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Biyani's Think Tank

Concept based notes

Plant Tissue Culture

[B.Sc. Biotech Part-III]

Ms. Meesha Srivastava

Revised by: Neha Joshi Dept of Science

Biyani Girls College, Jaipur

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Published by :

Think Tanks

Biyani Group of Colleges

Concept & Copyright :

Biyani Shikshan Samiti

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Ph : 0141-2338371, 2338591-95 Fax : 0141-2338007

E-mail : [email protected]

Website :www.gurukpo.com; www.biyanicolleges.org

ISBN: 978-93-81254-31-1

Edition : 2011

Price :

Leaser Type Setted by :

Biyani College Printing Department

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.

Plant Tissue Culture 3


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.

I look forward to receiving valuable suggestions from professors of various educational

institutions, other faculty members and students for improvement of the quality of the book.

The reader may feel free to send in their comments and suggestions to the under mentioned

address.

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

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Syllabus

PLANT TISSUE CULTURE AND BIOTECHNOLOGY

BT - 803

Section -A

- Historical background and terminology used in cell & tissue culture.

- Basic techniques of cell and tissue culture, surface sterilization, aseptic tissue transfer,

concept of totipotency.

- Nutritional requirement of cell in vitro, various types of nutrient media.

Section -B

- Someatic embryogenesis and organogenesis in plant .

- Variability in tissue cultures, somaclonal and other variations.

- Isolation of cells, single cell cultures and cloning.

Section -C

- Micropropagation and cloning of plants, applications of micropropagation in

agriculture, horticulture & forestry.

- Haploid production. various techniques, applications.

Section -D

- Production of disease free plants by tissue culture methods.

- Protoplast isolation and culture, fusion of protoplast .

- Somatic hybrids, selection methods. gene expression in somatic hybrid .



History of Plant Tissue Culture

Q.1. Explain in brief the history of plant tissue culture.

Ans. The history of plant tissue culture begins with the concept of cell theory given by

chleiden & chwann, that established cell as a functional unit. This concept was

experimentally tested by Haberlandt who gave the idea of culturing plant cells.

The significant contributions of selected scientist are as:-

(a) G Haberlandt

Gave the idea of culturing isolated plant cells in the nutrient solution.

He isolated mesophyll cells with knop’s nutrient solution.

Haberlandt described the cultivation of mesophyl cells of Lamium in purpureum

and Eichhornia crassipes.

Using pieces of mature potato tubers he observed that cell division almost

without exception when the explants contained a vascular strand.

He is also known as Father of Plant Biotechnology.

(b) P. Nobecourt

He is French plant pathologist.

He gave the possibility of cultivating. Plant tissues for unlimited period.

(c) P.R. Gautheret

He used piece of cambium cut form tree, attempts were made on liquid medium

Which failed to grow but on soild medium the very healthy callus was grown .

(d) Philip R. white

He reported for first time success full continues cultures of tomato root tips and

obtained indefinite growth of roots.

Knop’s salt solution later replaced by vitamins pyrodoxine, thiamine and

nicotinic acid.

(e) F.C. Steward

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.

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(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

Q.1 What is somatic embryogenesis? Give its application.

Ans. Plant cells are totipotent and can produce whole new plants under favourable

conditions of nutritional and plant growth regulators. These somatic embryos were

similar to zygotic embryos in development and structure. The origin of somatic

embryos morphologically developed through the 3 stages:-

(a) Globular

(b) Heart

(c) Torpedo

Embryogenesis is a two step process:-

(1) Induction of embryogenesis

(2) Development of embryo, ultimately leading to germination.

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.

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Embryogenesis cells are small, is diametric in shape, filled with dense

cytoplasm and have a conspicuous nucleus.

When somatic embryos, (early stage or developed), are transferred on induction

medium they give rise to somatic embryos. This method of obtaining embryos

recurrently is known as repetitive or cyclic embryogenesis. This method is

useful for continuously obtaining embryos in large no for example Atropa

belledona, Ranunculus.

