Methods in Plant Tissue Culture
ABOUT THE BOOK: The overwhelming response to the First Edition of the book
that led to its running out of print in this short duration encouraged to bring
out its Second edition. Many new chapters have been included and existing
chapters of first edition have been improved in light of available current
literature. Plant tissue culture is the technique of growing plant cells,
tissues and organs in an artificial prepared nutrient medium static or liquid,
under aseptic conditions. It has advanced the knowledge of fundamental botany,
especially in the field of agriculture, horticulture, plant breeding, forestry,
somatic cell hybridization, phytopathology and industrial production of plant
metabolites, etc. The term tissue culture is actually a misnomer borrowed from
the field of animal tissue culture. It is a misnomer because plant
micropropagation is concerned with the whole plantlet and not just isolated
tissues, though the explant may be a particular tissue. The term's plantlet
culture or micropropagation, therefore, are more accurate.
The nursery trade commonly accepts this method of propagation and it has had a
significant impact on commercial horticulture. Applications of plant tissue
culture vary from the curious gardener multiplying plants in a modest home
kitchen, to the renowned scientist working in an elaborate laboratory. It
reaches from the orchid hobbyist who has learned to multiply a few personal
favorites, to a million-dollar industry producing houseplants, ornamentals, and
secondary products.
Plant tissue culture, in recent years, has created unprecedented opportunities;
therefore, Government of India has established at least 32 institutions where a
strong infrastructure for training and research in plant tissue culture is going
on. Post-graduate M.Sc./M.Tech and Ph.D. programmes in biotechnology (including
plant tissue culture) have also been started in at least 28 selected
universities. At undergraduate and post-graduate levels, Indian universities
have also introduced courses in their syllabi. I would like to acknowledge the
valuable help received by me from various sources.
AUTHOR: Dr. U. Kumar | ISBN: 9798177540771 | YEAR: 2007 | PAGES: 444 | SIZE: 18.5 X 24.5 X 2 cm. | BINDING: Hard | LANGUAGE: English
CONTENTS:
1. PLANT TISSUE CULTURE: AN OVERVIEW
a. Callus Tissue and Organogenesis
b. Principles of Growth
c. Why Growth Occurs?
i. Kinetics of Growth or Measurement
and Pattern of Growth
ii. What Happens, When Growth Occurs?
d. The Botanical Basis for Tissue Culture
i. Power of Regeneration in Plants
e. Why Plants Regenerate?
f. Plant Tissue Culture: Principles
i. Callus Culture
ii. Meristem Culture
iii. Organ Culture
iv. Anther and Microspore Culture
g. The Concept of Totipotency of Cells
i. The Autonomous Organelles: Their
Behaviour during Growth Induction
ii. Morphogenesis
h. Genetic Approach of Plant Tissue
i. Culture
j. Importance of Tissue Culture
k. Technique
2. HISTORY OF PLANT TISSUE AND CELL CULTURE
a. Discovery of Microscope
i. Nature of a Cell
ii. Charles Darwin
iii. Justus von Liebig
iv. Vochting's Work
v. Rechinger's Work
vi. 20th Century Development
vii. Knop and Prdifer developed
viii. synthetic media
ix. F. C. Steward
x. H. E. Street
xi. C. O. Miller
xii. F. Skoog
xiii. Toshio Murashige
xiv. Vasil and Hilderbrandt
xv. New Era
b. Tissue Culture: New Technologies
i. Liquid Cell Suspension Cultures
ii. Pollen Culture
iii. Protoplasts Fusion
iv. Mutations Research in Plant
Tissue
v. Culture
vi. Discovery of Synthetic Seeds
vii. Production of Secondary
viii. Metabolites and Hairy Root
ix. Cultures
x. For Plant Pathological Studies
xi. Genetic Engineering
c. Tissue Culture Researches in India
d. Research Promotion and Priorities in
e. India
i. Bioinformatics
ii. Gene Banking, Conservation and
iii. Exchange in India
3. GENERAL METHODOLOGY OF PLANT TISSUE CULTURE
a. Media Preparation : Area/Room
i. Cabinets or Shelves
b. Culture Media, Washing Powder/Liquid Detergent,
Disinfectants
c. Aseptic Transfer Chamber Area
d. Environmentally Controlled Culture
e. Room
f. Analytical Room
g. Acclimatization Room
h. Miscellaneous Items
i. Specifications of Laboratory Equipments
j. Aseptic Technique
k. Sterilization of Plant Tissues
l. Cleaning (Preparation of Glasswares/ Plastic wares (Autoclavable)
m. Sterilization
n. Surfactants
i. Control of Bacterial and Fungal
Contaminants by Antibiotics in Plant Tissue Culture
o. In Vitro Environment
p. Pre-treatment to Explant Tissues Prior to Culture
i. Why Aeration is Essential?
