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Tissue Culture Practices in Horticulture

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 Tissue Culture Practices in Horticulture
Article ID: 32263
Udit Joshi , D.K. Rana2, Pawan Singh Rana3, Ajay Dhyani4
1

Department of Horticulture, H.N.B. Garhwal University Srinagar (Garhwal) Uttarakhand1,2,3, and Department
of Floriculture and Landscaping, G.B.P.U.A&T Pantnagar Uttarakhand4.

Background
Technological revolutions have changed m our lives in every field of life from livelihood, communications, and
food habits. An important filed of life which has not seen technological revolutions to the extent as that of
searchers, Agriculturists,
Horticulturists, and Farmers have come up with innovative ways and ideas to bring some phenomenal changes
in the field of agriculture but even there were some massive changes as that of the reputed Green Revolution,
it has not been simple enough to meet the increasing demand of the growi
the edge of the agriculture revolution that was never witnessed before in the decades. The beginning of
agriculture technologies with the advancement in genetic research and other biotechnological programs has
bought new opportunities to mankind. Many of the uncontrolled problems have come to the human reach.
Biotechnology is the broad area of biology that helps to manipulate the living cells and their molecules and that
can be used to improve the crop production and other abiotic and biotic resistance of crops. The major
techniques used in biotechnology are Tissue culture (one of the most widely used techniques) PCR (polymerase
chain reaction), gel electrophoresis, cell culture, protein engineering, etc.

Introduction
Plant Tissue Culture, Cell Culture, or Micropropagation is a collection of biotechnological techniques used to
grow the plant cells in the sterilized conditions to get the desirable traits or qualities in a short period. Nowadays
it is the widely used method to produce clones through micropropagation in the controlled sterilized conditions.
A single specimen plant is capable of producing the entire crop population. The parts taken from the mother
plant is capable of producing a quick mature plant. The method greatly reduces the chances of transmitting
disease and other pathogens. The explants can be taken from the shot tip, leaf, stem, flower bud, lateral bud,
root tissues, etc. the technique relies on the ability that many plant cells can regenerate into the whole plant.
This ability of plants is known as totipotency. In -vitro conditions are provided to get the optimum pre-requisite
plant growth. The regeneration ability of the ex-plants helps it to initiate multiply and root thus producing a cell
into a mature plant. The prepared plantlets are later on hardened in the climatically controlled conditions that
are in greenhouse or polyhouse etc. depending upon the type of plant species become ready for planting in the
varying time frame. This technique of plant propagation is easy and reliable also reduces the labour requirement
and the time-space. The new varieties used by this method can help to increase production rates.
Scientists have been experimenting to make evolution in the micropropagation technological program. The
experiments have been conducted by scientists in various horticultural crops such as coconut, palms. Mango,
orange, etc. tissue culture has also bought a scope to propagate fine varieties of flowers and other fruit trees.
The plants that are sterile or do not propagate easily can be multiplied by tissue culture techniques.
Commercialization by this method has already taken place. In crops such as Ornamental crops Orchids,
Gladiolus, Gerbera, Carnation, etc. have been grown commercially. And in the case of fruit crops such as
Bananas are propagated by tissue culture method. Eucalyptus and teak among forest trees are also grown
commercially by the tissue culture method. The technological advancement has also reached to the medicinal
plants. Biotechnology is the area with huge potential and is expanded to the diverse sciences of health and
agriculture and has tremendous potential for food, fibre medicine, etc.

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Micropropagation
Micropropagation is the method of producing numerous progeny plants from a single plant. The single cell of
the plant by providing the optimum conditions can be grown into the full-fledged plant. The whole process
takes place with many metabolic events and facilitates the production of a new plant from a single cell of the
old plant. The events that take place during the whole process are:
Differentiation: In biological terms, differentiation can be used in various senses. The differentiation can be
defined as the process in which a distinct type of cells arises from the precursor cell and mature or become
different from one another. The meristematic cells get converted into more types of cells, tissues, and organs
are later differentiated from each other to form a new plant.
De-differentiation: It refers to the transformation of cells into the highly organized tissue or the unorganized
tissues into the organized tissues.
Re-differentiation: It is the process of differentiation occurring in the undifferentiated tissues.
Regeneration: The plants can regenerate or can form into the entire new plant from the single callus culture
known as regeneration. The plant part that is removed physiologically or is isolated has this ability to regenerate.
It is the genesis of the plant to develop from the single callus culture is called regeneration.

