LABORATORY WRITTEN REPORT

Activity No. 2
Title: Basics of Plant Tissue Culture

Name: Abdul, Fatima Khadija O. Subject: SSED13 – Cell and Molecular Biology
Course & Year: BSED III – Sciences

I. Rationale
In the field of Cell and Molecular biology, plant tissue culture is significant
because this technique is use in the application of cell behavior, plant modification
and improvement, pathogen-free plants and germplasm storage and etc. In addition,
according to Thorpe (2007), Cell cultures have remained an important tool in the
student of basic areas of plant biology and biochemistry and have assumed major
significance in the studies in molecular biology as well as agricultural biotechnology.
Plant tissue culture is a method by which plant cell can be cultured artificially
or propagated (under sterile conditions) in order to grow plants or plant organs. It is
referred as an aseptic culture of cells, tissues, organs and their components under vitro
in a detailed physical and chemical conditions. It helps in yielding and in the
propagation of genetically homogeneous, disease-free plant material. In addition,
there are 6 types of Plant Tissue Culture – seed and seedling culture, embryo culture,
organ culture, callus culture, cell suspension culture, and the protoplast culture. In
contrary, one of the applications of Plant tissue culture is widely used in forestry and
floriculture. Micropropagation can also be used to conserve or endangered plant
species. Moreover, according to BiologyDiscusion.com, Tissue culture is sometimes
referred to as ‘sterile culture’ or ‘in vitro’ culture. By this technique living cells can be
maintained outside the body of the organism for a considerable period.
Thus, in order for the different biological fields, studies and researchers to be
feasible, plant tissue culture is needed. This laboratory experiment entitled, “Basic
Plant Tissue Culture” stretches its topic on the brief history of development of in vitro
techniques of the plant cell culture, different requirements for establishing tissue
culture laboratory, the application and uses of root culture and the likes.
 II. Objectives:
At the end of this laboratory report, students should be able to
1. Illustrate the different requirements in establishing tissue culture in the
laboratory;
2. Explain the advantages of callus culturing in biology
III.Answers to Questions:
1) Write brief history of development of in vitro techniques of the plant cell culture.
(As stated by BiologyDiscussion.com …)

Year Historical Events

1898 Gottlieb Haberlandt first attempted to culture plant tissues ‘in vitro’. He used cells
from palisade tissues of leaves, cells from pith, epidermis and epidermal hairs of
various plants for culture in media -containing Knop’s solution, aspergine, peptone
and sucrose.
1901 A cell which can develop into a whole organism by regeneration is called a to tipotent
cell which was coined by Morgan.
1922 Robin, together with Maneval cultured roots and maintained the culture for 20 weeks
by sub culturing. Gautheret later used medium supplemented with B-vitamins and
IAA based from his previous discoveries
1937 White recognized the importance of B-vitamins for growth of root cultures. Nobecourt
obtained some growth in culture of carrot root explants. He also noted root
differentiation in tissue culture.
1939 White, Nobecourt, and Gautheret cultured successfully a plant callus tissue which can
be generated artificially in vitro. Gautheret said that carrot culture required Knop’s
solution supplemented with Bertholots’ salt mixture, glucose, gelatine, cysteine HC1
and IAA.
1941 Van Oberbeck cultured embryos of Datura on a medium supplemented with coconut
milk. Importance of coconut milk and 2-4D as nutrient was recognized. The
stimulatory property of coconut milk is due to the presence of zeatin.
1954 Studies on cell suspension culture were carried out by Muir, Hildebrandt and Riker.
1953 Muir developed paper raft nurse technique for single cell culture.
1960 Cocking recorded release of protoplasts from root tip cells by using fungal cellulase in
0.6M sucrose. He was able to culture isolated protoplasts, which regenerate new cell
walls and produce cell colonies and ultimately plantlets.
1974, A method of microspore culture of Nicotiana and Datura was developed by Nitsch. He
1977 was able to double the chromosome number and obtained homozygous diploid plants.
Late 70’s It was evident that plant tissue culture technique can be successfully used in various
field of agriculture, such as, production of pathogen free culture, production of
secondary products, clonal propagation, mutant culture, haploid breeding and genetic
engineering.