During somatic embryogenesis in cell suspension embryos of different sizes are

produced, this can be achieved by sieving or fractionation of suspension with

appropriate sieve size. Such cultures may be fully synchronized for their

growth.

a) Composition of Medium - levels of sucrose and nitrogen is to be monitored

Reduced nitrogen is not require for the induction and oxoid nitrogen alone in high

amount is sufficient for induction of somatic embryogenesis However, reduced

nitrogen in the embryo development medium supports embryo development.

Increased osmotic conc. by sucrose affect the embryo development.

(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.



ADVANTAGES OF SOMATIC EMBRYOGENESIS :-

1) Rapid multiplication through cell culture and use of bioreactors.

2) Presence of bipolar structure avoids the rooting steps required in organogenesis.

3) Possible to induce dormancy and store the culture for long duration.

4) Possibilities of encapsulation and other methods of packing and direct delivery

system can be employed.

5) Provides an important resource for analysis of molecular and biochemical

events that occur during induction and maturation of embryo.

6) Isolation of specific storage protein is possible.

7) It shortens the breeding cycle of deciduous trees and increases the germination

of hybrid embryos where delayed germination of seed is a significant handicap

in rooting of the plants of horticultural intrest.

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Factor’s Affecting Somatic Embryogenesis

Q.1 What is micro propagation?

Ans. Introduction:-

The technique of culturing plant became a wide subject embracing

morphology,physiology, biochemistry, molecular biology and genetic engineering

multiplication of plant through plant tissue culture can be achieved by any of the

following methods depending on the objectives. The basic concept is to achieve rapid

multiple without creating un wanted somaclonal variation.

Micro propagation is defined as production of miniature planting material in

largenumber by vegetative multiplication through regeneration.

Axillary budding- It is the development from pre-existing meristems on nodal

regions to ensure genetic stability of the regenerants.

Adventitious budding-De novo formation of adventitious buds (not from pre-

existing meristems) may occur directly from the tissues of the explant.

The technique of micro propagation is as:-

It is divided into four stages:-

Stage I-

Selection and establishment of Aseptic cultures.

i. In this, selection of typical, healthy, disease free mother plants.

ii. Selection of plant is followed by preparation of explants, surface sterilization

and transfer to appropriate media. iii.Sterilization is carried out through soaking

in a calcium hypo chlorite.

iv. Main aim is to attain an aseptic culture of the plant.

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).

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5) This production is unaffected by seasonal variations as uniform conditions are

maintained (no seasonal effect).

6) No care is required between two subculture as compared to conventional

vegetative propagation system like watering, weeding(less care).

7) Small glass house space is required because of miniature size of plant lets

8) Mother plant or genotype of stock plant can be stood and maintained in vitro

without damage to environmental factor and stock plants.

9) Being sterile transport across countries is permissible without difficulties

(transport across countries does not require phytosanitory regulation).

10) Miniature storage organs(tubers, corns, tuberous, roots) can be produced for

genotype storage and subsequent plantation which is also called as Germplasm

storage.

11) It is possible to mechanize whole process of vegetative propogation for large

scale plantations. 12)The plants which are difficult to propagate vegetatively by

conventional method can be propagated by this method.

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.

Androgenic Methods - It is a method of haploid production which is done from the

male gametophyte of an angiosperm plant i.e. microspore(immature pollen).

The underlying principle is to stop the development of pollen cell whose fate is

normally to induce a gamete (Sexual cell) and to force its development directly into a

plant. -Haploid can be obtained by the culture of excised anthers and pollen.

Anther Culture

Young flower buds with immature anthers which have the microspores are surface

sterilized and rinsed with sterile water.

One of the anthers is crushed in acetocarmine to know the stage of pollen

development.

Anthers at appropriate stage are inoculated in the nutrient media.

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.

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Anther tissue debris is removed by filtering the suspension through a nylon sieve

having a pore deameter of 40 inch.

This suspension is then centrifuged at low speed.

This suspension is then centrifuged at low speed.

