KULLIYYAH OF ENGINEERING

DEPARTMENT OF BIOTECHNOLOGY
BTE 3212 | BIOTECHNOLOGY ENGINEERING LAB IV

Semester I 14/15

PLANT TISSUE CULTURE

By
Group members (Section 1):

1126494

Nurul Alia binti Fazil (introduction & objectives)

1124650

Nurhafatin Emira binti Shahrul Nazron (procedures)

1121448

Nurfarhana binti Abdul Hanid (results & calculation)

1127380

Fatin Nabilah binti Murad (discussion)

1123746

Suhadahafiza binti Shafiee (conclusion)

EXPERIMENT 2: PLANT CELL AND TISSUE CULTURE; PREPARATION OF EXPLANTS FOR CALLUS
CULTURE AND PLANT CELL SUSPENSION CULTURE.

INTRODUCTION
Plant cell tissue cultures are widely used in plant biology as a convenient tool for the
investigation of a wide range of phenomena, bypassing the structural complexity of the plant
organism. The homogeneity of an in vitro cell population, the large availability of material,
the high rate of cell growth and the good reproducibility of conditions make suspensioncultured cells suitable for the analysis of complex physiological processes at the cellular and
molecular levels. Moreover, plant cell cultures provide a valuable platform for the production
of high-value secondary metabolites and other substances of commercial interest. Here we
describe how to initiate and maintain plant cell cultures starting from explants obtained from
in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and
regeneration of plants via organogenesis and somatic embryogenesis are also presented. There
are several types of plant cell and tissue culture such as callus culture, organ culture and plant
cell suspension culture.
A plant contains many organs like meristem, cortex, phloem, epidermis that consists
of structural unit called cell. The process of plant organs that used in in-vitro conditions to
generate new plant is called organogenesis. In organogenesis of plant included two different
stages first is de-differentiation (callus formation) and other is re-differentiation (budding on
callus) of organ or explant. Organogenesis of plant is require to gain how a organ generated or
developed to an whole plant. Organogenesis is method to regeneration of plant in for of
organs from callus.
Formation of organ in callus depends on medium constitutes because medium of tissue
culture also contain growth regulators witch determine to shoot regeneration or root
regeneration. for this auxin and cytokinin ratio used in a appropriate ratio witch responsible
for callus regeneration. more concentration of cytokinin compare auxin generated to shoot
part in callus and more quantity of auxin generated to roots in callus. plant growth regulators
like auxin, cytokinin, gibberelin, ethylene, abscisic acid, etc are affect to callus regeneration
according ratio of them in medium.
Cell suspension is prepared by transferring a fragment of callus to the liquid medium
and
agitating
them
aseptically
to
make
the
cells
free.
Single cells can be isolated from either callus or any other part of the plant and cultured in
liquid medium using both mechanical and enzymatic methods. Mechanical methods involve
grinding of the tissue to a fine suspension in a buffered medium followed by filtration and
centrifugation to get rid of cell debris. The enzymatic method uses the enzymes (pectinase or
macerozyme) to dissolve the middle lammela between the cells. After the isolation of the
cells, they are cultured by batch cultures or continuous cultures.As the medium is liquid in
nature, the pieces of callus remain submerged which creates anaerobic conditions. To

