Course name: Biotechnology I

Course Code: BIOT3113

Title: Introduction to Tissue Culture
Date: 26/09/2022

INTRODUCTION

Plant clone propagation is accomplished by a process called plant tissue culture. Plant tissue
culture uses a set of in vitro techniques which are methods done in a control environment such as
in a test tube. Plant tissue culture also utilizes a variety of technologies (plant biotechnology).
Plant tissue culture is especially important to plant clone propagation which produces several
identical plants which may have a particularly positive qualities, such as the production of high-
quality crops, grow plants that become adult plants quickly, multiply plants without seeds or the
pollinators. Furthermore, gene-modified plant cells can also be employed in plant tissue culture
to regrow entire plants. Plant tissue culture can be used to generate new plants from mature plant
tissue.

Protoplasts preparations is a tissue culture technique is used to combine plant cells of different
species which many be sexually incompatible through a process called somatic hybridization.
Another use of plant tissue culture is phytosanitary improvement which involves the mass
propagation of pest-free plants. The explants of these specific plant-free plants are removed and
used for tissue culture. An explant is a part of the plant which a new plant can be produced from.
In addition, tissue culture can be used to produce pest and stress resistant plants using in vitro
systems which use the already naturally resistant plant or induce this resistance using chemical or
physical methods which cause advantageous mutations. Plant tissue culture may also be used for
intro vivo germplasm conversation. The goal of germplasm conservation is to protect vulnerable
and endangered plant species for future development and reproduction.

The most common application of tissue culture is micropropagation. In fact, plant tissue culture
method in vitro is also referred to as micropropagation. Micropropagation is a technique used to
produce many from an explant or small piece of the stock plant in a relatively short time. The
explant may be a leaf, shoot tip, root, lateral bud or root; the explant selection is dependent on
the species. Using the explant, usually means that the whole plant is not destroyed. As soon as
the plant is placed in culture, lateral buds, adventitious shoots, and shoots that are differentiated
straight from calluses. They all grow and multiply, greatly increasing the number of shoots that
can be rooted. In a single year, one explant can produce thousands of plants. Actively dividing
cultures, once established, provide a constant source of microcuttings that can produce plants
under greenhouse conditions without seasonal interruption. Explant selection, sterilization,
initiation into culture, establishment, and in vitro plantlet multiplication through a succession of
subculturing are all parts of stock plant care and in vitro plantlets' roots and
acclimatization/hardening are all steps involved in micropropagation.
Micropropagation usually produces more resilient plants than conventional methods, like seeds
or cuttings, which speeds up growth. Micropropagation may produce thousands of propagules
while having an abnormally high fertility rate when compared to standard methods.
Micropropagation has additional benefits over traditional planting techniques, such as disease-
free crop production, annual plantlet generation, germplasm preservation, multinational trade,
and genetic stability.

Sterile technique is the strictest variation of aseptic technique. It aims to create an environment
free of any microorganisms. In operations and other major, invasive treatments when infection
could be particularly dangerous, sterile technology is used. A sterile chamber, gloves, gowns,
hats, instruments, masks, handwashing, and aseptic fields are also necessary. The use of
appropriate aseptic procedures stops from unwanted microbes from accidentally entering the
environment and/or contaminating a culture. In tissue culture, contamination come as either
microorganisms on the surface (exogenous) or in the tissue (endogenous, systemic) of explants,
or improper laboratory practices. If these contaminants are left in culture the plants may become
exposed to infections from non-pathogenic and pathogenic bacteria. Apart from contaminants on
the explant and in the laboratory, microbes that may contaminate the culture include mites, trips.
Three ideal techniques used when implementing the aseptic technique include the Vertical and
Horizontal Laminar Flow as well as the Mobile micropropagation unit, MMU.