q. Isolation of Plant Material
i. Age of the Plant Tissue
r. Callus Tissue and Organogenesis
i. Suspension Cell Culture from
Callus in vitro
s. Micropropagation through Organogenesis
t. Culture Media and Their Preparation
i. Nutrition of Callus Cultures
u. Media Components
i. Inorganic Salts
ii. Plant Growth Regulators
iii. Vitamins
iv. Carbon Source
v. Gelling Agent
vi. Amino Acids and Amides
vii. Antibiotics
viii. Natural Complexes
v. Additional Requirements
w. Sterilization of Media
i. Use and Storage of Coconut Water
x. Related Procedures
i. Ultraviolet Light
ii. Preparation of Phenol
iii. Silanization of Plastic and
iv. Glassware
y. Initiation of Embryogenesis in
z. Suspension Culture
i. Initiation of Embryogenic Tissue
aa. Establishment of Embryogenic
bb. Suspension Culture
i. Maturation of Embryos
cc. Important Parameters for Consideration
i. Simplification of the Technique
ii. Different Causes of Failures (and
Remedies)
4. CULTURE MEDIA INGREDIENTS, PREPARATION AND RELATED PROBLEMS
a. Media Components
i. Inorganic Salts
ii. Organic Chemicals
iii. Growth Regulators
iv. Other Growth Regulating
Substances
v. Amino Acids
vi. Antibiotics
vii. Chemically Undefined Constitutes
of Tissue Culture Media
viii. Natural Complexes
b. Additional Requirements
i. Ultraviolet Light
ii. Preparation of Phenol
iii. Working with 32P Labelled
Compounds
iv. Silanization of Plastic and
Glassware
v. Preparation of Dialysis Tubing
c. Chemical List
d. Preparation of Media
i. Stock Solutions
ii. Stock Solutions for Begonia Rex
Media
iii. MS Salts Stocks
iv. Vitamin stock (store in
refrigerator)
v. Auxin stock (250ml)
vi. Cytokinin stock (250 ml)
e. Media Mixing
f. Liquid Media
g. Media Problems
h. Air Requirements
i. Vitrification
j. Refrigeration
k. Symptoms, Causes, and Solutions
l. Culture Media for Various Plants
i. Hippeastrum Media
ii. Hyposix media
iii. Anthurium Media
iv. Dieffenbachia Media
v. Syngonium media
vi. Lolium x Fescue media
vii. Freesia Medium
viii. Iris (rhizomatous) medium
ix. Iris (bulbous) media
x. paragus media
xi. Hemerocallis media
xii. Hosta media
xiii. Lilium media
xiv. Cattleya medium
xv. Cymbidium media
xvi. Miltonia, Odontoglossum, and
xvii. Oncidium medium
xviii. Catharanthus media
xix. Panax media
xx. Begonia media
xxi. Nandina media
xxii. Epiphyllum media
xxiii. Mammillaria medium
xxiv. Dianthus media
xxv. Chrysanthemum medium
xxvi. Kalanchoe media
xxvii. Brassica media
xxviii. Cucumis media
xxix. Rhododendron media
xxx. Saintpaulia media
xxxi. Juglans media (DKW)
xxxii. Juglans media (somatic
xxxiii. embryogenesis)
xxxiv. Eucalyptus media
xxxv. Phlox media
5. METHODS OF STERILIZATION AND DISINFESTATION
a. The Pattern of Microbial Death
b. Effectiveness of Antimicrobial Agent Activity
i. Population Size
ii. Population Composition
iii. Concentration/Intensity of an
iv. Antimicrobial Agent
v. Exposure Time
vi. Temperature
c. Sterilization
i. Sterilization by Heat.
