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Plant Tissue culture

Plant Tissue culture

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The term ‗Plant Tissue Culture‘ broadly refers to the in vitro cultivation of plants, seeds, plant parts etc. on nutrient media under aseptic conditions. Haberlandt (1854-1945) attempted to cultivate plant tissue culture cells in vitro. He is regarded as the father of plant tissue culture.
The term ‗Plant Tissue Culture‘ broadly refers to the in vitro cultivation of plants, seeds, plant parts etc. on nutrient media under aseptic conditions. Haberlandt (1854-1945) attempted to cultivate plant tissue culture cells in vitro. He is regarded as the father of plant tissue culture.

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Published by: Shree Krishna Adhikari on Mar 05, 2013
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Biotechnology
Plant tissue culture
5
 
Prepared By: Shree Krishna Adhikari, Bsc.Ag 7 
th
semester. IAAS, Rampur Campus
2012
 
B. PLANT TISSUE CULTURE:
The term ‗Plant Tissue Culture‘ broadly refers to the
in vitro
cultivation of plants, seeds, plant partsetc. on nutrient media under aseptic conditions.Haberlandt (1854-1945) attempted to cultivate plant tissue culture cells
in vitro
. He is regarded as thefather of plant tissue culture.
Chapter-1
PLASTICITY AND TOTIPOTENCY
Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration.Plants, due to their sessile nature and long life span, have developed a greater ability to endure extremeconditions and predation than have animals. Many of the processes involved in plant growth anddevelopment adapt to environmental conditions. This plasticity (
ability of the plants to sustain adverseenvironment with their metabolism, growth and development 
) allows plants to alter their metabolism,growth, and development to best suit their environment. Particularly important aspects of this adaptation,as far as plant tissue culture and regeneration are concerned, are the abilities to initiate cell division fromalmost any tissue of the plant and to regenerate lost organs or undergo different developmental pathwaysin response to particular stimuli. When plant cells and tissues are cultured
in vitro
they generally exhibit avery high degree of plasticity, which allows one type of tissue or organ to be initiated from another type.In this way, whole plants can be subsequently regenerated. This regeneration of whole organisms dependsupon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of theparent plant. This maintenance of genetic potential is called
totipotency
.Cultured tissue must contain competent cells or cells capable of regaining competence (dedifferentiation).
e.g. an excised piece of differentiated tissue or organ (Explant) → dedifferentiation → callus(heterogenous) → redifferentiation (whole plant) = cellular totipotency.
 Plant cell culture and regeneration do, in fact, provide the most compelling evidence for totipotency. Inpractical terms though, identifying the culture conditions and stimuli required to manifest this totipotencycan be extremely difficult and it is still a largely empirical process.1957 Skoog and Miller demonstrate
d that two hormones affect explants‘ differentiation:
 
 – 
 
Auxin: Stimulates root development
 – 
 
Cytokinin: Stimulates shoot development
• Generally, the ratio of these two hormones can determine plant development:
 
 – 
 
↑ Auxin ↓Cytokinin = Root development
 
 – 
 
↑ Cytokinin ↓Auxin = Shoot development
 
 
BIO-TECHNOLOGY 
Prepared By: Shree Krishna Adhikari, Bsc.Ag 7 
th
semester. IAAS, Rampur Campus
2012
 – 
 
Auxin = Cytokinin = Callus development
Chapter- 2
STEPS IN GENERAL TISSUE CULTURE TECHNIQUES
 – 
 APPLICATION OF PLANT TISSUE CULTURE IN CROPIMPROVEMENT
Plant tissue culture is the aseptic method of growing cells and organs such as meristems, leaves, roots etceither in solid or liquid medium under controlled condition. In this technique small pieces of viabletissues called ex-plant are isolated from parent plants and grown in a defined nutritional medium andmaintained in controlled environment for prolonged period under aseptic condition. The generaltechnique of plant tissue culture involves four main stages. They are:-
 
