PAPER

ORGAN CULTURE

Submitted as one of the requirements for passing the course

Plant Tissue Culture

By :

Group 2

Fadhillah Rahma Purba (4193342004)

Mamanda Silitonga (4193342002)

BESP 2019

BIOLOGY EDUCATION STUDY PROGRAM

FACULTY OF MATH AND SCIENCE

2022
 FOREWORD

Praise be to Allah SWT who has provided smoothness in the preparation of this paper so that this
paper can be completed. The author would also like to thank all those who have helped in the
preparation of this paper and the various sources that we have used as data and facts in this
paper.

The author admits that the author is a human being who has limitations in many ways. Therefore
nothing can be solved perfectly. Similarly, this paper has been completed. Not everything can be
described perfectly in this paper. The author does it as much as possible with the ability that the
author has. Therefore, the author is willing to accept criticism and suggestions. All criticism and
suggestions will be accepted by the author as a stepping stone that can improve the author's paper
in the future.

So that the next paper can be completed with better results.

Medan, March 2022

Writer
PRELIMINARY
Plant tissue culture is a technique to grow cells, tissues or slices of plant organs in the
laboratory on an artificial medium that contains aseptic (sterile) nutrients to become a whole
plant. Sterile conditions are an absolute condition for the successful implementation of tissue
culture, so this condition must be maintained during the culture process. Even if only one fungal
spores or only one bacterial cell enters the culture medium, the culture work will fail and no new
plants will be produced. Plant tissue culture is based on the theory of cellular totipotency which
states that each plant cell has the capacity to regenerate to form a whole plant. New plants
obtained in this way are identical to their parents, and are called plantlets. The number of new
plants produced is not only one, but can be tens to hundreds (from one planting material or
explant) so that tissue culture techniques are used as a method of plant propagation. The plant
propagation method carried out by tissue culture techniques is classified as vegetative
propagation, meaning that it does not involve fertilization between eggs and male sex cells as
well as the formation of seeds in plants, that is why the resulting plantlet is identical to the
parent. Plant propagation by tissue culture techniques is also called micropropagation or micro-
propagation. The word 'micro' refers to the initial planting material used, namely explants that
are small (micro = small), can even reach ≤ 1 mm in meristem cultures.
Tissue culture is used for several purposes, including:
1. Genetic Transformation/Genetic engineering
Genetic transformation/genetic engineering requires tissue culture techniques if gene
transfer is done in vitro. The technology of producing transgenic plants in vitro absolutely
requires tissue culture. Growing transformation targets in the form of 'callus', 'protocorm
like bodies' and 'in vitro buds' to become transgenic plantlet candidates, requires tissue
culture expertise.
2. Multiplying GM (Genetically Modifi ed)
Plants or what is known as transgenic plants. Transgenic plants have agronomic
characteristics that are specifi k according to the gene of interest inserted. Propagation of
this plant must be carried out vegetatively so that the resulting saplings are genetically
identical to the mother.
Propagation of hybrid plants that have superior properties

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 Hybrid plants are the result of a cross between two plants that each carry a specifi
character, so that the character it has is a combination or combination that comes from
two elders. Hybrid plants must be propagated vegetatively to maintain the superior
properties they possess.
3. Make it easier to ship plants in sterile containers
Long-distance plant delivery can be made easier if the plants sent are relatively small in
size and pathogen-free
4. Propagate plants whose seeds are difficult to germinate (Orchids, Nepenthes)
Orchids and Nepenthes plants are types of plants that have seeds that are difficult to
germinate under normal conditions in soil media as are generally other types of
angiosperms (seed plants)
5. Produce virus-free plants from meristem cultures
A meristem is a part of a plant whose cells are meristematic (actively dividing). The
meristem is located at the end of the shoot (apical as well as axillary) and the tip of the
root. The meristem culture that produces the first virus-free plants was introduced by
George Morrell in the 1960s.
6. Protoplas Fusion
The purpose of protoplast fusion is to cross (crossing) plants carried out in vitro.
7. Embryo Rescue
Some plant species do not develop after fertilization. For cases like this, embryo culture
is carried out. Embryo rescue through embryo culture is called embryo rescue.
8. Produce double haploid plants through microspores culture
The production of homozygous double haploid plants through microspores cultures or
anther cultures has a very important meaning for the field of plant breeding because this
method can shorten the time required by conventional plant breeders.

There are several types of tissue culture as follows:

1. Meristem cultures (meristem cultures) Meristems are parts of plants whose cells are
meristematic and actively dividing. On the plant body the position of the meristem is at
the tip of the shoot (apical and axillary shoots) which functions to increase the length of
the shoot, at the tip of the root it functions to increase the length of the root and at the
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 stem cambium which causes an increase in the diameter of the plant. In graminae (grass)
tribes, there are special meristems called 'intercalary meristems' whose position is in the
stem nodes (nodes) which causes an increase in the length of the stem internodes. In
tissue culture, the meristem commonly used as explant material is the shoot tip meristem
(apical and axillary). Meristem culture using very small explant material, measuring 1
mm.
2. Culture of shoot tip (shoot-tip cultures) Culture of shoot tip using explants of apical buds
or axillary buds, measuring 3-20 mm, includes some leaf primordia and vascular tissue.
Explants of axillary buds can be in the form of 'nodal segments' (slices of books) because
in the slices of books (the books are the places where the leaves grow) there are axillary
buds. Explants were planted on shoot induction media (media containing cytokinins) to
produce 'pre-existing' shoots in the explants.
3. Embryo cultures What is meant by embryo culture is culturing zygotic embryos in vitro.
Zygotic embryo is the result of fertilization between an egg cell and a sperm cell nucleus
that occurs in the double fertilization process of angiosperms.
4. Protoplast cultures and fusion (Protoplast cultures and fusion) The term protoplast refers
to plant cells without a cell wall or cell contents that are wrapped only by a plasma
membrane. The protoplast of a cell can be separated from its cell wall enzymatically or
mechanically. Protoplasts that have been separated from their cell walls can be
regenerated into whole plants.
5. Microspore cultures Microspores are male sex cells (gametes) in angiosperms and can be
found in flower buds of plants. Microspores can be said to be immature pollen (pollen
that has not yet matured physiologically). Naturally, microspores will develop into pollen
or pollen. This pollen will later develop into sperm nucleus 1 and sperm nucleus 2 in
double pollination of angiosperm plants. However, in microspore culture, microspores
are deflected in the direction of their development into embryos, not pollen.
6. Callus Cultures and Suspension Cultures Callus is a collection of undifferentiated cells.
Callus forms on scars or incisions in plant organs. In vitro callus will form on the incision
/ wound of the organ being cultured, but in some plant species, callus can form on the
interior (interior). In suspension culture, the formed callus will be taken and cultured in
liquid media to form a liquid culture or suspension culture. Callus crumbs easily

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 separated to form a cell culture. Cell culture is performed with agitation or a shaker for
oxygen supply. In plant propagation through in vitro culture, cell culture (through callus)
is used in indirect embryogenesis, but several studies have shown that tillers produced by
cell culture are genetically unstable, so this method is rarely used.

7. Seed Cultures Seed culture is carried out for seeds of plants that cannot be germinated ex
vitro or if they can germinate ex vitro, the percentage of germination is very low. This is
because the seeds are very small and have little or no endosperm (food reserves).

Organ culture is divided into 2, namely shoot culture and root culture.

1. Root Culture

Root culture can be defined as radical tip culture cut from aseptically germinated seeds in a
liquid medium in which the seeds are induced to grow independently under controlled
conditions.

Principles of root culture : Root culture can be successfully initiated from the tip of the radicle
cut from aseptically germinated seed. Whole in vivo plants are not suitable for isolation of intact
root tips because plant roots are buried deep in the soil. Again, the root tips of young seedlings
are very sensitive to toxic sterilants. So it is better to avoid sterilizing the tip surface of young
roots for the formation of root cultures.

Root tip cultures are generally maintained in a mobile liquid medium. In culture, the root tips are
induced to grow like the root system of a whole plant. Clones from excised roots can also be
made from single root cultures by repeatedly cutting and transferring the main root tips or lateral
ends to fresh media in each subculture at specific time intervals. The growth of the cut roots can
be expressed in terms of fresh and dry weight, increase in main axis length, number of laterals
raised and total lateral length per culture.

Clone Initiation:
Root material originating from one root tip can be duplicated and maintained in continuous
culture. Genetically uniform root cultures are referred to as clones of isolated roots.

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Importance of Root Culture in Relation to Basic Information:

(1) Root culture has increased our knowledge of carbohydrate metabolism, the role of mineral
ions, vitamins, etc. in root growth.

(2) Root culture has provided basic information regarding the dependence of roots on shoots for
growth hormones.

(3) Root clones are suitable for studying the effect of various compounds on root growth.

2. Shoot culture/ Shoot tip culture

Shoot culture is for vegetative propagation of plants. Ynag is the in vitro propagation of plants in
one of the micropropagation techniques carried out by culturing explants containing shoot
meristems. Shoot tip culture using explants of apical buds or axillary buds, measuring 3-20 mm,
includes several leaf primordia and vascular tissue. Explants of axillary buds can be in the form
of 'nodal segments' (slices of books) because in the slices of books (the books are the places
where the leaves grow) there are axillary buds. Explants were planted on shoot induction media
(media containing cytokinins) to produce 'pre-existing' shoots in the explants. Many shoots can
appear (shoot multiplication), but it can also produce only one shoot from one explant. If it
produces a lot of shoots, the shoots are subcultured into the media. The effect of apical
dominance can be removed by using PGR, especially cytokinins into the medium, which will
produce a large number of shoots. Shoot size affects the success of this culture, larger shoots
develop faster and are more resistant so that more axillary shoots are produced. Robbins in 1922
was the first to perform shoot culture, after which development was slow. It was only in the
1970s that many research results using this technique were published.

In shoot culture, medium with high cytokinin concentration is widely used to encourage axillary
shoot formation. The bud culture technique is more profitable to apply if it has the aim of
eliminating genetic changes, but the multiplication rate is lower than shoot culture. For large-
scale potato propagation in order to obtain fusarium-free shellfish, this culture has been applied
in PAU IPB (Wattimena, 2000).

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Plant propagation through shoot culture has been widely carried out in commercial tissue culture
laboratories. The factors that led to the use of shoot culture techniques for commercial purposes
are:

1. The shoot culture method can be applied to many types of plants with the same principle.
2. It is possible to obtain control of the resulting shoots free of viruses.
3. Plants produced are genetically uniform and according to their type
4. In many plants, have a high rate of propagation

Why Epical Meristem is free from viruses?

Apical meristems in the infected plants are generally either free or carry a very low concentration
of the virrues

The various reasons attributted to the escape of the meristems by virus invasion are:

1. Virrues move readily in a plant body throught the vascular system whish is meristem is
absent
2. A high metabolic activity in the actively dividing meristematic cells does not allow virus
replication
3. A high endogenous auxin level in shoot apices may inhibit virus multiplication

There are several stages of Culture of shoot tip culture according to Murashige:

Stage 1 is the stage of culture formation when explants can develop into single shoots or double
shoots. At this stage the explants were supplemented with cytokinins such as BA, kinetin and
2iP.

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In stage 2 the aim is to multiply the propagules and for this followed the proliferation of axillary
buds as it maintains higher genetic stability.

Stage 3 aims to regenerate adventitious roots from shoots obtained in stage 2. A number of
studies have shown that NAA followed by IBA, IAA, 2,4-D and other auxins are used to induce
root formation.

The application of using shoot culture is as follows:

 Large scale production of Orchids

 Production of pathogen indexed plants
 Long term storage of germsplasm
 Micropropagation
 Generation of trancenic plants
 Quarantine

3. FACTORS AFFECTING ORGAN CULTURE

1. SOURCE OF MATERIALS

The source of the material must be chosen properly. Preferably, successful in vitro cultures have
been observed using vascular cambium, storage organs, root pericycle, endosperm, cotyledons,
leaf mesophyll, and provascular tissue. Also, the choice of explant depends on the purpose of the
culture. So it is better to choose the best explant before the culture is performed.

2. SOURCE MATERIAL STERILIZATION

Surface sterilization of explants is an important step that must be considered. Explants collected
from external sources may be filled with spores or other microbial cells. And, if the explants are
not properly sterilized prior to culture, it will cause contamination in all cultures and lead to
severe culture loss. Also, if any tissue is damaged or dead, clean it thoroughly before culturing
the explants on the culture medium.

3. EXPLANT PREPARATION

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The size and shape of the explant are important factors in organ culture. If the size of the explant
is not optimal, it can cause callus induction failure and loss of culture. Cultivators generally
prefer the use of large explants to obtain viable cultures.

4. Culture Media

The type and composition of the culture media are some of the important factors that affect
organ culture. Culture is more influenced by the level of plant hormones—both endogenous and
exogenous—in the medium. Callus or organ cultures are generally grown on solid media but
sometimes liquid media are also preferred. sometimes using liquid media refers to a condition
when plants cannot grow in solid media. This can occur due to uneven distribution of nutrients,
improper gas exchange, and accumulation of toxic waste products that can develop between the
callus and the media.

5. SUBCULTURING

The culture should be transferred to fresh media after some time. Storing cultures on one
medium for a longer time leads to waste buildup, depletion of nutrients from the media, and
drying of the media. Thus, it is recommended that cultures should be subcultured every 4-6
weeks while incubating at 25°C.

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 CONCLUSION

Plant nurseries are an effort in providing plant seeds where there is a production process.
Nurseries need seeds that have superior varieties, uniform, free of pathogens, free of pests.
Tissue Culture is one of the techniques used to produce seeds in large quantities, relative time
and similar to the parent. The use of shoot tip culture method is divided into 2, namely shoot
culture and root culture. Shoot culture is for vegetative propagation in vitro, one of the
micropropagation techniques is culturing explants containing shoot meristems. Shoot tip culture
using explants of apical buds or axillary buds, measuring 3-20 mm, includes several leaf
primordia and vascular tissue. Whereas root culture is a radical tip culture cut from aseptically
germinated seeds in a liquid medium in which the seeds are induced to grow independently
under controlled conditions. Root culture can be successfully initiated from the tip of the radicle
cut from aseptically germinated seed. Root tip cultures are generally maintained in a mobile
liquid medium. In culture, the root tips are induced to grow like the root system of a whole plant.
Clones from excised roots can also be made from single root cultures by repeatedly cutting and
transferring the main root tips or lateral ends to fresh media in each subculture at specific time
intervals. The growth of the cut roots can be expressed in terms of fresh and dry weight, increase
in main axis length, number of laterals raised and total lateral length per culture.

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 BIBLIOGRAPHY

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