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 various factors affecting the androgenesis are :-

1. Genotype: - For successful culture, the genotype of anther is predominant.

2. Physiological status of the donor plant:-The physiological status of the plant at the

time of excision of anther influences the sporophytic efficiency of microspores.

3. Stage of pollen- Selection of anthers at an :-Appropriate stage of pollen development.

Anthers with microspores ranging from tetrad to the binuecleate stage are responsive.

4. Pretreatment of anthers :- As the androgenesis is the deviation from the normal

development so for the induction certain treatments are given:

(a) Cold treatment: -It is given between 30 to 60C for 3 to 15 days. As a result weak and

nonviable anthers and microspore are killed and the material gets enriched.

- This treatment retards aging of the anther wall.



(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.

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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 various factors affecting the androgenesis are :-

1. Genotype: - For successful culture, the genotype of anther is predominant.

2. Physiological status of the donor plant:-The physiological status of the plant at the

time of excision of anther influences the sporophytic efficiency of microspores.

3. Stage of pollen- Selection of anthers at an :-Appropriate stage of pollen development.

Anthers with microspores ranging from tetrad to the binuecleate stage are responsive.

4. Pretreatment of anthers :- As the androgenesis is the deviation from the normal

development so for the induction certain treatments are given:

(a) Cold treatment: -It is given between 30 to 60C for 3 to 15 days. As a result

weak and nonviable anthers and microspore are killed and the material gets

enriched.

- This treatment retards aging of the anther wall.

(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 budswhich

can be collected from regenerated plants. Acetocarmine is used for staining of cells.

3. Number of nucleoli:- Haploids contain one nucleoli whereas diploids have 2

nucleoli

4. Flow cytometric analysis:- Nuclear DNA content reflects the ploidy state of the

donor which is determined by flow cytometery.

Depolarization

Haploids can be diploidized to produce homozygous plants by following method :-

1. Colchicine Treatment

2. Endomitosis(Chromosome duplication without nuclear division)

Significance and uses of Haploids

a) Development of pure homozygous lines.

b) Hybrid development.

c) Induction of mutation.

d) Induction of genetic variability.

e) Generation of exclusively male plants.

f) Cytogenetic Research.

g) Significance in the early release of varieties.

h) Hybrid sorting in plant breeding.

) Disease resistance.

j) Insect resistance.

k) Salt tolerance.

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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

Ovaries are removed and surface sterilized.

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.

Q.1 What is somaclonal variation?

Ans. The growth into whole plants is an asexual process, involving only mitotic division of

the cell. As expected that the process will produce genetically uniform plants orclonal

multiplication is possible through callus regeneration.



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.

- Gametoclonal variation has been introduced for variation observed in gametic

cells.

- The two methods of obtaining somaclonal variation are:-

(a) Without in vitro selection An explant is cultured on a suitable medium.

The basal medium is supplemented. With growth regulators which support the

differentiation of callus.

These cultures are subcultured and then transferred to shoot induction medium for

plant regeneration.

The plants so regenerated are transferred to pots grown to maturity and analyzed for

variants.

Somaclonal variants for various characters are not selected with directed approach as

both dominant and homozygous recessive traits can be directly selected.

(b) With in vitro selection

In vitro culture of higher plants can be used for selection of mutants.

Selection for resistance is the most common method for mutant selection, resistant

cells in a large population can be selected by their ability to grow in the presence of

an inhibitor.

The dedifferentiated culture(callus) is subjected to selection against inhibitors like

antibiotics, amino acid analogs etc.

These compounds are put in the medium at a concentration such that some cell

population survives and can be further grown on a selective medium.

If plants are resistant to the inhibitor, then stable transmission of that character is

analayzed in subsequent generations.

In this approach, variants for a particular character are selected rather than the general

variation obtained in first case where selection is done at the plant level.

Various factors influencing the somaclonal variation are:-

a) Genotype - It can influence both frequency of regeneration and the frequency of

somaclones.

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b) Explant source - It is a critical variable.

c) Duration of cell culture - Most long term cultures are chromosomally variable.