Initiation of culture
 
Multiplication (or) sub culture
 
Development and differentiation
 
Hardening
1. Intiation of culture
The most important factor in tissue culture technique is the maintenance of aseptic condition. For thispurpose the culture medium generally, a GR-free (growth regulator free) medium is used. Immediatelyafter preparation the culture vessel has to be plugged and autoclaved at 121
O
C 15 psi (pounds per sq.inch) for an about 15-20min. The plant material has to be surface sterilized with a suitable sterilent. Thetransfer area should also maintained free of micro-organisms. Strict precautions are to be taken to preventthe entry of micro-organisms. The plug of a culture vessel is removed carefully to transfer plant materialto the nutrient medium during sub culturing. After inoculation the cultures are incubated in culture roomunder controlled condition at 25 + 12
O
C temperature and 1000 lux light intensity generated by florescenttube and at a constant photoperiod regulated by automatic timers.
2. Multiplication / Subculture
After 2-3 weeks the explants show visible growth by forming either callus (or) differentiated organs likeshoots, roots (or) complete plantlets, depending upon the composition of the medium. Periodically sub-culturing of callus (or) organs (or) plantlets to the fresh medium is done to multiply the callus (or) organs(or) to obtain large number of plantlets from the callus.
3. Development and Diffentiation / organogenesis
The concentration of phytoharmones in the medium are altered to induce differentiation in callus. A highcytokinins to auxin ratio induces shoot formation (
Caulogenesis
) (basal medium + low cytokinins / GA3medium is used before they can be rooted. Higher concentration (>2 mg/l BAP) of cytokinins induceadventitious shoot buds and retard shoot growth. Very high auxins to cytokinin ratio induces root
 
Biotechnology
Plant tissue culture
7
 
Prepared By: Shree Krishna Adhikari, Bsc.Ag 7 
th
semester. IAAS, Rampur Campus
2012
 
formation (
Rhizogenesis
). The development of organ structures like shoot, roots etc. from the culturedcells (or) tissues is known as
Organogenesis
. Alternatively media composition can also be altered toinduce the development of somatic embryos and the process is known as
somatic embryogenesis
.Further, an entire plantlet can be induced to grow on culture media by manipulating the phyto-hormonebalance correctly and the process is called
Regeneration
. The regeneration may be either direct or callusmediated. The
in vitro
induced shoots must be transferred to the culture media that supports rootinduction.
4) Hardening:
The
in vitro
cultured rooted plants are first subjected to acclimatization before transferring to the field.The gradual acclimatization of 
in vitro
grown plant to
in vivo
conditions is called hardening. The plantletis taken out from the rooting medium and is washed thoroughly to remove entire agar from the surface of plantlet as agar may attract microbes to grow and destroy the plantlets. The plantlet is now kept in a lowminimal salt medium for 24-48hrs and transferred to a pot that contains autoclaved sterilized mixture of clay soil, coarse sand and leaf moulds in 1 : 1 : 1 ratio proportion. The pot containing plantlet is coveredgenerally with the transparent polythene cover having holes for aeration to maintain the humidity. Theplantlets are maintained for about 15-30 days in this condition. The plantlets are then transferred to thesoil and are ready for transfer either to the green house or main field.
Steps in plant tissue culture technique
 
Selection of plant
 
Isolation of explant
 
Sterilization of explant
 
Inoculation of explant
 
Incubation
 
Initiation of callus
 
Sub culturing
 
Regenaration
 
Hardening
 
Transfer of plantlets to Green house or open field
Applications of plant tissue culture in crop improvement
1. Micro propagation helps in mass multiplication of plants which are difficult to propagate throughconventional methods.2. Some perennial crop plants like ornamental and fruit crops can not be propagated through seeds. Thevegetative propagation like grafting, budding are tedious and time consuming. In such crops micropropagation helps in rapid multiplication.3. Rapid multiplication of rare and elite genotypes such as Aromatic and Medicinal plants. Isolation of 
invitro
mutants for a large number of desirable character Eg:- Isolation of biochemical mutants and mutants

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