ii. Thermal Death Point
iii. Susceptibility to Moist Heat
iv. Susceptibility to Dry Heat
v. Uses of Temperature at 100 °C
vi. Types of Autoclaves
d. Sterilization of Media
i. Sterilization by Filtration
ii. Micro filters
e. Air Sterilization
i. Laminar Flow Biological Safety
ii. Cabinets
f. Sterilization by Radiation
g. The Transfer Room and Sterile Technique
i. Cleanliness
ii. Sterilization of Air
iii. Sterile Technique for Explants
iv. Sterile Technique for Transfer
h. Disinfection
i. Chemical Disinfectants
ii. Classification of Chemical
iii. Disinfectants
i. Methods of Laboratory Sanitation
i. Possible Source
ii. Human Element
iii. Testing for Solid-Surface
Biological
iv. Contaminants
v. Preparation ofMicrobial-
vi. Enrichment Agar
vii. Preparing Swab Samples from A
viii. Solid Surface
ix. Preparing an Agar Streak Plate
x. How to Observe Contaminants
xi. Microbiological Stain
xii. Testing Laboratory Air
6. ASEPTIC TECHNIQUES AND PREPARATION OF EXPLANTS
a. Sterilization of Plant Tissues
i. Cleaning (Preparation of
Glasswares/Plastic wares (Autoclavable)
ii. Sterilization
iii. Surfactants
b. Control of Bacterial and Fungal Contaminants by
Antibiotics in Plant
c. Tissue Culture
d. In Vitro Environment
e. Pretreatment to Explant Tissues Prior to Culture
f. Explants and Their Preparation
i. Age of the Plant Tissue
g. Size of the Explant
i. Which Plant is to be selected for
Explant?
ii. How to Obtain Explants?J
h. Further Considerations
i. Cleaning and Treatment of Explant..
7. PLANT TISSUE CULTURE SOME RELATED ASPECTS
a. Bio Village ConceptI
i. Qualifications Required to Join
the
ii. Training Coursei
iii. Employment for Rural YouthJ
b. Efforts of Public Research Institutes
c. Production Criteria and Economics
i. Selection of Crops for
ii. MicropropagationJ
iii. Selection of location
d. Optimising Size of Production
e. Available Facilities and Production Potential
f. Economic Analysis of the Project
g. Production Management
h. Order Processing
i. Planning for Production
i. MultirateJ
ii. PassageJ
iii. Operator Efficiency
j. Production Monitoring
k. Contamination: Economic Assessment
l. Calculation of Cost Price
m. Quality Control in Commercial Plant
n. Tissue Culture
8. HISTOLOGICAL AND PHOTOGRAPHIC TECHNIQUES FOR PLANT TISSUE CULTURE
a. Histological Techniques
i. General Considerations for
Paraffin Studies
ii. Immobilization of Specimens for
Paraffin Sectioning
iii. Dissection and mounting of
specimens
b. Preparation of Specimens for Scanning Electron Microscopy
c. Photographic Methods for Plant Cell and Tissue Culture
i. Type of Photography for Plant Cell
and Tissue Culture Research
ii. Macrophotography
iii. Microphotogruphy with
Stereomicroscopes
iv. Focusing and Cropping the Image
v. Types of the Films
vi. Composition
9. PROTOPLAST TECHNOLOGY
a. Importance of Protoplast Isolation and Culture
b. Isolation of Protoplast from Various Plant Parts
i. Protoplast Isolation: Enzymatic
method
ii. Protoplast Isolation: Mechanical
method
c. Enzymic Isolation of Protoplasts: Method
d. Methods of Protoplasts Culture
i. Laboratory Facilities
ii. Enzyme Mixture and Osmotic
Stabilizer (Osmoticaj
e. Purification of Isolated Protoplasts
i. Sedimentation and Washing
ii. Flotation
iii. Other Purification Methods
f. Protoplast Viability Testing
i. Protoplast Culture: General Steps
g. Protoplast Culture: Culture Media
i. Protoplast Culture Media for PC I
Group
ii. Murashige and Skoog Medium
iii. Protoplast Culture Media PC II
Group