LITERATURE REVIEW

Sterile procedures are needed for plant cell, tissue, and organ culture purposes. For tissue culture
methods to be successful, aseptic or sterile conditions must be maintained (Gamborg & Phillips,
1995). The maintenance of sterility in areas where plant tissue culture is conducted greatly
reduces the risk of culture contamination which negatively impacts the culture though the
introduction of pest and infections. The Laminar Flow and Mobile Micropropagation Units and
their equivalent are usually used to carry out plant tissue cultures. The majority of plant tissue
culture procedure is performed in these spaces with a horizontal airflow that moves from the
back to the front. Furthermore, Gamborg & Phillips (1995) state that there are three different
categories of sterilization. These include the preparation and use of sterile culture media and
petri dish or test tubes. The aseptic techniques employed when obtaining the explant material
from the original plant and ensuring that is free of contaminants such as microbes and dirt.
Lastly, the maintenance of aseptic conditions of the culture would be the final category of
sterilization highlights.

The central purpose of the journal article written by Krikorian et al. (1984) entitled Aseptic
Culture Techniques for Banana and Plantain Improvement was to discuss solution-based
strategies to create Sigatoka' disease-tolerant or -resistant bananas. Moreover, use of aseptic
culture methods to create and/or propagate these pathogen-tolerant clones was a constant theme.
Now, the 'Black Sigatoka' disease affects the banana leaves resulting black spotting; this disease
plaque both “cooking” and “desert” bananas. As a result the economic sectors especially the
industrial banana plants suffered great loss and countries who relied on the starchy staple
(plantains) had to find other more expensive and less reliable sources.
Krikorian et al. (1984) suggest that tissue culture techniques to propagate banana can be used to
create a model to be applied to other crops. It is these molecular biological approaches and
aseptic culture that are frequently used to improve crops and other commercially significant
plants. The improvement includes things like a general rise in the quantity of a certain highly
desirable or exceptional genotype or phenotype through clonal propagation.

In the paper a critical issue faced what the lack of scientific knowledge of the Musa (Musaceae
genus-edible banana and plantain-producing flowering plants) genome which limited the extent
to which genetic engineering could be used to address and solve this 'Black Sigatoka' disease
problem plaguing the bananas. As such, Krikorian and Cronauer (1984) explored and assessed if
aseptic meristem, cell, and protoplast culture methods have any potential to improve banana and
plantain quality. The article concluded on the point that meristem culture has a legitimate
purpose and has the possibility of being beneficial, but it cannot take the place of cell and
protoplast cultivation systems.

MATERIAL AND METHODS

Materials included:

Petri dishes
MS (Murashige and Skoog) Media
Lamina Flow
Mobile Micropropagation unit
70% isopropyl alcohol
Parafilm

Methods

1. The class was split into groups of 5-6 students, and each were assigned a group number
and two of four areas to work in around the lab and/or outside. Another 5-6 student
utilized two of four areas in around the lab and/or outside. The final group consisted of
10-12 students.
2. The four areas where the experiment would be conducted where identified. For the
purpose of this experiment two of the areas were considered the dirty areas (Bench top
(BT) and Outside lab (OL)) and the other two were the clean (sterile) areas (Laminar
Flow (LF) and Mobile Micropropagation Unit (MMU)).
3. Four closed inverted petri dishes filled with ½ MS Media were labelled as Laminar Flow
(LF), Mobile Micropropagation Unit (MMU), Bench top (BT), Outside lab (OL)
respectively.
4. The hands of the student who handed the petri dish labelled LF were sprayed with
alcohol and then the petri dish was sprayed.
5. The petri dish was placed in the middle of the vertical lamina flow and opened for one
minute. Afterwards, the petri dish was closed and then removed from the laminar flow.
Parafilm was used to wrap around the edges of the petri dish to create a seal.
6. The petri dish was then appropriately labelled with the group number, group members
initials, type of media used and the date.
 7. Another petri dish labelled MMU was sprayed after the student’s hands were sprayed
with alcohol. Alcohol was also sprayed in the MMU.
8. Extreme caution was taken to enter the MMU which. The plastic tarp was wrap securely
around the student’s hand as the petri dish was open in the MMU for one minute, then
closed and parafilm was used to wrap around the edges of the petri dish. The petri dish
was then appropriately labelled.
9. This procedure was repeated for the petri dishes labelled BT and OL. Where the petri
dish and students’ hands were sprayed with alcohol and the petri dish was open over the
bench top and outside of the lab on the corridor respectively for a minute each.
10. Both BT and OL petri dishes were wrapped with parafilm and appropriately labelled.

