Professional Documents
Culture Documents
Published by :
Think Tanks
E-mail : acad@biyanicolleges.org
ISBN: 978-93-81254-31-1
Edition : 2011
Price :
While every effort is taken to avoid errors or omissions in this Publication, any
mistake or omission that may have crept in is not intentional. It may be taken note of that
neither the publisher nor the author will be responsible for any damage or loss of any kind
arising to anyone in any manner on account of such errors and omissions.
Preface
I am glad to present this book, especially designed to serve the needs of the s-tudents.
The book has been written keeping in mind the general weakness in un-derstanding the
fundamental concepts of the topics. The book is self-explanatory and adopts the “Teach
Yourself” style. It is based on question-answer pattern. The language of book is quite easy
and understandable based on scientific approach.
Any further improvement in the contents of the book by making corrections, omission
and inclusion is keen to be achieved based on suggestions from the readers for which the
author shall be obliged.
I acknowledge special thanks to Mr. Rajeev Biyani, Chairman & Dr. Sanjay Biyani,
Director (Acad.) Biyani Group of Colleges, who are the backbones and main concept
provider and also have been constant source of motivation throughout this Endeavour. They
played an active role in coordinating the various stages of this Endeavour and spearheaded
the publishing work.
Note: A feedback form is enclosed along with think tank. Kindly fill the feedback
form and submit it at the time of submitting to books of library, else NOC
from Library will not be given.
Meesha Srivastava
Syllabus
PLANT TISSUE CULTURE AND BIOTECHNOLOGY
BT - 803
Section -A
Section -C
Section -D
He is Known as of the pioners of plant tissue culture & contributed by giving the
requirment of plant tissue culture & developing techniques for the different
application.
Used coconut water for the first time and obtained good result from it.
Gave the somatic embryogenesis concept form cell suspension of carrot cells.
(f) J. Reinert
Gave the concept of totipotency of cells.
Conducted experiment on parenchymat our cells of carrot root in complex
medium.
Worked on embryogenesis on carrot cells.
(g) Folke Skoog
Done pioneering work with auxin, a plant growth hormones.
Also work with cytokinin, he also should that number of cytokinins occur
naturally.
He was also pioneer in investigating on how to control formation of roots, stem
and leaves.
(h) Toshio Murashige
He worked on nutrition of plant cells using tobacco pith cells.
He formulated the whites medium which was known as Murashige & Skoog
medium.
Developed the micro-propogation technique.
Worked on somatic embryo. formation using carrot and citrus plants.
(i) Guha & Maheshwari
First time development of haploids through anther and pollen culture.With the
development of the technique plant tissue culture and nutritional requirement of
plant cell, it was possible to develop news technologies by culturing plant
organs such as
Anther
Ovary
Ovule
Petal
Leaf
Meristem
Leading to establishment of new research lines as:-
Haploids
Virus free Plants
In-Vitro fertilization
Embryo rescue etc.
Somatic Embryogenesis
It is of two types:-
(a) Direct Embryogenesis
(b) Indirect embryogenesis.
Indirect Embryogenesis.:-When explants produce callus and the callus forms then
its known as indirect embryogenesis.
Direct Embryogenesis.:-
When embryogenesis. Occur directly on the explants without the production of
callus it is known as direct embryogenesis.
The exogenously supplied auxin is required in appropriate concentration for the
induction of somatic embryogenesis from callus or explants.
In direct embryogenesis cells of explanted tissues are already determined for
embryonic development and termed as pre-embryogenic determined
cells(PEDC’s).
In indirect embryogenesis cells require redetermination through a period in
culture and termed as induced embryogenesis determined cells(IEDC’s).
Embryogenesis occurs from a single cells or from a group of cells.
(b) Auxins - 2,4D appear to be required for embryo induction but adveresely affect
embryo development.
(c) Cytokinins-
Except zeatin other cytokinins suppress embryogenesis.
(d) Ethylene-
Suppresses embryogenesis.
(e) Abscisic acid-
Suppress abnormal development of embryos.-Imparts dormancy and help in the
formation of cotyledonary stage somatic embryo.
APPLICATIONS:-
(i) It provides potential in the form of somatic buds. It can be used for the production of
synthetic seeds.
(ii) Somatic embryos provides organized culture system, such cultures produce organ
specific or differentiation related compounds in higher amounts compared to cell
culture of that species.
Stage- II
Multiplication of Propagate
i. In this rapid multiplication of the regenerative system for obtaining large
number of shoots.
ii. For this medium and tissue factors are optimized empirically.