Thus variation increases with increasing duration of culture.

d) Culture conditions - Growth regulators can influence the frequency of

karyotypic alteration in cultured cells.

Hypothesis related DNA modification to various mutational events leading to

somoclanal variation.

Tissue Culture Environment

Cell physiological disturbances

(eg. Nucleotide pool imbalance)

DNA modi fications

(eg. Hypo/hyper-methylation)

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.

Specific base modification

Single gene mutationbase modifications

Transposable element Quantititativeactivation

chromatin structure changes



Late replication- induced chromosome breakage.

1. Rearrangements dependent on heterochromatin, distribution.

2. Chromosome type, break fusion, bridge cycle.

Disadvantages of somaclonal variation:-

a) Variation is cultivar dependent.

b) Frequencies of change vary.

c) Many changes are desirable.

d) Some changes are unstable.

e) Many changes are not novel.

f) Characters of interest may not change.

Advantages of somaclonal variation:

i. A rapid source of variation is available.

ii. Some changes occur at higher frequency.

iii. Agronomic tariff can change.

iv. Novel variants can rise.

v. Improved plants through somaclones.

Various methods of assessment are:-

A. Phenotypic parameters

Quantitative eg. Leap size, plant height etc.

Qualitative eg. Branching pattern, flower colour.

B. Physiological parameters

- Protein patterns by electrophoresis for an enzyme, or total content.

- Secondary products formation eg alkaloid and steroid etc.

C. Genetic parameters

- Chromosome number and structure.

- Giemsa/C-Banding pattern of chromosomes

- RFLP, RAPD analysis for alteration in DNA segments.

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Q.3 What is organogenesis ?

Ans. Introduction- Potentiality of a plant cell to regenerate the entire organism (Plant) is

termed as totipotency. This potentiality has been used for culturing of protoplast,

cells, tissues and organs in vitro.

Organogenesis :-

This is a process by which cells and tissues are forced to undergo changes which lead

to the production of a unipolar structure, namely a shoot or root primordium, whose

vascular system is often connected to a parent tissue.

(i) History - controlled experiments of organogenesis by white 1939, he obtained shoots

on callus of a tobacco, these finding were extended by skoog 1944. Who showed that

auxin could stimulate rooting and inhibit shoot formation. Further studies of skoog

and co-workers conclusively established a balanced combination of auxin and

cytokinin controls the root and shoot formation he was also associated with discovery

of cytokinin.

There are several advantages of plantlet regeneration through plant biotechnological

method using organogenesis or embryogenesis the advantages are:-

a) Efficiency of process (formation of plantlet in four steps).

b) Potential for production of higher nos. of plantlet and the morphological and

cytological uniformity of the plantlets.

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

24


Multiple Choice Questions

1. Plant tissue culture technique is a redefined method of ________

a) Hybridization

b) Vegetative Propagation

c) Asexual Reproduction

d) Selection

2. Polyethylene glycol is

a) Fusogenic chemical

b) Electrofusion stimulant

c) Callus stimulant

d) Differentiation stimulant

3. Somatic hybridization is achieved through

a) Grafting

b) Protoplast fusion

c) Conjugation

d) Recombinant DNA technology

4. The enzymes required to obtain wall-free / naked protoplasts are

a) Cellulase and Proteinase

b) Cellulase and Pectinase

c) Cellulase and amylase

d) Amylase and Pectinase

5. The first transgenic crop was

a) Pea

b) Tobacco

c) Flax

d) Cotton

6. A(n) __________ is an excised piece of leaf or stem tissue used in micropropagation.

a) microshoot

b) medium

c) explant

d) scion



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

8. Which of the following is considered as the disadvantage of conventional plant tissue

culture for clonal propagation?

a) Multiplication of sexually derived sterile hybrids

b) Less multiplication of disease free plants

c) Storage and transportation of propagates

d) Both (b) and (c)

