PAPER BIOTECHNOLOGY

Type of Tissue Culture and Explant Preparation (Sterilization,

Innoculation and Subculture)

Arranged by :

Naufan Hermawan (185040200111008)

Irene Magdalena S. (185040200111107)

Prima Hansen S. (185040200111236)

Eldo Iriyo C. M. (18504020111079)

Kirana Fadhilah (185040207111029)

Rury Maytawanti (185040207111087)

CULTURES OF ORGANIZED STRUCTURES

Organized growth contributes toward the creation or maintenance of a defined structure. The
growth of higher plants depends on the organized allocation of functions to organs, which in turn
become differentiated, modified, and specialized to enable them to undertake their essential
roles. Organ cultures are good examples of organized structures. Organ culture is used as a
general term for those types of cultures in which an organized form of growth can be
continuously maintained. These are the cultures of isolated plant organs including cultures
derived from root tips, stem tips, leaf primordia, or immature parts of flowers and fruits. Organ
cultures are divided into determinate and indeterminate organs (Chawla, 2011)

1. Culture of Determinate Organs

According to Robbin (1992), A plant with determinate growth stops growing once
it reaches its mature phase. Hence, those organs of plants that stop growing at maturity
and exhibit determinate growth are called determinate organ cultures. Usually
determinate organs cultures have their defined size and shape, e.g., leaves, flowers, and
fruits.
Advantages of tissue culture which is :
1.Molecular to mass level of work can be performed. The tedious and laborious
work such as weeding, spraying, watering, etc. can be skipped in plant tissue culture.
2. For rejuvenation of plant material and cultures, can be preserved for long time.
3. Production is independent on season and can continue throughout the year.
4. It is the best technique for the introduction of temporal characteristics and
studying the effects of several mutagens and to attempt immobilizations of cells, for
biotransformation or biochemical reactions.
5. It may provide a continuous, reliable source of phytopharmaceuticals and could
be used for the large-scale culture of plant cells.
6. Plant tissue culture can produce a large number of clones (with desirable
characteristics), draught resistant, flood resistant, disease-free plants, plants of high
nutritional values, and high quality plants with improved yields of secondary metabolites
(also used to study biogenesis of secondary metabolites).
7. The process offers uniform biomass available all the time, synthesis of
medicinal compounds difficult or impossible to synthesize chemically, independent of
soil conditions and change in climatic conditions.
disadvantages of tissue culture which is :
1. Requires lot of expensive and specialized procedures, facilities, and advance
skills.
2. Plants produced are dependent on nutritional sources in contrast to field
plants.
3. It is not obligatory to have callus within a short time. Sometimes callus will
not appear for months and for some plants callus does not appear.
4. Plantlets are initially small and have undesirable characteristics.
5. Plants do not function autotrophically in culture and have to undergo a
transitional phase before independent growth.
6. Chances of producing genetically aberrant plants may be increased.
7. Plantlets are more susceptible to contamination and water loss in the external
environment, since they are grown at a high relative humidity.
a. Leaf Culture Or Primordial Culture
Leaf culture may be described as the culturing of an excised leaf primordial or an
immature young leaf of the shoot apex aseptically in a chemically defined medium
where they grow and follow the development sequences under controlled conditions
(Tas et al, 2013)
b. Nucellus culture
Nucellus cultures are those cultures in which embryos are derived from the
nucellus or integument. Nucellus cultures are taken from pollinated flowers and widely
reported in the genus of Citrus. It is widely utilized to study the formation of embryos. It
is known to be the reliable technique for the production of virus-free citrus plants and can
be grown on casein hydrolysate supplemented White’s medium giving a callus. The
callus may then give rise to pseudobulbils, differentiating into embryos, which can
eventually develop into seedlings. The seedlings obtained from the nucellar tissue are of
parental type and thus may be important for maintaining purity of horticultural stocks
(Weathers, 1959)
c. Fruits Culture
There are various indications of fruit explant cultures that have shown better
results. The effect of juice from an “acidless” orange variety on in vitro growth of explant
(juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (Citrus
limon), grapefruit (Citrus paradisi), sweet orange (Citrus sinensis), and mandarin (Citrus
reticulata) fruits was studied by Einset. In his study he demonstrated the significant
growth stimulation effect of orange juice on various in vitro cultured fruits in basal
medium containing Murashige and Skoog salts, sucrose, myoinositol, thiamine, 2,4-D,
and kinetin. It was also discovered that this stimulation was not due to the presence of
citric acid since the addition of “acidless” juice was only responsible for stimulation
effects.
d. Stamens Culture
Stamens are the male reproductive part of a flower. The development of the male
reproductive system is a complex biological process, which includes the formation of the
stamen with differentiated anther tissues (in which microspores/pollens are generated),
then anther dehiscence, and subsequently pollination. Stamen specification and anther
development involve a number of extraordinary events such as meristem transition, cell
division and differentiation, cell-to-cell communication, etc., which need the cooperative
interaction of sporophytic and gametophytic genes.
e. Flower Culture
Flower culture may be described as the aseptic culture of excised flower buds or a
complete flower bud on a chemically defined nutrient medium where it continues
developing the full form in a culture vessel. In tissue culture, in vitro flowering serves as
 an important tool for many reasons. One of the most important ones is the ability to
shorten the life cycles of plants; other aims include studying flower induction and
initiation and floral development. Controlling the environment and media components
enables the manipulation of different variables that affect these processes. Flower
stamens contain anthers and anthers contain microspores. These microspores then
develop an exine around it, called a pollen grain. In other words, when microspores
mature they become pollen grains. Under in vitro conditions explants such as pollen or
anthers are usually utilized for the production of haploid tissues (Paramasivan et al, 2014)