iv. Prcroplast Culture Media PC III
Group
v. Agar Embeded Culture
vi. Microchambers
vii. Hanging Drop Cultures (HDC)
Techniques
viii. Multidrop Array (MDAj
Techniques
h. Growth and Division of Protoplast
i. Protoplast Culture: Regeneration
of Cell Wall
i. Development of Callus/Whole Plant... Determination of
Protoplast Plating
j. Efficiency
k. Technique for Isolation of Sub-protoplasts
l. Handling of Regenerated Plantlets
m. Protoplast Fusion
i. Protoplast Fusion : General Steps
n. Protoplast Culture: Regeneration of Plants
i. Organogenesis
ii. Embryogenesis
iii. Handling of Regenerated
Plantlets
o. Protoplast Culture: Uptake of Foreign Materials
i. Incorporation of Foreign DNA
ii. Incorporation of Nuclei
iii. Incorporation of Chloroplasts
iv. Incorporation of Cyanobacteria
v. Incorporation of Bacteria
vi. Incorporation of Virus
p. Incorporation of Non-Biological Materials
q. Protoplast Fusion Hybrids : Selection
i. Visual Selection
ii. Fluorescent Labels
iii. Fluorescence Activated Cells
Sorting
iv. Nutritional Selection
v. Drug Sensitivity and Resistance
r. Somatic Cell Hybridization or Cybrid or Cytoplast
s. Protoplasts for Isolation of Cell Components
i. Plasmalemma
ii. Chloroplasts
iii. Mitochondria
iv. Vacuoles
10. MICROPROPAGATION IN PLANTS
a. Material for Micropropagation
b. Advantages of Micropropagation
i. Plant Health
ii. Germplasm Storage
c. Types of Micropropagation
i. Micropropagation Through Shoot Bud
Proliferation
ii. Micropropagation Through
iii. Adventitious Buds
iv. Meristem Tip Culture
v. In Vitro Tuberization
vi. Somatic Embryogenesis
d. Methods of Micropropagation (General)
e. Important Considerations and Precautions
i. Problems and Contamination in
Culture
f. Methods of Micropropagation
g. Applications of Micropropagation
11. MICROPROPAGATION: COMMERCIAL LABORATORY PRODUCTION
a. Requirements
i. Media Preparation/dishes
ii. Storage of Media
iii. Transfer Room
iv. Culture Room(s)
v. Cold Storage
vi. Shipping/lunch Area
vii. Locker Room
viii. Offices
ix. Miscellaneous Storage
b. The Process
i. What to Grow
ii. Preparation of Stock Plant.
iii. Initiation of Culture (Stage I)
iv. Shoot proliferation (Stage II)
v. Media formuli
vi. Rooting (Stage III):
vii. Greenhouse acclimatization
(Stage IV):
viii. Scheduling of Production
12. SOMATIC EMBRYOGENESIS PRINCIPLES, CONCEPTS AND APPLICATIONS
a. Carrot as a Embryogenic System:
b. Somatic and Zygotic Embryos
c. Totipotency and Role of Auxin
i. Proembryogenic Masses and Role of
Auxin
ii. Acquistion of Totipotency
iii. Expression of the Programme in
the Absence of Auxin
iv. Methylated Cytosine Level and
Development
d. The Genetic Approach of Somatic Embryogenesis
e. The Process of Somatic Embryogenesis
i. Induction of the Embryogenic State
f. Recurrent Embryogenesis
i. Problems
ii. Maintenance
iii. Auxin vs Cytokinin for Induction
of Embryogenesis
g. How to Obtain Embryos from Embryogenic Cultures?
h. Embryo Maturation and the Development of Germinability
i. Auxin Levels
ii. Sucrose Level
iii. Heat Shock Treatment
iv. ABA Levels
v. Water Saturation
i. Genetic Control of Embryo Cloning
i. Genetic Variability
ii. Cytoplasmic Effects
j. Applications of Somatic
k. Embryogenesis
i. Mass Propagation
ii. Scale-up Potential
iii. Use of Bioreactors
iv. Protoplast Culture
l. Embryo Cloning and Gene Transfer
m. Somatic Embryogenesis in Trees
13. SOMACLONAL AND GAMETOCLONAL VARIANT SELECTION
i. Genetic Variations and Crop
ii. Improvement
a. Why Variations Occur?