9. What is meant by 'Organ culture' ?

a) Maintenance alive of a whole organ, after removal from the organism by partial

immersion in a nutrient fluid

b) Introduction of a new organ in an animal body with a view to create genetic mutation in

the progenies of that animal

c) Cultivation of organs in a laboratory through the synthesis of tissues

d) The aspects of culture in community which are mainly dedicated by the need of a

specified organ of the human body

10. Which method of plant propagation involves the use of girdling?

a) Grafting

b) Cuttings

c) Layering

d) Micropropagation

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)

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d) genesis of organs

13. In a callus culture

a) increasing level of cytokinin to a callus induces shoot formation and increasing level of

auxin promote root formation

b) increasing level of auxin to a callus induces shoot formation and increasing level of

cytokinin promote root formation

c) auxins and cytokinins are not required

d) only auxin is required for root and shoot formation

14. Protoplast are the cells devoid of

a) cell membrane

b) cell wall

c) both cell wall and cell membrane

d) none of these

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

17. Cell fusion method includes the preparation of large number of

a) plant cells stripped of their cell wall

b) single plant cell stripped of their cell wall

c) plant cells with cell wall

d) cells from different species

18. Subculturing is similar to propagation by cuttings because

a) it separates multiple microshoots and places them in a medium

b) it uses scions to produce new microshoots



c) they both use in vitro growing conditions

d) all of the above

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

20. What is/are the benefit(s) of micropropagation or clonal propagation?

a) Rapid multiplication of superior clones

b) Multiplication of disease free plants

c) Multiplication of sexually derived sterile hybrids

d) All of the above

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

22. Cellular totipotency is the property of

a) Plants

b) Animals

c) Bacteria

d) all of these

23. Agrobacterium based gene transfer is efficient

a) only with dicots

b) only with monocots

c) with both monocots and dicots

d) with majority monocots and few dicots

24. Who is the father of tissue culture?

a) Bonner

b) Haberlandt

c) Laibach

d) Gautheret

28


25. The production of secondary metabolites require the use of

a) Protoplast

b) Cell suspension

c) Meristem

d) Auxillary buds

26. Synthetic seed is produced by encapsulating somatic embryo with

a) Sodium Chloride

b) Sodium alginate

c) Sodium acetate

d) Sodium nitrate

27. Hormone pair required for a callus to differentiate are

a) Auxin and cytokinin

b) Auxin and ethylene

c) Auxin and absiccic acid

d) Cytokinins and gibberllin

28. DMSO (dimethyl sulfoxide) is used as

a) Gelling agent

b) Alkylating agent

c) Chelating agent

d) Cryoprotectant

29. The most widely used chemical for protoplast fusion, as fusogen is

a) Manitol

b) Sorbitol

c) Mannol

d) Polyethylene glycol

30. Cybrids are produced by

a) Fusion of two different nuclei from two different species

b) Fusion of two same nuclei from same species

c) Nucleus of one species but cytoplasm from both the parent species

d) None of the above

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

32. Part of plant used for culturing is called

a) Scion

b) Explant

c) Stock

d) Callus

33. Growth hormone producing apical dominance is

a) Auxin

b) Gibberellin

c) Ethylene

d) Cytokinin

34. A medium which is composed of chemically defined compound is called

a) Natural media

b) Synthetic media

c) Artificial media

d) None of these

35. To obtain haploid plant, we culture

a) Entire anther

b) Nucleus

c) Embryo

d) Apical bud

36. Somaclonal variations are the ones

a) Caused by mutagens

b) Produce during tissue culture

c) Caused by gamma rays

d) Induced during sexual embryogeny

37. Which of the following plant cell will show totipotency?

a) Xylem vessels

b) Sieve tube

c) Meristem

d) Cork cells

30


38. Which vector is mostly used in crop improvement?

a) Plasmid

b) Cosmid

c) Phasmid

d) Agrobacterium



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.

Aseptic Technique---Procedures used to prevent the introduction of fungi, bacteria, viruses,

mycoplasma or other microorganisms into cultures.

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).

Callus---An unorganized, proliferate mass of differentiated plant cells, a wound response.