overcome this problem, the suspension cultures are agitated by a rotary shaker which
disperses the cells and expose them to air.
The advantages of cell suspension cultures over the callus culture:
a) The suspension can be pipetted.
b) They are less heterogeneous and cell differentiation is less pronounced.
c) They can be cultured in volumes upto 1,500 litres.
d) They can be subjected to more stringent environmental controls.
e) The manipulations for the production of natural products by feeding precursors, is possible
OBJECTIVES
1. To cultivate adventitious shoot from explants (organogenesis).
2. To initiate callus from leaf.
3. To establish cell suspension cultures from callus.
METHODS
Plant material
Fectus Deltoidea
Chemicals
70% ethanol
Solid Media:
Murashige and Skoog basal medium (MS media) with 2.5g/l gelrite agar, 30g/l sucrose
(carbon source), 3ppm plant growth regulator (PGR) and:
a) 1 jar without KB; 3 ppm picoram, 3 ppm 2, 4-D, (Auxin)
b) 1 jar with KB; 3ppm BAP, 3ppm Kinetin, (Cytokinin)
Liquid media (suspension media):
Murashige and Skoog basal medium (MS media) with 3ppm PGR, 3ppm 2,4-D, total volume
100 ml. no agar, 30g/l sucrose, 3ppm picoram, pH 5.7
Apparatus/Equipment
Petridish

70% ethanol

Scalpel

Laminar flow hood

Bunsen burner

Parafilm

Forceps

Gyratory shaker at
125rpm

Temperature controlled
room at 25
2 Jars and 1 conical flask
Mother callus jar contains
the packed cells.

Procedure:
1. Three types of medium were prepared. The mixtures were done as stated above. Then
they were autoclaved at 121oC for 15 minutes to kill all bacteria (sterilization).
2. Aseptic techniques were applied throughout the experiment. Hands, tools, working
bench, aluminium foil coverings and mouth of jars and flask were disinfected with
either 70% ethanol or by flame.
3. Samples were taken from mother callus that was grown beforehand by the instructor.
The healthy parts of the callus (whitish-yellow masses) were weighed up to 0.5g into a
sterile Petri dish. The callus was then cut into small pieces. Three 0.5g samples were
prepared.
4. About four to five pieces of callus were transferred using sterilized forceps into each
of the three prepared medium. The pieces were gently spread in solid media jars, one
containing KB, and the other without KB with appropriate distance between pieces.
For the suspension flask, the callus pieces were simply dropped in 100ml of
suspension media.
5. All containers were sealed with parafilm and labelled. The solid media jars were kept
under artificial light and the suspension media was kept on gyratory shaker shaking at
125 rpm.
6. The cultures were then observed for four consecutive weeks for growth or
contamination. The observations were recorded.
RESULT
a) Cell suspension
Week
1
No
result
was
taken
in
week
1

Week 2

-the sample was at the

bottom of the beaker at
the liquid surface level.
- all the samples were
alive even though they
were small.

Week 3

Week 4

-all the samples were

alive.
-the changed of the
sample from previous
week were small.

-all the samples were alive.

-the size of samples increased
from previous week.
-no contamination occur
throughout the whole month
for liquid suspension.

b) Kb solid media
Week 1
No
result
was
taken in
week 1

Week 2

Week 3

Week 4

-contamination occur
-assumption made:
there were other
organism inside the
beaker and
contaminated the plant
cell

-contamination occur
-this was the expected
result from the 3rd week
-the samples were
grown but with small
scale which could
hardly observed

-contamination occur
-this was the expected result
from the 4th week
-the samples were alive and
grew but in slow growth.
-no great differences from
the previous week

c) (P2,4-D) Solid media

Week
1
No
result
was
taken
in
week
1

Week 2

Week 3

Week 4

-the samples were

contaminated
-assumption made:
there were other organism
inside the beaker and
contaminated the plant cell

-this was the expected

result obtained in
week 3
-the samples were still
alive

-this was the expected result

obtained in week 4
-the samples were still alive
-no great differences in size
compare to previous week.