i. Physiological Causes of Variation
ii. Genetic Causes of Variation
iii. Biochemical Causes of Variation
iii. Induced (or Directed) Causes of
Variation
iv. In Vitro Mutation
v. Applications of Tissue Culture-
derived Variation
vi. Induction of salt heavy metal
vii. tolerance through tissue culture
viii. The Mechanism of Somaclonal
Variation
b. Source Material and Culture Conditions
i. Determination of Cell Number
c. Forms of Somaclonal Variation
d. Detection and Isolation of Somaclonal Variants
e. Mutagenesis and Somaclonal Variation
f. Somatic Genetics of Nitrogen
g. Metabolism
h. Method for Isolation of Desired Variant Cells for NaCl-Tolerant
from Callus/ Suspension Cultures
i. Applications in Plant Breeding
j. The Future
14. FOURTEEN SYNSEEDSOR SYNTHETIC SEEDS
a. The Natural Seed
b. Development of the Concepts of Tissue Culture and
Artificial Seeds
c. Discovery of Synthetic Seeds
d. Uses and Limitations of Artificial Seeds
i. Potential Uses of Artificial Seeds
e. Production of Synthetic Seeds
f. Encapsulation or Coating of Synthetic Seed
i. Gel Complexation via a Dropping
Procedure
ii. Automate Encapsulation Processi
g. Mass Balance Concept
h. Artificial Seed : Steps of Commercial Production
i. Artificial Seeds : Hydrogel Encapsulation
j. Synthetic Seed and Forest Trees;
15. PLANT TISSUE CULTURE AND GERMPLASM CONSERVATION
a. Germplasm Conservation
i. Germplasm Conservation Using Cell
and Tissue Culture Objectives
ii. Germplasm Facilities
iii. In vitro Conservation
Limitations
b. Ex-Situ Conservation : Classic Approaches
c. Aonservation : New Approaches
i. Conservation: In vitro Propagation
ii. Concervation: Molecular
Technologies
d. Germplasm : Collection and Exchange
i. Problems with Conventional Methods
ii. Problems for recalcitrant seeds
iii. In Vitro Field Collection
iv. In vitro Germplasm Exchange
e. Slow-Growth Storage
i. Classic Techniques
ii. Alternative Techniques
f. Cryopreservation
i. History
ii. Cryostorage or Long-term
Storage...
iii. Classic Techniques
iv. Freezing Techniques
g. Cryopreservation : New Techniques
i. Principle
ii. Encapsulation-Dehydration
iii. Cooling
iv. Recovery
v. Desiccation
vi. Pregrowth-Desiccation
vii. Vitrification
h. Genetic Stability of In-Vitro Conserved Germplasm
i. Slow growth
ii. Cryopreservation
i. Cryopreservation : Methods
i. Collecting, Cleaning and Drying
j. Pollen and Spore Gene Banking
i. Technique of Pollen Conservation
k. Ex-Situ Conservation : Present Status
16. PLANT TISSUE CULTURE AND SECONDARY METABOLITE PRODUCTION
a. Production of Secondary Metabolites in Plants
b. Stages of Secondary Metabolites Production
i. Stress Products
ii. Genetic Engineering Products
c. Uses of Tissue Culture Techniques for Production of
Secondary Plant
d. Metabolites
e. Applications of New Culture Methods for the Producton of
Secondary
f. Metabolites
i. Hairy Root Culture
g. Elicitation of Product Accumulation
h. Biotransformation : Production of Phrmaceutical Compounds
i. Evaluation of Antimicrobial and Antifertility Activity
j. Biological Control on Production of Secondary Metabolites
k. Steroids
l. Ergot, Ergotism and Ergot Alkaloids
17. INDEXING FOR PLANT PATHOGENS
a. What is indexing?
i. Specific crops targeted
b. Plant Pathogen Indexing Techniques
i. Elimination of Bacterial and
Fungal
ii. Pathogen
iii. Indexing Bacteria and Fungi
iv. Indexing of Viruses
v. Bioassays
18. TECHNIQUES FOR CROP IMPROVEMENT
a. Banana and Plantain
b. Cellular Biotechnology
i. Embryo Culture
ii. Direct Adventitious Embryogeny
c. Somatic Embryogenesis
i. Embryogenic Cell Suspensions
ii. Somaclonal Variation
iii. Anther Culture
iv. Germplasm Exchange and
v. Conservation
d. Protoplast Culture
i. Procedure
ii. Cell Suspension and Protoplast
iii. Culture
iv. Protoplast Fusion
e. In vitro mutagenesis
f. Citrus
i. Micropropagation
ii. Anther culture
iii. Protoplast Culture
iv. Somatic Embryogenesis
g. Papaya
i. Micropropagation
ii. Axillary bud/shoot-tip culture
iii. Embryo Culture
iv. Ovule Culture
v. Anther Culture
vi. Protoplast Culture
vii. Suspension Culture
h. Somatic Embryogenesis
i. Callus Culture
ii. Indirect Organogenesis
iii. Indirect Somatic Embryogenesis
i. Hardening
j. Grape
i. Micropropagation
ii. Embryo Culture
iii. Anther Culture
iv. Somatic Embryogenesis
v. Somaclonal Variation
k. Cassava
i. Micropropagation
ii. Regeneration
iii. Elimination of Cassava Mosaic
iv. Cryo-preservation
v. Germplasm Collection