Stage - III
Plantlet Regeneration
i. Plantlets are produced through rooting of isolated shoots or germination of
somatic embryos.
ii. Shoots of appropriate length or age are required, which depends on the medium
Composition.
iii. High auxin concentration composition is used for the shoot development.
iv. Low salt strength of rooting medium facilitates the rooting.
v. In- vitro produced shoots are treated with auxins and transferred directly to pot
mixture.
Stage - IV
Preparation and transfer to field.
i. It is concerned with transfer of plantlets in pots their hardening and
establishment in soil.
ii. This stage is to prepare the propogule for these successful transfer to soil.
iii. Hardening of plants imparts some tolerance to moisture stress and plants
become autotrophic from heterotrophic condition.
iv. Stage organs are formed on plantlets their establishment in soil becomes easier.
v. These tuberous organs may require chilling treatment to germinate.
vi. When plantlets are taken out from the vessels adhering with running tap water
and plantlets are transferred in a soil.
vii. Plantlets are exposed to decreasing humidity by slowly exposing the plant or
reducing the mist period in the glass house.
viii. Hardened plants are then transferred to glass or poly houses with normal
environmental conditions.
ix. Plants are irrigitated frequently and their growth and variation are monitored
regularly.
Advantages of Micro-Propagation:-
1) Shoot multiplication can be achieved in small space so became miniature
plantlets can be produced.
2) Propagation is carried out under sterile condition. No damage is caused due to
insects and diseases and plantlets are produced from microbes(pathogens).
3) Virus free material is used (even through virus elimination by meristem culture)
a large number of virus free plants can be obtained.
4) Plant tissue culture is carried out under defined conditions of environmental,
nutritional and tissue system, therefore, it is a highly reproducible system under
the defined set of reproducible system under the defined set of
conditions(controlled conditions, reproducibility).
Disadvantages of micro-propagation:-
1) Micropropagation method involve expensive material like autoclave, laminar air
flow, contaolled culture room.
2) It is a skilled work so a decision making and technique knowledge are required
in the personnel.
3) Contamination cause severe damage to material and add to the cost of
production, affects time schedule delivery of the material.
4) Genetic stability is not confirmed in certain methods.
5) Explants taken are delicate so it takes longer.
6) Specific conditions for micro-propogation may be required. Therefore, each
material requires separate research method.
Q. What are haploids? Give a briefs description of anther and pollen (n) in this
culture.
Ans. Haploids are plants which has gametic chromosome
- Hapliods may be grouped into two broad categories:-
(a) Monoploids
- Which possess half the number of chromosomes from a diploid species.
(b) Polyhaploids
- It possess half the number of chromosomes from a polyploid species.
Haploid production through anther culture has been referred to as and rogensis while
gynogenesis is the production of haploid plants from ovary to ovule culture where the
female gamete or gametophyte is triggered to sporophytic development.
The anthers in later stage gradually turn brown and within 3-8 weeks they burst open
due to the pressure exerted by the growing pollen callus or pollen plants.
They attains a height of about 3-5 cm, the individual plantlets or shoots emerging
from the callus are separated and transferred to a medium that would support further
development.
Microspore culture
- Haploid plants can be produced through in vitro culture of male gametophyte cells ie
microspores or immature pollen.
- General procedure of culture is : -
Anthers are collected from sterilized flower buds in a small beaker containing basal
media.
Microspores are then squeezed out of the anthers by pressing them against the side of
beaker with a glass rod.
Anther tissue debris is removed by filtering the suspension through a nylon sieve
having a pore deameter of 40 inch.
The supernatant containing fine debris is discarded and the pollen of pellet is
resuspended, in fresh media.
The microspores obtained are then mixed with an appropriate culture medium.
Final suspension is then pipetled into small Petri dishes. (For deation, thin layer of
liquid is made).
Each dish is then sealed with parafilm to avoid dehydration and is incubated.
(b) Hot Treatment :- Explants are subjected to 30C for 24 hrs or 40C for 1 hr. stimulates
embryogenesis.
(c) Chemical Treatment: Chemicals induce parthenogenesis example-
Chloroethylphosphonic acid.
5. Culture Media :- Presence of sucrose, nitrate, ammonium salts and amino acids are
essential components to be present in a culture medium. Activated charcoal also
enhances the percentage of androgenic anthers in some.
Pollen embryogenesis can be induced on an mineral sucrose medium.