Morphogenesis: It is defined as the biological process that causes the organization of cells, tissue, or organism
to develop its shape or form is referred to as morphogenesis. The morphogenesis under in vitro environment
can be achieved by two routes: de novo origin of the organ primordia is differentiated to form the shoots or
roots from the cultured tissue is termed as organogenesis. and de novo and formation of the embryos with the
distinct roots and shoot poles lie on the opposite ends formed by somatic cells cultured in vitro known as
somatic embryogenesis. The seed coat or endosperm is not formed in somatic embryogenesis.
Organogenesis: It is defined as the means of development or formation of adventitious organs or primordia
from the undifferentiated mass of cells. Organs in plants such as root, shoot, leave flowers, etc. The first report
of induction in shoot organogenesis in vitro was by White (1939) utilizing a tobacco hybrid; and the first root
formation in carrot callus was observed by Nobecourt (1939). The regulatory mechanism behind the science of
organogenesis was not identified until the late 1950s. Skoog and Miller (1957) experimented on the chemical
that controls the new shoot formation in the pieces of tobacco stems grown in culture. They recognized the
regulatory mechanism of the balance between auxin and cytokinin. In their research, they concluded that a high
level of auxin with as compared to cytokinin favored the root formation whereas the reverse favoured the shoot
formation in plants. Utilizing this concept, it has now become easy to achieve organogenesis in a large number
of plant species by culturing the explants, calli, and cell suspension in the definite medium. In organogenesis,
the shoot or root may form first reliant upon the nature of growth hormones in the basal medium. The start of
shoot and root from the explants or callii is termed as caulogenesis (caulm = stem) and rhizogenesis (Rhizo =
root) respectively.

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Type of Culture
Embryo culture Seed culture Meristem culture

Cell culture Plant tissue culture Protoplast culture

Callus culture Bud culture Organ culture

Fig.1 Various types of plant tissue cultures

Callus Culture
A callus is an undifferentiated mass of mainly unorganized plant cells. The callus culture is composed of
parenchymatous cells that undergo division. When the media of explants is cultured with a sufficient number
of auxins it results in a culture that pro
with the type of explant. It mainly depends on the physiological state of the explant tissues. Callus can be
maintained for a very long time by alternating sub-culturing to a new medium. Callus cultures can be used for
different purposes by changing the hormone concentrations in the media be it for the regeneration of plantlets,
preparation of single cells or suspension cultures, or for protoplasts preparation and genetic transformation
studies. In various cases, it becomes a compulsion to go through a callus phase before regeneration via somatic
embryogenesis or organogenesis. Cultures are also suitable for somaclonal variants generation (genetic or
epigenetic) and can also be used in the case of in vitro selection of cells and tissue variants. The callus is grown
after that on a new medium which is known as subculturing. An S-shaped or sigmoid pattern of growth of callus
culture is obtained when subculture regularly on agar medium.
There are five phases of callus growth:
1. Preparation of cell division (Lag Phase).
2. Highest cell division stage (Exponential Phase).
3. The rate of cell division gets slow but the rate of cell expansion gets increased (Linear phase).
4. A decrease in rates of cell division and elongation (Deceleration phase).
5. Constant number and size of cells (Stationary phase).
The growth of the callus can be monitored by various fresh weight measurements, which are more convenient
while observing the growth of cultures over time in a non-destructive manner. Generally, weight measurements
based on dry weight give more accuracy than fresh weight measurements, but at the same time, this method
requires the sacrifice of various sets of samples. Measurement-based on the mitotic index of cell division rates
requires an extensive sampling for reducing error in sampling and at the same time are not feasible to perform.

References
1. Amasino, R. (2005). 1955: Kinetin Arrives. The 50th Anniversary of a New Plant Hormone. Plant physiology, 138(3), 1177 1184.
2. Chawla, H. S. (2009). Introduction to Plant Biotechnology. Oxford & IBH Publishing Company Pvt. Ltd. (India). Pp:33-43.
3. Prasad, S. (2004). Impact of Plant Biotechnology on Horticulture. Agrobios (India). Pp:22-27.
4. Skoog, F., Miller, C. O. (1957). Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symp. Soc. Exp. Biol. XI: 118
13.

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