DISCUSSION
The specific differences in the regeneration potential of different organs and explants
have various explanations. The significant factors include differences in the stage of the cells
in the cell cycle, the availability of or ability to transport endogenous growth regulators, and
the metabolic capabilities of the cells. The most commonly used tissue explants are
the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These
tissues have high rates of cell division and either concentrate or produce required growth
regulating substances including auxins and cytokinins. Shoot regeneration efficiency in tissue
culture is usually a quantitative trait that often varies between plant species and within a plant
species among subspecies, varieties, cultivars, or ecotypes. Therefore, tissue culture

regeneration can become complicated especially when many regeneration procedures have to
be developed for different genotypes within the same species. The three common pathways of
plant tissue culture regeneration are callogenesis (shoot culture or nodal culture),
organogenesis and non-zygotic (somatic) embryogenesis
The propagation of shoots or nodal segments is usually performed in four stages for
mass production of plantlets through in vitro vegetative multiplication but organogenesis is a
common method of micropropagation that involves tissue regeneration of adventitious organs
or axillary buds directly or indirectly from the explants. Non-zygotic embryogenesis is a
noteworthy developmental pathway that is highly comparable to that of zygotic embryos and
it is an important pathway for producing somaclonal variants, developing artificial seeds, and
synthesizing metabolites. Due to the single cell origin of non-zygotic embryos, they are
preferred in several regeneration systems for micropropagation, ploidy manipulation, gene
transfer, and synthetic seed production.
In this plant cell and cell tissue culture which is the plant cell suspension culture from
callus is highlighted. The media that had been used in this experiment were liquid and solid
media. Theoretically, the suspension cultures are fast growing on the liquid media rather than
solid media. This is because the cell culturing uses all the surface of media. So that all the
nutritious that contain in liquid media are taken for the cell growth. The medium in this
experiment were divided into three types they are liquid media, solid media with and without
sucrose. As we can see from the result, there are only the cells in the liquid media growth
until the week forth. It is might be because the condition of the suspension media was the
same like in vivo for that particular plant cell. Hence, the cells were easy to adapt with their
new surrounding and can growth without any problem. The good aseptic also had been done
so that is why the cells were not contaminated. For solid media with kb or without kb the
contamination occurs. The cells were still alive but with the present of other organisms. The
solid media was contaminated since the first week. This is because of many factors that can be
said firstly it might be because of chemical contamination where it would be came from the
media itself, water that had been used, sera, endotoxins, storage vessels and incubators itself.
The cells also might be contaminated because of biological contamination which are would be
from bacteria, mould, or yeasts. The others factor that might lead to contamination are the
disturbance from virus, insects, protozoa, mycoplasmas and cross-contamination by other cell
cultures.
There are some precautions that should be taken in order to avoid the contamination.
The antibiotic should be given to the cell by adding it into the media so that the cells would
not contaminate easily. Besides, a good aseptic technique should be applied so that it will
reduce the contamination problem. Next, the laboratory should always be cleaned and any
accidents during culturing should be avoided as well. By having all the precautions the
contamination would be reduced definitely.

CONCLUSION
In conclusion, all the objectives in this experiment are accomplished which are to
cultivate adventitious shoot from explants (organogenesis), to initiate callus from leaf and to
establish cell suspension cultures from callus.
However the results obtained are not successful because of many factors as what had
been discussed. Tissue culture and plant regeneration are an integral part of most plant
transformation strategies, and can often prove to be the most challenging aspect of a plant
transformation protocol. Therefore a very cautious step in handling the experiment is needed.
REFERENCES

Walden, R. and Wingender, R. (1995). Gene-transfer and plant-regeneration

techniques. Trends in Biotechnology, 13, 32431.
Gamborg, O. L. (2002). Plant tissue culture. Biotechnology. Milestones. In vitro
Cellular and Developmental BiologyPlant, 38, 8492.
Dodds, J. H. and Roberts, L. W. (1995). Experiments in plant tissue culture.
Cambridge University Press, Cambridge.