Summary of Bananas and Plantains (Botany and Crop History)

In the video entitled Bananas and Plantains (Botany and Crop History) a brief description of the
various species of bananas in the Musa family was given. A description and purpose of the
various types were highlighted as well as the climate in which they thrive in. The video also
spoke about the evolutionary changes of bananas. These “new age” bananas originated from the
Musa acuminata and Musa balbisiana species and the difference between the two wild type
species was explained in detail. Musa acuminata clonal bananas are called desert bananas and
Musa balbisiana produces clones referred to as plantains. Additionally, the parts of the banana
plant from corms to crown (top), as well as the growth and development the banana plant was
discussed. The video mentioned that Bananas originated from Southeast Asia and then to the
world. The economic, commercial, social and nutritional importance of bananas was also
covered in the video. In the second video entitled Bananas and Plantains (Propagation and
Breeding) spoke to the various methods of plant tissue cultures. The procedure, advantages,
disadvantages, purpose and result of plant tissue culture.

RESULTS

 Illustrations of the Aseptic Technique and Petri dish method

Figure 1: Student spraying hands with 70% isopropyl alcohol to handling petri dish or entering
clean/sterile areas.

Figure 2: Student spraying the petri dish with 70% isopropyl alcohol before placing it in the
Mobile Micropropagation Unit (MMU) to ensure that the plate is free from contaminants.
Figure 3: Student holding the petri dish towards the back of the Mobile Micropropagation Unit
(MMU) where the cleanest air is in the unit.

Figure 4: Student holding the labelled petri dish for one minute outside the lab.

Figure 5: Student holding the petri dish open for one minute in the middle of the Laminar Flow
where the clean air is in the unit.
Figure 5: Student holding the petri dish open for one minute over the bench top.

Figure 6: Student wrapping the petri dishes with parafilm to create and airtight seal to keep out
contaminants.
Figure 7: Four closed inverted petri dishes filled with ½ MS Media labelled as Laminar Flow
(LF), Mobile Micropropagation Unit (MMU), Bench top (BT), Outside lab (OL) after one
minute exposure in each of the listed locations (2 cleans and 2 dirty areas) respectively after the
initial procedure was carried out; no growth resulted.

Figure 8: Plate that was exposed to the surroundings outside the laboratory after 3 days of
incubation showing no growth.
Figure 9: Plate that was exposed to the surroundings inside the Mobile Micropropagation Unit
after 3 days of incubation showing no growth.

Figure 10: Plate that was exposed to the surroundings inside the Laminar Flow after 3 days of
incubation showing no growth.
Figure 10: Plate that was open over the bench top after 3 days of incubation showing no growth.

After the third day of incubation the plates where accidently sterilized by a laboratory technician.
Therefore, no results (observations) were obtained for Day 7 of the experiment. Furthermore,
when the plates were observed at Day 3 the plates had no growth present when the experiment
ended pre-maturely. Any growth that would have resulted would have been halted as the
potential microbes would have been killed during the sterilization process. For this reason, this
laboratory report as it relates to the parts of the discussion aspect will be based on expected
results and theoretical assumptions of what would have occurred if the experiment was fully
carried (full 7 days).

