambir

sterilization is done with alcohol. The explants are to be put inside the alcohol, then

burn it on top of the spiritus. This is to kill unwanted bacteria on the explant. This is

done three times. Afterwards, explant is to cut with a scalpel that’s already

sterilized. (All of the tools are to be burned on top of the spiritus to be sterilized.)

and a pointy long tweezers that’s already dipped in alcohol. Then, put the explant

inside the solid medium, and close it tightly with a tin foil, paper and a rubber band

around the jar.

Figure of physical sterlization

Sterilization Chemically :

Chemical sterilization is done for soft plants that burn easily. This is done

without burning / fire. It’s done with materials, such as tween, Benlate, Clorox 1%.

The first step in chemical sterilization is to clean the explant thoroughly and rinse

with running water. Then the explants are inserted inside the Erlenmeyer with

Benlate and shake it softly for 5 minutes. After that, throw the benlate away, and

wash the explant with sterilize aquadest while shaking the Erlenmeyer softly for 1

minute. This step is done twice. Afterwards, the explant is sterilized with 1% Clorox

and 80 Tween (2 drops). The next step is to throw away the Clorox and the explants

are washed with sterilized aquadest. Then as usual, shake it softly for 1 minute. This

step is done three times. Then cut the explant into small pieces, and choose parts

with young meristem tissue. (This can be seen with the color of the leaf and height

of the root. If the leaf is light green, then choose that part. If the root is short with

little root on the tip of it, then choose that part also.) After it is cut, the each of the

explant is put inside the solid medium.

The materials used for chemical sterilization has different functions. Benlate,

for example functions to kill fungi. Clorox functions to kill off bacteria that might

contaminate the explant. Tween is for the explant’s pores to open up, this can

absorb the nutrient in the medium. The alcohol functions as an anti bacteria and

fungi (It kills of unwanted contimantion.) Alcohol can also go deep in the plant’s cell

wall which can kill microbial organisms.

Explants that were sterilized physically and chemically (left to right)

The next experiment that we conducted is acclitimatization of moon orchid

(anggrek). This process is to adjust to a gradual change in its environment, (such as a change

in temperature, humidity, photoperiod, or pH) allowing it to maintain performance across a

range of environmental conditions. The medium that we used is called medium fern, which

is made of fern. This replicates soil for the moon orchid.

The first step in the last experiment is to prepare or pick a moon orchid that’s

already growing. The medium with the moon orchid is given water (running water) for the

moon orchid’s root to loosen itself from the medium. After the moon orchid is removed,

move it to the pot that’s already been prepared. The moon orchid that was removed was

washed until all of the body’s moon orchid is gone. Afterwards, submerge it with benlate

solution for five minutes. Then dry the moon orchid. It is to be planted in a pot that’s

already been given a medium called medium fern. The last step is to give it water and close

it with plastic (which was given holes).

We were to observe these the results of our group’s experiments (Explants and

moon orchid). For every three days, we observed it and saw if the explants grew or if it were

contaminated.

Leaf Explant

The first observation of Leaf I and leaf II was not contaminated. However in

the second observation, leaf I grew fungi, while the second leaf was still

uncontaminated. Then the third and fourth observation, we saw that both

of the leaves were contaminated.

Root Explant

The first observation of root explant I is that it grew bacteria. It grew even

more in the second, third and last observations. However the second root

explant was not contaminated.

Stem Explant

The first observation of stem explant I was not contaminated. However the

stem explant II was contaminated with fungi. Then the second and third

observations that we saw was that both of them grew fungi.

Carrot Explant

The carrot explant was contaminated with bacteria in the first observation.

It grew even more bacteria in the second, third, and last observation.

Garlic Explant

The first observation of garlic explant I and II was that they were not

contaminated. However in the second, third, and last observations showed

that the garlic grew more contamination of bacteria.

Potato Explant

The first observation showed that both of the potato explants experience

bacterial contamination. Then the second, third, and last observation

showed that it experience browning, bacterial, and fungi contamination.

Contaminated Stem Uncontaminated root Contaminated Potato

Contaminated Leaf Contaminated carrot

Types of contamination :

Bacteria

Bacteria can appear on an explant because of unwanted contamination. Even if

the explant and tools that were used on the experiment were sterilized, there

will always be contamination from the air. Contamination from the air we

breathe (because of talking, sneezing, etc) can affect the chances of our explant

being contamination. From the observation, we can tell if the explant

experience bacterial contamination from white mucous that appeared on the

explant.

Fungi

Fungi contamination can appear from too much water or too much liquid in the

explant. A fungus grows on a low humidly and wet environment. This is why the

explant experience fungi contamination – because the medium turned into

liquid or there are too much water in the medium. From the observation, we

can tell if the explant experience fungi contamination from a white cotton like

organism appearing on the explant or near the explant. Brownish green fungi

can also appear on the explant. These type of fungi appear when it mixes with

liquid or water.

Browning

Browning contamination appears because off unwanted water and or liquid.

Browning can also appear because the explant has too much antioxidant such as

polyphenol. For example, an apple ; it has a large amount of polyphenol, thus

when we leave it in a free environment (without covering it), the apple will

change into a brownish color – like it’s rotting. From the observation, we can tell

if the explant experience browning contamination from color formation. It will

change into a brown color ( or it looks like its rotten).

Most of our explants experience contamination in browning, bacteria, fungi, or even

a combination of the three. This maybe because of unsterilized medium or not a good

X. Conclusion :

Yogyakarta, 17 Maret 2007

Pembimbing Praktikan

Drs.P. Ari Kriswardana Elisa Aster Nugroho