Determination of tn tp bod & cod

INTRODUCTION
Determination of Biological Oxygen Demand (BOD5)
Biological oxygen demand is a test which determines the amount of organic material
in wastewater by measuring the oxygen consumed by microorganisms in decomposing
organic constituents of the waste. The higher the BOD, the more oxygen will be demanded
from the waste to break down the organics. The BOD test is most commonly used to measure
waste loading at treatment plants and in evaluating the efficiency of wastewater treatment.
The BOD test is performed by incubating a sealed wastewater sample for the standard 5 day
period, then determining the change in dissolved oxygen content. The bottle size, incubation
temperature, and incubation period are all specified. The temperature of water also contribute
to high BOD levels. Increased water temperature will speed up the bacterial decomposition
and result in higher BOD levels.
Most wastewaters contain more oxygen demanding materials than the amount of DO
available in air saturated water. Therefore, it is necessary to dilute the sample before
incubation to bring the oxygen demand and supply into appropriate balance. Because bacterial
growth requires nutrients such as nitrogen, phosphorous, and trace metals, these are added to
the dilution water, which is buffered to ensure that the pH of the incubated sample remains in
a range suitable for bacterial growth. Complete stabilization of a sample may require a period
of incubation too long for practical purposes; therefore, 5 day period has been accepted as the
standard incubation period.
When BOD levels are high, dissolved oxygen (DO) levels decrease because the
oxygen that is available in water is being consumed by the bacteria. Hence, because of less
dissolved oxygen in the water, fis and other aquatic organisms may not survive.
Total Nitrogen (TN)
The Total Nitrogen in water is comprised of dissolved inorganic (nitrites, nitrates and
ammonia) and organic nitrogen. High organic nitrogen levels are due to decomposition of
aquatic life and sewage run off while inorganic levels are enhanced by erosion and residential
runoff (fertilizers). Total nitrogen is sometimes regulated as an effluent parameter for
municipal and industrial wastewater treatment plants, but it is more common for limits to be
placed on an individual nitrogen form, such as ammonia. Treatment plants that have a total

nitrogen limit will usually need to nitrify and denitrify in order to achieve the limit of total
nitrogen.
Total nitrogen is the sum of nitrate (NO3), nitrite (NO2), organic nitrogen and
ammonia (all expressed as N). Note that for laboratory analysis purposes, Total
Kjeldahl Nitrogen (TKN) is a test performed that is made up of both organic nitrogen and
ammonia. Excess nitrogen in water may cause eutrophication by stimulating rapid growth of
algae and other aquatic plants. Other conditions such as sunlight and turbidity of water are
also fulfilled. Therefore, determination of nitrogen is essential to protect the water bodies
from eutrophication and other toxicity.
Total Phosphorus (TP)
Phosphorus is also one of the nutrients which is frequently present in water in various
forms. Total Phosphorus (TP) is the form of analysis typically cited as an effluent parameter
for municipal and industrial wastewater treatment plants. Total phosphorous in effluent is a
measure of the remaining dissolved phosphate plus any insoluble phosphorous carried over
into the effluent in the form of precipitates or within microbes. Digestion using chemicals,
heat or enzymes may be required to separate phosphorous from the precipitate or microbe.
Following digestion, all phosphorous is converted into dissolved (ortho) phosphate for
analysis. Total phosphorous is not, however, the form of analysis that is the most useful for
process monitoring or control purposes because the total phosphorus analysis does not
identify the original form of phosphorous in the effluent. Total phosphorous is typically
analyzed strictly for compliance monitoring purposes in wastewater.
Excess amount of phosphorus in water also may cause eutrophication by stimulating
rapid growth of algae and other aquatic plants, same like nitrogen.

OBJECTIVES

To determine the amount of oxygen necessary for biological oxidation of wastewater.

To determine the total nitrogen in water and wastewater.

To determine the total phosphorus in water and wastewater.