DISCUSSION

1. What is the sterile technique and how does it differ depending on the clean area
used? (2 marks)

Ans: The strictest type of aseptic technique, is the sterile technique which strives to eliminate all
bacteria from the surroundings. Although the basic procedure for the aseptic technique is almost
the same for all situations they vary slightly. For instance, in the Mobile Micropropagation Unit
it is best to work closes to the back of the unit as there is where the cleanest flow of air enters the
system. Additionally, inside of the unit is sprayed using 70% isopropyl alcohol and the plastic
shield is kept sealed as the operator works. In contrast, when working in the Lamina Flow it is
best work in the middle as there is where the air is cleanest. Both techniques require the use of
70% isopropyl alcohol to spray the hands of the operator and the equipment in order to kill
unwanted microbes and other contaminants.

2. Based on the petri dish experiment, what area is suitable to conduct tissue culture
and why? Explain the results you got. (2 marks)
Ans: Although the experiment was not carried to term (full 7 days), one could predict that the
most suitable area to conduct the tissue culture would be the Lamina flow. The Laminar flow is
designed to displace contaminants from an area while supplying clean air. This is done in order
to maintain a clean and contaminant-free conditioned area, there should be little to no entrapment
of room air and other containments. The mobile micropropagation unit (MMU) works in a very
similar system however the plastic tarp of the MMU must be sealed properly as the operator
works. For an inexperience operator this proves to be a difficult task and gaps between the tarp
and the desk is maybe created as the operator moves their hands. For this reason, one could
expect that the MMU would be slightly more contaminated than the Laminar flow resulting in
more growth observed on the plate. Therefore, the laminar flow would be the most sterile area to
conduct tissue culture. As the bench space is open to the surrounding, it is expected that any
microorganisms in the lab would mostly be seen growing on the agar plate the same is also
expect of the plate opened outside. However, the outside would have the most growth of
microbes and other contaminates as the outside surrounding are not cleaned and sterilized as
strictly as the laboratory would be.

3. Describe the basic requirements of a standard tissue culture laboratory. Indicate the
reason for those requirements. (4 marks)

Ans: The basic requirements of a standard tissue culture laboratory are as follows:

- Sterile work area: Plant tissue culture techniques must be performed in a sterile

environment to minimize the spread of microorganisms and lower the risk of
infection such as a Lamina flow.

- Explant: An explant is a piece of a plant used in the plant tissue culture procedure to
create a whole new plant. Explants can be collected from stems, roots, leaves,
flowers, fruits, seeds, etc. they are used for culturing.

- Nutrient Medium: For plant tissue culture, some common media, such as Murashige
and Skoog's (MS) medium, can be used successfully. 

4. What tissue culture methods are used to obtain clonal plants? Explain (2 marks)

Ans: Micropropagation is a method of plant propagation that involves cultivating very small bits
of plant tissue (explants) in a lab environment to create new clonal plants. These explants are
acquired from a carefully selected and prepared mother plant.

5. What tissue culture methods are used to obtain new varieties? Explain (2 marks)

Ans: Somaclonal variation is a helpful technique in plant breeding, allowing for the production
of crops with unique features by utilizing diversity in tissue culture using somatic cells to
produce genetically variable plants. In sugarcane, banana, and potato, somaclonal variation is
an mutation induction performed in vitro have been employed to improve specific features,
primarily the resistance to illnesses and other faults that restrict the use of some significant
commercial types.Although not exclusively present, somaclonal variation is more prevalent in
plants that have grown from calluses. 

6. Describe protoplast culture (2 marks)

Ans: Protoplast culture is the process of cultivating isolated protoplasts from any plant part, such
as a root, shoot, leaf, or embryo, in an artificial medium under artificial circumstances that
encourage cell division and plant regrowth. Protoplasts are cultivated in a semisolid agar or
media which is in a liquid state. Protoplasts may occasionally be moved to an agar medium after
initially allowing their cell walls to form in a liquid medium.

7. Describe haploid culture. (2 marks)

Haplotype cells (pollens or ovules) are used in the in vitro culture process known as haploid
culture to produce a whole plant. This process is known as "parthenogenesis" and occurs
naturally in some plants, such as apples. Here, the embryo is created directly from an egg cell.
But haploid plants can only be produced by this natural mechanism.
8. Give four reasons for presoaking seeds prior to chemical mutagenesis. (2 marks)

Ans: The apical meristems in seed embryos, buds, and other explants, which are shielded,
respectively, by the seed testa, leaves, and cotyledons require time for the mutagen to penetrate
them during chemical mutagenic treatments. Additionally, the chemical mutagens
may spontaneously degrade during the mutagenic process, and some of the reactive chemical
species are lost in alkylation reactions with the content material of tissues and cells other than the
target meristematic cells, which are eventually reached by a depleted mutagenic solution several
hours later.  Due to the protective outer layers being thinner, the mutagen can penetrate more
quickly to the tissues of interest (meristems) (eg. seed testa is removed). Additionally, Seeds that
have been presoaked have more mutagen infused into the embryo. The damage induced by ethyl
methane sulphonate (EMS – a mutagen) within a particular treating period was shown to be
reduced by presoaking, but it had no effect on the biological action of EMS once it had been
absorbed.

9. What are the two wild species of banana? and state their country of origin. (2
marks)

Ans: To wild species of banana are Musa acuminata and Musa balbisiana. Musa acuminata
origin form Malaysia while Musa balbisiana originated in India.

10. In which year was banana and plantain first introduced to the West Indies? (2
marks)

Ans: Banana and plantain were first introduced to the West Indies in 1516.
 11. Commercial bananas may be vegetatively propagated, state two disadvantages of
this method. (2 marks)

Ans: Vegetative propagation is disadvantageous as it has a low coefficient of multiplication

which means many new plants are not produced using this method and transmission of disease
particularly viral in vegetative tissue.

12. Briefly describe the meristem tip and embryo culture techniques used in the
propagation of bananas. What are their advantages? Name two other crops
multiplied by either technique. (2 marks)

Ans: Meristem-tip culture's key objective is the removal of the organized shoot's apex from a
chosen donor plant for subsequent in vitro cultivation. Without the assistance of any adventitious
organs, only organized expansion of the apex directly into a shoot is permitted under the culture
conditions. The possibility of eliminating pathogenic organisms present in the donor plant from
the in vitro culture is a significant benefit of working with such a small explant and the genetic
stability of the method is another benefit, as it may be done without producing plantlets from
accidental organs. Crops that can be propagated using the meristem tip culture include
cauliflower and ornamental plants like Orchid Cymbidium. The process of growing embryos
from seeds and ovules in a nutritional media is known as embryo cultivation. In embryo
cultivation, the plant either grows directly from the embryo or indirectly by developing a callus,
which is followed by the development of shoots and roots. An advantage of the embryo culture
techniques includes its capability to identify the variables that control the primordial organs of
the seedling plant's growth. This technique is also useful in researching the molecular and
metabolic elements of dormancy and germination is useful it and   facilitates the investigation of
the numerous embryonic growth indicators. Plants that can be multiplied using the embryo
culture technique include Helianthus annuus (sunflower seeds) and Zea mays (maize).

13. . What is the purpose of bleach in tissue culture and state its mechanism of action?
(2 marks)

Ans: Sodium hypochlorite also referred to as "bleach" is one of the chemicals that is most
frequently used to sterilize the surface of explants. In tissue culture procedures, bleach is diluted
up to 10–20% for sterilizing. Dilution is necessary as high concentrations can damage the plant
tissue causing it to become nonviable. The solution will contain between 0.5 and 1.0% of bleach.
Each form of explant has a different sterilization time requirement. But typically, 30 to 40
minutes are sufficient to achieve sterilization.

14. The basic initiation medium for tissue culture is Murashige and Skoog. List five
constituents of this medium and their purpose in plant growth. (2 marks)

Ans: Murashige and Skoog Medium (MS) provides all the essential constituents need for plant
growth. Potassium dihydrogen phosphate serves as a source of phosphate. Microelements like
Boron, Manganese, Molybdenum, Copper and Zinc play vital role in plant metabolism. Sucrose
acts as a carbon source while cytokinin benzylaminopurine stimulates in vitro development of
new shoots. Boron plays a key role in carbohydrate metabolism. Thiamine, nicotinic acid,
pyridoxine, inositol act as enzymatic cofactors in universal pathways including glycolysis and
TCA cycle along with primary and secondary metabolism in the plants. Glycine serves as a
source of amino acid.