2. Culture of Indeterminate Organs

A plant with indeterminate growth can keep growing as long as the environment can
support it. Hence, plant organs that grow indefinitely by adding modules exhibit
indeterminate growth and are called culture of indeterminate organs (Morel and Martin,
1952).
a. Meristem culture
Cultivation of shoot apical meristem in vitro is known as meristem culture. Meristem lies
in the shoot tip beyond the youngest leaf or first leaf primordium. Culture of the extreme
tip of the shoot is used as a technique to free plants from virus infections. Explants are
 dissected from either apical or lateral buds. They comprise a very small stem apex
consisting of just the apical meristem and one or two leaf primordia (George et al, 2008)
b. Meristem And Shoot Culture
The growing points of shoots can be cultured in such a way that they continue
uninterrupted and organized growth. As these shoot initials ultimately give rise to small
organized shoots, which can then be rooted, their culture has great practical significance
for plant propagation. Recently, the meristem culture technique with shoot-tip culture
technique was studied for obtaining virus-free (yellow dwarf and leek yellow stripe
viruses) plants of two different garlic species (Allium sativum and Allium tuncelianum).
Results of a polymerase chain reaction assay for the detection of viruses proved that more
than almost 70% of the obtained cultures are virus free.
c. Shoot Or Shoot Tip Culture
The culture of larger stem apices or lateral buds (ranging from 5 to 10 mm in
length to undissected buds) is used as a very successful method of propagating plants.
The size and relative positions of two kinds of explants in the shoot apex of a typical
dicotyledon are considered in vitro.
d. Embryo Culture
Embryo culture is the culture of isolated immature or mature embryos. Zygotic or
seed embryos are often used advantageously as explants in plant tissue culture, for
example, to initiate callus cultures. This embryo develops properly when nourishing
tissue; endosperm was present in the seed during the development.
e. Seed Culture
Seed culture is an important technique when explants are taken from in vitro
derived plants and the propagation of orchids. Each explant requires a suitable
sterilization procedure, which can otherwise cause contamination and affects the
regeneration of plants. In such a case the culture of seeds to raise sterile seedlings is the
best method.
f. Endosperm Culture
 The endosperm is a short-lived structure and is consumed during the development
of embryo. It is the nourishing tissue in seeds. During the process of fertilization in
angiosperms the two male gamete/nuclei fuse separately with two different female nuclei
and this process is known as double fertilization.

CULTURE OF UNORGANIZED CELLS

a. Callus Cultures
A callus consists of an amorphous mass of loosely arranged thin-walled
parenchymatous cells arising from the proliferating cells of the parent tissue. Callus
culture provides information on the regeneration potentiality of the plant and acts as a
good source material for protoplast isolation. Callus can be used as inoculums for the
initiation of suspension cultures, serve as a better source of cells for the genetic
manipulation of plant genomes, and be maintained indefinitely in the in vitro
condition for longer periods of time (Iwase et al, 2011).
b. Protoplast Culture
A cell without a cell wall is called a protoplast. Protoplast fusion or somatic
hybridization is one of the most important uses of protoplast culture. This is
particularly significant for hybridization between species or genera, which cannot be
made to cross by conventional methods of sexual hybridization. Although somatic
hybridization was achieved first in animals and only later in plants, its significance
has been realized fully in plants because the hybrid cell can be induced to regenerate
into whole plants (Yang et al, 2008)
STERILIZATION

Based on Sultana (2007), methods of sterilization are :