RESULT
DAYS
0
1
2
3
4
5

pH
: 7.67
Temperature : 26.70C
DO saturation : 95.4 %

BOD5(mg/L)
0
10.6
11.6
12
12.6
13.2

BOD5 VS DAYS
14

BOD5 (mg/L)

12
10
8
6
4
2
0
0

DAYS

Maryam Lake Water Sample

BOD5 VS DAYS
14

BDO5 (mg/L)

12
10
8
6
4
2
0
0

3
DAYS

CAC Water Sample

BOD5 (mg/L)

BOD5 VS DAYS
14
12
10
8
6
4
2
0
0

DAYS

Gate 3 water sample

BOD5 (mg/L)

BOD5 VS DAYS
14
12
10
8
6
4
2
0
0

3
DAYS

Female Sport Complex water sample

BOD5 VS DAYS
30
BOD5 (mg/L)

25
20
15
10
5
0
0

3
DAYS

Zubair water sample

DISCUSSION
The Biochemical Oxygen Demand (BOD) test measures the ability of naturally
occurring microorganisms to digest organic matter, usually in a 5 day incubation at 20 C, by
analyzing the depletion of oxygen. This measures biodegradable organic matter. This is
normally expressed as O2 mg/L. The biochemical oxygen demand analysis is an attempt to
simulate the effect a waste will have on the dissolved oxygen of a stream by a laboratory test.
The BOD test gives an indication of the amount of oxygen needed to stabilize or biologically
oxidize the waste. The advantage of the BOD test is that it measures only the organics which
are oxidized by the bacteria. The disadvantage is the 5 day time lag and the difficulty in
obtaining consistent repetitive values. In the test about BOD5, it can be seen that the highest
reading was at female sport complex which is 0.64mg/L and the lowest reading was at gate 3
which is 0.16mg/L. hence, it can be said that the water river at female sport complex contain
more organic matters compared to the other places where the samples of water were taken.
The BOD5 for all samples are more than 7 so that, all water samples are might be
contaminated or they contain a lot of dead organism and plant. For the BOD track, the reading
on the fifth day is higher which is 13.2mg/L compared to BOD5 where the reading is only
7.67mg/L. the BOD tracks reading is high because nutrient was added into the vials in order
to make sure there is enough microbes to degrade the organic matters. By adding the nutrients
the microbes will growth well and consume more oxygen, so it is the explanation why the
reading for BOD track is higher.
The Chemical Oxygen Demand (COD) is a test incorporates the complete oxidation of
biodegradable organics via utilization of a strong oxidizing agent. It is such an alternative of
the BOD test. The advantages of doing the COD test are it is fast and reproducible. In this test
the reading for Tasik Maryam is the highest which is 66mg/L. Therefore, the Tasik Maryam
water is the most polluted river where the pollution comes from the inorganic matter. The
results are valid since the COD reading is higher than BOD reading. The reading of COD for
water sample at Zubair was under range. It might be because of two possibilities which are the
water itself contains not enough inorganic matters ( the concentration ) or the technical error.
However, the higher possibility goes to technical error because it might be some mistakes that
brings to the under range reading. It might be because of the vials not shaken properly during
the process or the transfering process from one place to another was done incorrectly.
Nitrogen compounds are of interest to wastewater treatment plant operators because of
the importance of nitrogen in the life cycles of plants and animals. Nitrogen is a nutrient and
occurs in many forms including ammonia, organic, nitrate and nitrite each of which may be
tested for in a variety of ways. Raw wastewater nitrogen is normally present in the organic
nitrogen and ammonia forms, with small quantities of the nitrite and nitrate forms.
Depending on the amount of nitrification which occurs within the plant, the effluent may
contain either ammonia or nitrate nitrogen. Under normal circumstances, the nitrite form of