Process of androgenesis - Haploid plantlets are formed in two ways:-
(a) Direct embryogenesis: Embryos originate directly from the microspores of anthers
without callusing.
(b) Indirect embryogenesis: It is also known as organogenic pathway, in this
microspores undergo proliferation to form callus which can be induced to
differentiate into plants.
- Process of androgenesis Shows microspores undergo divisions, which continues
until it a undergo various stages of development, stimulation those of normal zygotic
embryo formation. However when the microspore take organogenetic pathway, then
these all increase in size, exerting pressure and the contents are released in the form at
callus.
- These calluses differentiate into plantlets. The plants with developed shoots and roots
are then transferred to pots.
- The physical environmental conditions in which the cultures are to placed can
enhance differentiation. These are:-
(a) Incubation at 24-280C
(b) Light intensity of 500 Lux
(c) After induction kept at 14 hr day light at 2000-4000 Lux.
- For obtaining homozygous lines, the plants derived form their anther culture are
analysis for this physiology status. Some of these methods are :-
1. Counting of plastids in the stomata :- Count the number of plastids in the stomato
of a leaf.
2. Chromosome number:- It can be counted from pollen mother cells of buds
The supernatant containing fine debris is discarded and the pollen of pellet is
resuspended, in fresh media.
The microspores obtained are then mixed with an appropriate culture medium.
Final suspension is then pipetled into small Petri dishes. (For deation, thin layer of
liquid is made).
Each dish is then sealed with parafilm to avoid dehydration and is incubated.
- The physical environmental conditions in which the cultures are to placed can
enhance differentiation. These are:-
(a) Incubation at 24-280C
(b) Light intensity of 500 Lux
(c) After induction kept at 14 hr day light at 2000-4000 Lux.
- For obtaining homozygous lines, the plants derived form their anther culture are
analysis for this physiology status. Some of these methods are :-
1. Counting of plastids in the stomata :- Count the number of plastids in the stomato
of a leaf.
2. Chromosome number:- It can be counted from pollen mother cells of budswhich
can be collected from regenerated plants. Acetocarmine is used for staining of cells.
Gynogenic Haploids
Recent advances has lead to the induction of haploid from ovary and ovule
culture.
The Megaspores or female gametophytes of angiosperms can be triggered in
vitro saprophytic development.
In vitro culture of unplanted ovaries and ovules represent and alternative for
species
Ovaries can be cultured as pollinated and unpollinated.
Procedure
Before culturing the tip of distal part of the pedicel is cut off and the ovary is
implanted with the cut end inserted in the nutrient media.
When liquid medium is to be employed, the ovaries can be placed on a fitter paper
and inserted into the medium.
Factors affecting gynogenesis
1) Genotype
2) Growth condition of the donor plant
3) Stage of harvest of ovule
4) Embryo sac stage
5) Culture conditions
6) Seasonal effects
7) Physical factors.
This provided a basis of genetic manuplation in plants using callus. The origin of the
genetic variation is as we know the plant cells are totipotent it is possible to have
regeneration from single cells or protoplast, but in this process, a cell divides and
redivides several hundred times to produce callus and subsequently organs.
Earlier terms such as “calliclones” and “protoclones” were coined to indicate
variation arising in regenerated plants from stem and protoplast derived callus
respectively. A general term somaclones has been given for plants derived from any
form of somatic cell culture and somaclonal variation is the variation displayed
amongst such plants.
Specific base modifications chromatin structure changes Single gene mutation Transposable
element Quantitative base modifications activation
(a) Insertion tariff variant or substitution.
(a) Insertion tariff variant or substitution.
(b) Excisions (c) Chromosome breakage.
Q.4 What are basic tools and techniques and various sterilization methods of
plant tissue culture?
Ans. Various tools and techniques used are:-
1) ph meter
2) Autoclave -works on the principle of pressure cooker
3) Plant growth chamber.
4) Laminer Air Flow- works on the principle of HEPA.
5) Microscopy.
6) Colorimeter.
7) Centrifugation.
8) Chromatograply
1. Paper
2. Thinlayer
3. Two dimensional
4) Thermometer
5) Hygrometer
The methods of sterilization are:-
1. Laboratory - cleanliness:-
1) Minimize the air current in the working area as much as possible.
2) Stare pro
3) Separate area for cleaning.
4) Laboratory, three types of sterilization is used
a) a dry heatb) Wet heat
c) Filter sterilization
2. Sterilization of tools
Disinfectants are used for the sterlization of tools. Some of them are:-
(i) Ag (ii) Chlorine
3. Explants – Sterilization
A suitable sized explant can be sterilized by any one of the following:-
1) 1-4% saturated solution of calcium hypochlorite.