15. Identify three fungal diseases affecting bananas. (2 marks)

Ans: Three fungal diseases that affect bananas include black leaf streak disease also called black
Sigatoka, which is caused by Mycosphaerella fijiensism Fusarium wilt which is caused by
Fusarium oxysporum f. sp. Cubense and fruit rot which is caused by Botrtosphaeria ribis

16. What are the two methods of inducing mutation in vitro? (2 marks)

Ans: Ionizing radiation and chemical mutagens are two methods can be used to induce mutations
in seeds and other planting materials (explants).

17. State examples of the mutagens utilized in each method. (2 marks)

Ans: Ionizing radiation such as Gamma-rays radiation has been used frequently to create
mutations because it is known to result in chromosomal mutations such as inversions and
deletions. While ethyl methane sulfonate (EMS) is a chemical that causes mutations in plants. It
has been demonstrated that single base point mutations are mainly caused by EMS.

18. If you were given a job to carry out ionization mutation in seeds here at the
university, what facilities would you use to accomplish your job? (4 marks)
Ans: When using any form of ionization radiation caution must be taken to ensure that the
operator as well sample should not be over-exposed to the radiation which has serious
immplications. For the operator, acute health impacts like skin burns and acute radiation
syndrome (also known as "radiation sickness") can occur after exposure to extremely high doses
of radiation. As such, minimizing the amount of exposure time, keeping a safe distance from the
origin of radiation and erecting a barrier between the and the source are a few safety protocols
when dealing with ionizing radiation. Although, ionization radiation can be used to induce
mutations in seed high exposure to gamma irradiation can cause the seeds to develop metabolic
abnormalities that prevent them from germinating or surviving for more than a few days. For this
reason, caution must be taken to ensure that the seeds are not exposure to extreme levels of
radiation or for prolonged periods of time.
 References
Brennan, D. (2021, April 25). What to know about aseptic technique. WebMD.

https://www.webmd.com/a-to-z-guides/what-to-know-about-aseptic-technique

Gamborg, O. L., & Phillips, G. C. (1995). Sterile Techniques. In Plant Cell, Tissue and Organ

Culture (pp. 35–42). Springer Berlin Heidelberg.

Grout, B. W. (1990). Meristem-tip culture. Methods in Molecular Biology (Clifton, N.J.), 6, 81–

91. https://doi.org/10.1385/0-89603-161-6:81

Krikorian, A. D., & Cronauer, S. S. (1984). Aseptic culture techniques for banana and plantain

improvement. Economic Botany, 38(3), 322–331. https://doi.org/10.1007/bf02859010

Mitchell, S. (2022). BIOT3113 Biotechnology 1 Tissue Culture laboratory Manual. Plant Tissue

Culture, 2-11.
Oladosu, Y., Rafii, M. Y., Abdullah, N., Hussin, G., Ramli, A., Rahim, H. A., Miah, G., &

Usman, M. (2016). Principle and application of plant mutagenesis in crop improvement:

a review. Biotechnology, Biotechnological Equipment, 30(1), 1–16.

https://doi.org/10.1080/13102818.2015.1087333

Tapingkae, T., Zulkarnain, Z., Kawaguchi, M., Ikeda, T., & Taji, A. (2012). Somatic (asexual)

procedures (haploids, protoplasts, cell selection) and their applications. In Plant

Biotechnology and Agriculture (pp. 141–162). Elsevier.

Tran, N. (2021, September 9). 4 methods of sterilization used in plant tissue culture. Lab

Associates; Lab Associates B.V. https://labassociates.com/4-methods-of-sterilization-

used-in-plant-tissue-culture