Chemically Defined Medium---A nutritive solution for culturing cells in which each

component is specifiable and ideally of known chemical structure.

Clone---Plants produced asexually from a single source plant.

Clonal Propagation---Asexual reproduction of plants that are considered to be genetically

uniform and originated from a single individual or explant.

Contamination---Being infested with unwanted microorganisms such as bacteria or fungi.

Culture—A plant growing in vitro.

32


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.

Gibberellins---A plant growth regulator that influences cell enlargement. Endogenous

growth forms of gibberellin include Gibberellic Acid (GA3).

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.

Hormones---Growth regulators, generally synthetic in occurrence, that strongly affects

growth (i.e. cytokinins, auxins, and gibberellins).

Internode---The space between two nodes on a stem

In vitro---To be grown in glass (Latin). Propagation of plants in a controlled, artificial

environment using plastic or glass culture vessels, aseptic techniques, and a defined growing

medium.

In vivo---To be grown naturally (Latin)

Media---Plural of medium

Medium---A nutritive solution, solid or liquid, for culturing cells.

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---The transfer or transplantation of cells or tissues with or without dilution or

division, form one culture vessel to another.

Passage Number---The number of times the cells or tissues in culture have been subcultured

or passaged.

Pathogen---A disease-causing organism.

Pathogenic---Capable of causing a disease.

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.

Regeneration---In plant cultures, a morphogenetic response to a stimulus that results in the

products of organs, embryos, or whole plants.

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 cutting Cutting made from sections of roots alone.

Root hairs Outgrowths from epidermal cell walls of the root specialized for water and

nutrient absorption.

34


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.

Somaclonal Variation---Phenotypic variation, either genetic or epigenetic in origin,

displayed among somaclones.

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.

Sterile Techniques---The practice of working with cultures in an environment free from

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.

Undifferentiated---With plant cells, existing in a state of cell development characterized by

isodiametric cell shape, very little or no vacuole, a large nucleus, and exemplified by cells

comprising an apical meristem or embryo.

36


B.Sc./M.Sc. (Part III) EXAMINATION, 2011

(Faculty of Science)

(Common to Three and Five Year Integrated Course)

BIOTECHNOLOGY

PAPER BT- 803

(Plant Tissue Culture and Biotechnology)

TIME ALLOWED: THREE HOURS

Maximum Marks-50

1) No supplementary answer -book will be given to any candidate. Hence the

candidates should write the answer precisely in the main answer-book only.

2) All the parts of one question should be answered at one place in the answer-book.

One complete question should not be answered at different places in the answer

book.

.

Attempt FIVE questions in all, including Question No. 1 which is compulsory selecting one

question from each Unit.

1. Fill in the blanks:- i. Main role of growth hormone in cultural materials is …………. .

ii. Somatic embryogenesis was first induced in ………, ……..and……of…..by……. .

iii. Somaclonal variation has been proved as an alternative tool to ……. For

generating…… .

iv. Fusion of nucleated or enucleated cells produces ………… .

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? ……….. .

vii. Gelling agent / Soldifying agent is obtained from ……….. .

viii Haploid plants are useful in :-



a) ……….

b) ……….

c) ……….

ix. Plant materials are surface sterilized by ………… ,………. .

x. Somatic embryos are also called ………. . They are similar to ………., except

that they originate from ………. and ………. In size. 1x10=10

UNIT I

2. Describe the histological background of Tissue Culture Technique in detail.

Or

3. Write notes on the following:-

a) Totipotency

b) Various types of Nutrient media.

UNIT II

4. Define somatic embryogenesis and organogenesis in plants. Explain their

applications.

Or

5. Define somaclonal variation. What is its importance? Explain with suitable examples.

UNIT III

6. Describe application of micropropagation in agriculture, horticulture and forestry.

Or

7. What are haploids? How do you produce through tissue culture technique? Mention

their applications.

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.

38


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

http://en.wikipedia.org/wiki/Plant_tissue_culture

http://www.accessexcellence.org/LC/ST/st2bgplant.php

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