2) 1% solution of bromine water
3) 70% ethyl alcohol
4) 0.1 - 0.2% mercuric chloride
5) 7% of sodium hypochlorite
6) 10% hydrogen peroxide solution
7) 1% silver nitrate solution
2. Polyethylene glycol is
a) Fusogenic chemical
b) Electrofusion stimulant
c) Callus stimulant
d) Differentiation stimulant
7. Protoplasts can be produced from suspension cultures, callus tissues or intact tissues by
enzymatic treatment with
a) cellulotyic enzymes
b) pectolytic enzymes
c) both cellulotyic and pectolytic enzymes
d) proteolytic enzymes
11. Which of the following is used in the culture of regenerating protoplasts, single cells or very
dilute cell suspensions?
a) Nurse medium
b) Nurse or feeder culture
c) Both (a) and (b)
d) None of these
12. Organogenesis is
a) formation of callus tissue
b) formation of root and shoots on callus tissue
c) both (a) and (b)
d) genesis of organs
15. Which breeding method uses a chemical to strip the cell wall of plant cells of two
sexually incompatible species?
a) Mass selection
b) Protoplast fusion
c) Transformation
d) Transpiration
16. The phenomenon of the reversion of mature cells to the meristematic state leading to the
formation of callus is known as
a) Redifferentiation
b) Dedifferentiation
c) either (a) or (b)
d) none of these
19. The ability of the component cells of callus to form a whole plant is known as
a) Redifferentiation
b) Dedifferentiation
c) either (a) or (b)
d) none of these
21. When plated only in nutrient medium, how much time is required for the protoplast to
synthesize new cell wall?
a) 2-5 days
b) 5-10 days
c) 10-15 days
d) 15-17 days
29. The most widely used chemical for protoplast fusion, as fusogen is
a) Manitol
b) Sorbitol
c) Mannol
d) Polyethylene glycol
31. Callus is
a) Tissue that forms embryo
b) An insoluble carbohydrates
c) Tissue that grows to form embryoid
d) Un organized actively dividing mass of cells maintained in cultured
Key Terms
Adventitious---Developing from unusual points of origin, such as shoot or root tissues, from
callus or embryos, from sources other than zygotes.
Agar---a polysaccharide powder derived from algae used to gel a medium. Agar is generally
used at a concentration of 6-12 g/liter.
Aseptic---Free of microorganisms.
Autoclave---A machine capable of sterilizing wet or dry items with steam under pressure.
Pressure cookers are a type of autoclaves.
Auxin---A group of plant growth regulators that promotes callus growth, cell division, cell
enlargement, adventitious buds, and lateral rooting. Endogenous auxins are auxins that occur
naturally. Indole-3-acetic (IAA) is a naturally occurring auxin. Exogenous auxins are auxins
that are man-made or synthetic. Examples of exogenous auxins included 2,4-
Dichlorophenoxyacetic acid (2,4-D), Indole-3-Butyric acid (IBA), α-Naphthaleneacetic acid
(NAA), and 4-Chlorophenoxyacetic acid (CPA).
Chemically Defined Medium---A nutritive solution for culturing cells in which each
component is specifiable and ideally of known chemical structure.
Cytokinin---A group of plant growth regulators that regulate growth and morphogenesis and
stimulate cell division. Endogenous cytokinins, cytokinins that occur naturally, include zeatin
and 6-γ,γ-dimethylallylaminopurine (2iP). Exogenous cytokinins, cytokinins that are man-
made or synthetic, include 6-furfurylaminopurine (kinetin) and 6-benzylaminopurine (BA or
BAP).
Differentiated---Cells that maintain, in culture, all or much of the specialized structure and
function typical of the cell type in vivo. Modifications of new cells to form tissues or organs
with a specific function.
Explant---Tissue taken from its original site and transferred to an artificial medium for
growth or maintenance.
Horizontal laminar flow unit---An enclosed work area that has sterile air moving across it.
The air moves with uniform velocity along parallel flow lines. Room air is pulled into the
unit and forced through a HEPA (High Energy Particulate Air) filter, which removes
particles 0.3 μm and larger.
Media---Plural of medium
Micropropagation---In vitro Clonal propagation of plants from shoot tips or nodal explants,
usually with an accelerated proliferation of shoots during subcultures.
Node—A part of the plant stem from which a leaf, shoot or flower originates.