2) What are the requirements for establishing tissue culture laboratory?

 a) Aseptic conditions
The tissue culture laboratory should be well sterilized against
pathogens. It will help maintain good health of the callus, cell or protoplast
cultures resulting in recovery of healthy plants from such cultures. In
addition, the explant and glassware should be properly sterilized before
their entry into the tissue culture laboratory.
b) Control of temperature
Temperatures in the tissue culture laboratory varies from the types of
calluses which varies from species to species. However, high temperature
adversely affects the growth of the callus. Thus, air conditioning is
essential.
c) Proper culture media,
The medium has to be modified depending on the requirement of
particular species.
d) Sub-culturing
It is the process of Transferring of tissue or callus from old culture
media to fresh culture media. It is an integral part of this process in order
to maintain good health of the callus or tissues to avoid depletion of
nutrients in the culture media.

3) Define explants. How will you induce callus from it?

It is the small piece of plant tissue that is used for tissue culturing which are
cultured and regenerated in a nutrient medium under a sterile conditions. Explants
may be a cell, a protoplast, a tissue, or an organ. In contrary, plants naturally
develops callus or tumors, an unorganized cell masses which is due to their
response to various biotic or abiotic stimuli and based from the historical
discoveries, the combination of two-growth-promoting hormones, auxin and
cytokinin, induces callus from plant explants in vitro.

4) Why the calluses need dark condition for the development?

Callus culture is usually done in the dark to avoid differentiation of the callus.

5) Write some advantages of callus culturing?

 Some of the advantages of using callus culturing are:
 Cell suspension culture in moving liquid medium can be initiated from callus
culture;
 It could be a source of chromosomal variations (Somaclona Variation),
secondary Metabolites , tissue for cell Suspension culture, and tissue for plant
regeneration;
 Helps in production of secondary plant products
 Starting materials for vegetative propagation of plants.

6) What are the application and uses of root culture?

According to Saupe in his work, ‘Plant Organ Culture – Culturing Excised
Tomato Roots’, root cultures can be used in many ways including studies of
carbohydrate metabolism, mineral nutrient requirements, essentiality of vitamins
and other growth regulators, differentiation of the root apex and gravitropism.

IV. Conclusion
In the process of understanding this laboratory activity, the following
objectives; illustrate the different requirements in establishing tissue culture in the
laboratory and explain the advantages of callus culturing in biology were obtained.
In establishing a tissue culture there are various requirements needed in order
for it to be feasible. These are aseptic conditions in the laboratory to help maintain
good and stable life of the callus; control of temperature to avoid differentiation of the
callus; usage of proper culture media depending on the requirements of the callus; and
sub-culturing of the callus or tissue to prevent nutrient depletion. In contrary, the
advantages of callus culturing in biology could be seen in the number of studies it is
used. In addition according to Saupe, advantages of using callus culturing is that they
grow rapidly, are relatively easy to prepare and maintain, show a low level of
variability and can be easily cloned to produce a large supply of experimental tissue.
References:

Animated Biology With Arpan (2020) Plant Tissue Culture Overview. Retrieved from
https://www.youtube.com/watch?v=dFrx-t5J0PA&feature=share
Apsnet.org. (n.d) Plant Tissue Culture. Retrieved from
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Arora, S. (n.d) Project Report on Plant Tissue Culture. Retrieved from
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Bolthajira, M. (2014) Callus Culture ppt. retrieve from
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%20tissue%20culture%20has%20been,the%20leaves
Saupe, S. G. (n.d) Plant Organ Culture – Culturing Excised Tomato Roots. Retrieved from
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%20can%20be%20used,the%20root%20apex%20and%20gravitropism.
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