1. Heat sterilization
Heat sterilization is the most widely used and reliable method of sterilization,
involving destruction of enzymes and other essential cell constituents. The process is
more effective in hydrated state where under conditions of high humidity, hydrolysis and
denaturation occur, thus lower heat input is required. Under dry state, oxidative changes
take place, and higher heat input is required.
The efficiency with which heat is able to inactivate microorganisms is dependent
upon the degree of heat, the exposure time and the presence of water. The action of heat
will be due to induction of lethal chemical events mediated through the action of water
and oxygen. In the presence of water much lower temperature time exposures are
required to kill microbe than in the absence of water. In this processes both dry and moist
heat are used for sterilization.
a. Dry Heat Sterilization: Examples of Dry heat sterilization are:
It employs higher temperatures in the range of 160-1800 C and requires exposures
time up to 2 hours, depending upon the temperature employed. The benefit of dry
heat includes good penetrability and non-corrosive nature which makes it applicable
for sterilizing glasswares and metal surgical instruments. It is also used for sterilizing
non-aqueous thermostable liquids and thermostable powders. Dry heat destroys
bacterial endotoxins (or pyrogens) which are difficult to eliminate by other means and
this property makes it applicable for sterilizing glass bottles which are to be filled
aseptically.
b. Hot-air oven Dry heat sterilization
Moist heat sterilization involves the use of steam in the range of 121-1340 C. Steam
under pressure is used to generate high temperature needed for sterilization. Saturated
steam (steam in thermal equilibrium with water from which it is derived) acts as an
 effective sterilizing agent. Steam for sterilization can be either wet saturated steam
(containing entrained water droplets) or dry saturated steam (no entrained water
droplets).
2. Gaseous Sterilization
The chemically reactive gases such as formaldehyde, (methanol, H.CHO) and
ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a colorless, odorless,
and flammable gas. The mechanism of antimicrobial action of the two gases is assumed
to be through alkylations of sulphydryl, amino, hydroxyl and carboxyl groups on proteins
and amino groups of nucleic acids. The concentration ranges (weight of gas per unit
chamber volume) are usually in range of 800- 1200 mg/L for ethylene oxide and 15-100
mg/L for formaldehyde with operating temperatures of 45-63°C and 70-75°C
respectively.
3. Liquid Sterilization
a. Peracetic Acid liquid sterilization: Peracetic acid was found to be sporicidal at low
concentrations. It was also found to be water soluble, and left no residue after rinsing.
It was also shown to have no harmful health or environmental effects. It disrupts
bonds in proteins and enzymes and may also interfere with cell membrane
transportation through the rupture of cell walls and may oxidize essential enzymes
and impair vital biochemical pathways. The disadvantages of this method of
sterilization are that the devices must be immersible, must fit in the appropriate tray,
and must be able to withstand the 55°C temperature the process uses.
b. Hydrogen Peroxide Sterilization: This method disperses a hydrogen peroxide solution
in a vacuum chamber, creating a plasma cloud. This agent sterilizes by oxidizing key
cellular components, which inactivates the microorganisms. The plasma cloud exists
only while the energy source is turned on. When the energy source is turned off,
water vapor and oxygen are formed, resulting in no toxic residues and harmful
emissions. The temperature of this sterilization method is maintained in the 40-50°C
range, which makes it particularly well-suited for use with heat-sensitive and
 moisture-sensitive medical devices. The instruments are wrapped prior to
sterilization, and can either be stored or used immediately
c. Disinfectants

Plant tissue culture is a useful technology for plant propagation. The knowledge has been
provided to agriculturists worldwide. According to Thepsithar (2013) Unfortunately,
most agriculturists cannot carry out plant tissue culture laboratory by themselves due to
high production costs. One of the major problems is expensive equipment especially an
autoclave, a sterilizing apparatus. Therefore, the development of techniques, using
chemicals or plant essential oils or in combinations for eradicating microorganisms
causing agents of contamination, to replace the autoclaving method for establishing
sterile culture medium will be the best procedure for in vitro culture. The use of
disinfectants, fungicides and bactericides such as chlorine, sodium hypochlorite, calcium
hypochlorite. The report showed the efficiency of plant essential oils in combinations of
disinfectants as sterilising agents to prevent and eliminate microorganisms in MS
medium to obtain sterile condition without autoclaving. Further experiments are needed
to establish appropriate concentrations of single essential oil or various combinations as
sterilizing agents in MS medium used for plant tissue culture

4. Radiation Sterilization
Many types of radiation are used for sterilization like electromagnetic radiation
(e.g. gamma rays and UV light), particulate radiation (e.g. accelerated electrons).The
major target for these radiation is microbial DNA. Gamma rays and electrons cause
ionization and free radical production while UV light causes excitation. Radiation
sterilization with high energy gamma rays or accelerated electrons has proven to be a
useful method for the industrial sterilization of heat sensitive products. But some
undesirable changes occur in irradiated products, an example is aqueous solution where
radiolysis of water occurs.
5. Filtration Sterilization
 Filtration process does not destroy but removes the microorganisms. It is used for
both the clarification and sterilization of liquids and gases as it is capable of preventing
the passage of both viable and non viable particles. The major mechanisms of filtration
are sieving, adsorption and trapping within the matrix of the filter material. Sterilizing
grade filters are used in the treatment of heat sensitive injections and ophthalmic
solutions, biological products and air and other gases for supply to aseptic areas. They are
also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves
and freeze driers. Membrane filters are used for sterility testing.

INOCULATION

Based on Baday (2018), replanting or the so-called subculture is one of the stages
of the method in tissue culture, which is a technique that is carried out between stages of
culture. Subculture or overplanting is the transfer of a very small planlet (young plantlet)
from the old medium into a new medium that is carried out aseptically in the file or
Laminar Air Flow (LAF).
obtain nutrients or nutrients for its growth (Basically the subculture is a relatively
easy stage of activity compared to other activities in tissue culture. Subculture is carried
out for several reasons:
a.Plants already meet or are as tall as a bottle
b.Plants have been in the bottle for a long time so that growth is reduced
c.Plants begin to lack nutrients
d.The media in the bottle is drying
Subculture activities are carried out according to the type of plant being cultured.
Each plant has different characteristics and speed of growth. So the method and time of
subculture also varies. Plants that must be subcultured or relatively quickly are banana-
pisangan, allocasia, and caladium. For plants that are propagated by seed culture, embryo
culture, both somatic and microsporal embryos, as well as multi-shoot budding, then
subcultures can be carried out by separating plant saplings from their colonies or
thinning. Examples of plants are orchids, bananas, and other plants that are one type of
growth. For plants whose growth type is by elongation of stems, subculture can be done
by cutting down existing plants. But if there is a plantlet that is still too small and high
risk to be cut, then the subculture is sufficient to do it by separating it from its parent and
replanting it separately. Examples of plants are teak, chrysanthemum, and other plants
that have the same growth characteristics. we can calculate the speed of crop production
by knowing the speed of the plants to multiply until they are ready to be sub-culture.
Sub culture activities must be carried out on explants caused by several things,
including: The growth of explants is quite fast and has filled the entire culture bottle, The
growing media has dried which is marked by the reduction in the volume of jelly or the
liquid media has run out, Explants need to be further propagated for the purpose of the
next stage of propagation, Eksplan requires a new arrangement of media in order to
experience further differentiation, Eksplan or callus that is time to be transferred into the
new culture media must be carried out immediately and should not be too late. Sub-
culture that is late can cause the growth of explants or callus will stop or experience
browning or even contamination by fungus or bacteria. Such an explant is unlikely to be
saved because fungal or bacterial spores can spread very quickly.
SUBCULTURE

In Biology, a subculture is a new cell or microbiological culture made by transferring

some or all cells from a previous culture to fresh growth medium. This action is called sub-
culturing or passaging the cells. Sub-culture is used to prolong the life and/or expand the number
of cells or microorganisms in the culture. In plant tissue culture, Sub-culture is process in which
the plant tissue or explants is first subdivided and transferred into fresh culture medium.

According to Talati (2017), Subculture is the process by which the tissue or explant is
first subdivide, and then transferred into fresh culture medium. It is from a culture of a certain
volume into fresh growth medium of equal volume. This allows long-term maintenance of the
cell line.

One of the example of tissue culture is in vitro subculture. According Vujovic (2012)
Generally, subculture effect on multiplication rate of in vitro cultures varies from one species to
another. A decrease in multiplication potential during long-term growth and repeated
subculturing of shoots on medium of constant hormonal composition was reported in six
ornamental species and cultivars of Rosaceae. Given that plant propagation by tissue culture is
usually aimed at high multiplication rate and its maintenance during in vitro growth, the
objective of this study was to assess the effect of repeated subculturing on shoot multiplication of
different fruit rootstocks grown on media of constant hormonal composition. A shoot
multiplication protocol could have a large impact on our ability to rapidly multiply in vitro
desirable fruit rootstocks and ensure plant availability throughout the year accordingly. In this
study, the decline in shoot multiplication rate was observed over the repeated subcultures, which
implies necessity for further investigation in order to find the proper method of restoring
proliferation capacity of in vitro shoots and/or delay the decline in rate for several subcultures.
Given that the decline in multiplication index was already observed after the second subculture,
it is neccesary to determine if and when cytokinin concentration should be reduced, or if
hormon-free medium can be empoyed to delay the decrease.
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