nitrogen will not be present in large quantities due to its rapid oxidation or conversion to
nitrate. The presence of large concentrations of ammonia in a stream or lake can create a large
oxygen demand. This demand is caused by the conversion of ammonia to nitrate. High
concentrations of nitrate in wastewater treatment plant effluent can cause algae to grow in
large quantities. Dead and decaying algae can cause oxygen depletion problems which in turn
can kill fish and other aquatic organisms in streams.
Phosphate is commonly found in agricultural fertilizers. It gets into waterways when
carried by runoff during precipitation of non- point source. The combination between
phosphorus with ammonium nitrate can cause severe eutrophication of lakes and ponds which
the occurrence of excessive growth of algae and will diminished oxygen. The higher the
amount of phosphorus, the higher the water sample being polluted. This required the
extensive treatment if want to use it for any purposes. The same goes for nitrogen, if high
amount of nitrogen the extensive treatment should be taken out because it is harmful to human
where it can cause a potentially fatal blood disorder in infants that called methemoglobinemia
or baby-blue.
The higher the total nitrogen and total phosphorus the higher the BOD reading. Hence,
the higher the growth rate and lead to the higher the death rate. But if the total nitrogen and
total phosphorus is not too high it might be because of the temperature of that placed.
Temperature plays an important role as when there is low temperature the photosynthesis is
not occurred vigorously so that low amount of oxygen dissolved. Besides that, the type of the
biomass in the water not contain more nitrogen and phosphorus also influenced the reading of
both materials.
The total nitrogen (TN) and total phosphorus (TP) are high in river near to Zubair
which are 2.9mg/L and 5.72mg/L for TN and TP respectively. This is the reason for why the
BOD reading of water sample taken from Zubair is quite high which is 0.63mg/L. The water
sample taken at Gate 3 river, the total nitrogen is too high which is up to 89.9mg/L. The
appearance of the water sample is cloudy and the fish in that river might be killed because of
the oxygen depletion. The oxygen was used by the nitrogen to transform to nitrate and nitrite.
Nitrate can get into water directly as the result of runoff of fertilizers containing nitrate. Some
nitrate enters water from the atmosphere, which carries nitrogen-containing compounds
derived from automobiles and other sources. More than 3 million tons of nitrogen is deposited
in the United States each year from the atmosphere, derived either naturally from chemical
reactions or from the combustion of fossil fuels, such as coal and gasoline. Nitrate can also be
formed in water bodies through the oxidation of other forms of nitrogen, including nitrite,
ammonia, and organic nitrogen compounds such as amino acids. Ammonia and organic
nitrogen can enter water through sewage effluent and runoff from land where manure has
been applied or stored.
There might be some error that occurs during the experiment which leads to the
inaccurate result. One of the most important error that might be happened is timing that
should be followed in a certain steps was not followed properly. Time is the most important
things so that if it was not followed properly and accurately, the inaccuracy in the result might

be occurred. Besides that, the preparation of the materials used is not that accurate. It might be
because of human error such as parallax error. Thus, the precaution to avoid the parallax error
should be taken seriously.
CONCLUSION
In conclusion, all the objectives in this experiment are accomplished which are to
determine the amount of oxygen necessary for biological oxidation of wastewater, to
determine the amount of chemical oxygen demand of wastewater, to determine the total
nitrogen in water and wastewater and to determine the total phosphorus in water and
wastewater.
From the results obtained, the BOD5 for all samples are more than 7 so that all water
samples might be contaminated or contained a lot of dead organism and plant. While the
highest COD reading is belongs to the water sample taken from Tasik Maryam which is
66mg/L. This COD result proved that Tasik Maryam is a polluted river. The highest TN
reading obtained from the water sample taken at the Gate 3 river which is 89.9mg/L and the
total phosphorus (TP) is high in river near to Zubair which is 5.72mg/L. These are the overall
results obtained from this experiment.
REFERENCES

Metcalf and Eddy, Inc. 1991. Wastewater Engineering, Treatment, Disposal and
Reuse, 3rd Ed. New York: McGraw-Hill.

Sawyer, Clair N., McCarty, Perry L., Parkin, Gene F. 1994. Chemistry for
Environmental Engineering, New York, McGraw-Hill.

Golterman H L, 2004. The Chemistry of Phosphate and Nitrogen Compounds in

Sediments. Kluwer Academic Publishers.Dorsrecht.