Passage Number---The number of times the cells or tissues in culture have been subcultured
or passaged.
Petiole---A leaf stalk; the portion of the plant that attaches the leaf blade to the node of the
stem.
Plant Tissue Culture---The growth or maintenance of plant cells, tissues, organs or whole
plants in vitro.
Root apex The apical meristem of a root; very similar to the shoot apical meristem in that it
forms the three meristematic areas: the protoderm (developing into the epidermis); the
procambium (which develops into the stele); and the growth meristem (which forms the
cortex).
Root cap A thimble like mass of cells covering and protecting the apical meristem of a root.
Root culture The culture of isolated root tips of apical or lateral origin to produce in vitro
root systems with indeterminate growth habits. Root culture was among the first kinds of
plant tissue cultures, and is still largely used in the study of developmental phenomena, and
mycorrhizal, symbiotic and plant-parasitic relationships.
Root hairs Outgrowths from epidermal cell walls of the root specialized for water and
nutrient absorption.
Root nodule A small round mass of cells that is located on the roots of plants and contains
nitrogen-fixing bacteria.
Root zone The volume of soil or growing medium containing the roots of a plant. In soil
science, the depth of the soil profile in which roots are normally found.
Rootstock The trunk or root material to which buds or scions are inserted in grafting. See
stock.
Rotary shaker Rotating apparatus with a platform on which, containers can be shaken, such
as Erlenmeyer flasks containing cells in liquid nutrient medium.
Shoot Apical Meristem---Undifferentiated tissue, located within the shoot tip, generally
appearing as a shiny dome-like structure, distal to the youngest leaf primordium and
measuring less that 0.1 mm in length when excised.
Somaclones---Plants derived from any form of cell culture involving the use of somatic plant
cells.
Stage I---A step in in vitro propagation characterized by the establishment of an aseptic
tissue culture of a plant.
Stage II---A step in in vitro propagation characterized by the rapid numerical increase of
organs or other structures.
Stage III---A step in in vitro propagation characterized by preparation of propagules for
successful transfer to soil, a process involving rooting of shoot cuttings, hardening of plants,
and initiating the change from the heterotrophic to the autotropic state.
Stage IV---A step in in vitro plant propagation characterized by the establishment in soil of a
tissue culture derived plant, either after undergoing a Stage III pretransplant treatment, or in
certain species, after the direct transfer of plants from Stage II into soil.
Sterile--- (A) Without life. (B) Inability of an organism to produce functional gametes. (C) A
culture that is free of viable microorganisms.
Subculture--- With plant cultures, this is the process by which the tissue or explant is first
subdivide, then transferred into fresh culture medium.
Tissue Culture---The maintenance or growth of tissue, in vitro, in a way that may allow
differentiation and preservation of their function.
Totipotency---A cell characteristic in which the potential for forming all the cell types in the
adult organism are retained.
BIOTECHNOLOGY
Maximum Marks-50
iii. Somaclonal variation has been proved as an alternative tool to ……. For
generating…… .
v. Name the scientist who first isolated the protoplasts of plant tissue by using cell wall
degrading enzymes. ……… year …….. .
vi. Who first indicated that organogenesis could be chemically controlled? ……….. .
a) ……….
b) ……….
c) ……….
x. Somatic embryos are also called ………. . They are similar to ………., except
that they originate from ………. and ………. In size. 1x10=10
UNIT I
UNIT II
UNIT III
UNIT IV
8. Explain in detail:-
a) Role of tissue culture in producing disease free plants.
b) Define somatic hybrid. Narrate the selection method and gene expression in
somatic hybrid.
Or
9. Describe the whole technique of isolation of protoplast, culture them and fusion of
protoplast. Mention its application also.
Bibliography
1. Plant Tissue Cultures: S.S. Bhojwani and M.K. Razdan , Elsevier Science, The
Netherlands.
2. An Introduction to Plant Tissue Culture : M.K. Razdan
3. Cell Culture Methods and Cell biology, Vol. 4: D. W. Barens
4. Cell and tissue culture - laboratory procedure : A. Doyle.
5. Plant Tissue culture - A practical Approach : R.A. Dixon, IRL Press.
6. Biotechnology in Agriculture and forestry : Y.P.S. Bajaj, Narosa.
7. Plant cell and Tissue Culture : Rienert and Yeoman.
8. http://en.wikipedia.org/wiki/Plant_tissue_culture
9. http://www.accessexcellence.org/LC/ST/st2bgplant.php
10. http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm