Plant Tissue Culture and Biotechnology

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Plant Tissue Culture and Biotechnology L.
Ohnoutkova and R. N. Trigiano
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Way Too Much To Do In This Time Slot

• Brief review of anatomy/morphology of plants
• Tissue culture basics some definitions
• Some historical considerations
• Basics of tissue culture laboratory details
• Techniques and Uses

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BASIC ANATOMY

• ROOTS
• STEMS
• LEAVES
• MERISTEMS
• FLOWERS

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A
B
Cortex
Epidermis
Endodermis
Vascular cambium
Phloem
Pericycle
Endodermis
Vascular tissue
Xylem
Cortex
C
D
Cortex
Epidermis
Xylem
Vascular tissue
Phloem
Pith
Pith
Cortex
Endodermis
Pericycle
Exodermis
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SP
Vasc. tiss.
A
B
IFC
PF
SX
PP
E
FC
Pith
IFC
PX
Cortex
Pith
Cortex
C
D
X
P
L
Adv. root
Vasc. bundles
Coll.
Cortex
Fund tiss.
Vasc. tiss.
Pith
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A
B
Vein
Midvein
Epidermis
Buliform cell
Epidermis
X
Mesophyll
X
P
P
Vein

PL
Mesophyll
S
Stoma with guard cells
C
D
AM
Shoots
Shoots
E
Mesophyll
Leaf segment
Root
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A
B
Stem
L
Ax. bud
PET
PET
LP
AM
TR
Stem
VT
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A
B
STY
SEP
OVY
PLA
PET
LOC
CAR
OVL
OVL
CAR
OVY
FIL
PET
FIL
REC
SEP
C
D
EPI
TEP
END
TAP
STA
OVY
POL
VAS
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Plant Tissue Culture - Definition The growth
and development of plant seeds, organs, explants,
tissues, cells or protoplasts on nutrient media
under sterile (axenic) conditions.
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• Explant - Definition 
• This means to simply cut-out a very small piece
  of leaf or stem tissue, or even isolate
  individual cells, and place them in a tissue
  culture container.
• The tissue has to be surface-sterilized so it
  will not have any contaminating bacteria or
  fungus. 
• It is then placed inside the tissue culture
  vessel (dish, jar, etc.)containing a gel called
  agar.  In the agar is dissolved all the sugar,
  nutrients and plant growth regulators the explant
  needs.

 
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• Characteristic of Plant Tissue Culture Techniques
• Environmental condition optimized (nutrition,
  light, temperature).
• Ability to give rise to callus, embryos,
• adventitious roots and shoots.
• Ability to grow as single cells (protoplasts,
  microspores, suspension cultures).
• Plant cells are totipotent, able to regenerate a
  whole plant.

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Totipotency or Totipotent
The capacity of a cell (or a group of cells) to
give rise to an entire organism.
Differentiation (De-)
The physiological and morphological changes that
occur in a cell, tissue, or organ during
development.
Organogenesis
The development of tissues and/or organs from
individual cells not from pre-existing meristems.
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A Historical Account of Milestones in the Field
of Plant Tissue and Cell Culture

• 1904 First attempt to embryo culture of selected
  Crucifers. Hannig B., Bot. Zeitung, 62 45-80.
• First In vitro culture of tobacco used to study
  adventitious shoot formation. Skoog F., Am. J.
  Bot., 31 19-24.
• 1949 Culture of fruits In vitro. Nitsch J. P.,
  Science, 110 499.
• 1962 Development of MS medium. Murashige T. and
  Skoog F., Physiol. Plant., 15 473-497.
• 1967 Haploid plants from pollen grains of
  tobacco. Bourgin J. P. and Nitsch J. P., Ann.
  Physiol. Veg., 9 377-382 10 69-81.
• 1977 Successful integration of T-DNA in plants.
  Chilton M. D. et al., Cell, 11 263-271.
• 1985 Infection and transformation of leaf discs
  with Agrobacterium tumefaciens and regeneration
  of transformed plants. Horsch R. B. et al.,
  Science, 227 1229-1231.

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Laboratory Organization  

• General laboratory and media preparation area
• Transfer area
• Culturing facilities
• Washing facility

Spacious media prep laboratory with good water
supply and machine-aided media dispensing
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Large autoclave
Adequate storage room for media usually at 2-6C
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Transfer room
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Growth room that controls light, temperature,
humidity. Also needs to be isolated from all
other operations of the laboratory.
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Whats in Tissue Culture Medium?

• Water
• Mineral Salts
• Carbon Source(s)
• Vitamins
• Other Complex Addenda
• Plant Growth Regulators

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• The Plant Tissue Culture Protocols are part of
  Sigma's growing offer in Plant Biotechnology. We
  have added helpful information in each protocol
  including
• Media Preparation
• Media Formulation
• Sterilization Techniques
• Storage

http//www.sigmaaldrich.com/Area_of_Interest/Life_
Science/Plant_Biotechnology/Tissue_Culture_Protoco
ls.html
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WATER
Glass Distilled Heat Condensation Cartridge
System -- Filtered
Nanopure System
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Tissue Culture Medium -- Minerals
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Major Mineral Nutrients (mM)

• Nitrogen as Either Nitrate (NO3) and Ammonium
  (NH4)
• KNO3, NH4NO3, Ca(NO3)2 etc.
• Calcium as CaCl2 or Ca(NO3)2

• Magnesium as MgSO4

• Potassium as KCl or K2HPO4

• Phosphorus as K2HPO4 or KH2PO4 or Na Salts

• Sulfur as Many SO4

20-50 of Osmotic Potential
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Minor Mineral Elements (uM)

• Boron (B)
• Cobalt (Co)
• Iron (Fe --Usually Chelated with NaEDTA)
• Manganese (Mn not Mg)
• Molybdenum (Mo)
• Copper (Cu)
• Zinc (Zn)
• Iodine (I)

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Carbon Sources

• Cane Sugar Sucrose (Fructose and Glucose)

• Corn Sugar Fructose

• Maltose, Glucose Sorbitol, Raffinose and Other
  Sugars

Typically Added Between 20 and 40 g/l
20-50 of the Osmotic potential of the medium
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Vitamins and Other Organic Compounds
Vitamins usually added to medium at 0.2-1.0 mg/L
Vitamin B1 or Thiamine is considered essential
-- Carbohydrates
Vitamin C Antioxidant
Yeast extract Source of Many B Vitamins
Rarely Used
Inositol or myo-inositol Really a Sugar Alcohol
-- Membranes
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Vitamins and Other Organic Compounds
Coconut Milk (Really the Water) Source of PGRs
(Kinetin and/or Zeatin) Varies Greatly
Casein Hydrolysate or Peptone (Amino Acids),
Ammonium, etc.
Polyamines Somatic Embryogenesis, Root
Formation
Activated Charcoal, PPVP, Ascorbic and Citric
Acid -- Polyphenols
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Plant Growth Regulators

• Auxins -- IAA, IBA, NAA, 2,4-D, TDZ, Dicamba,
  etc.

• Cytokinins Kinetin, BA, 2iP, Zeatin,
  Thidiazuron, etc.

• Gibberellic Acids -- More Than 60 Forms GA 4 7
  Most Commonly Used

• Abscisic Acid -- Cis and Trans Forms

• Ethylene The Only Gaseous PGR

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Discovery of auxin - Went
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Prunus species
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Plant Growth Regulators

• Used in Concentrations of 0.001 10 uM

• Many Can Be Autoclaved (Especially Synthetic Such
  as 2,4-D, Dicamba, TDZ, BA), But Others Degrade
  With Heat and Should Be Filter-Sterilized (IAA,
  Kinetin, Zeatin, etc).

• Most Have Interactions With Each Other -- Can
  Cause a Multitude of Effects

• Can be Prepared in Water, KOH, Ethanol, DMSO

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Preparation of Plant Growth Regulators
Example Benzyladenine or BA

• Dissolve 100 mg of Either BA (Benzyladenine) in 5
  ml of 95 Ethanol or 1.0N KOH
• Bring Volume to 100 ml with Water
• Yields 1mg/ml
• Store at 4C (Lasts Over a Year if Not
  Contaminated)

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Working Conc. (mg/L)
Powder Storage
Liquid Storage
Mol. Wt.
µM for 1mg/L
Solvent
Diluent
 
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Add 30 g Sucrose and Plant Growth Regulator(s)
Bring to One Liter with Distilled Water
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Adjust pH to About 5.8 and Then Add 8g Agar per
Liter of Medium
OR
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Agar and Alternatives (Support)

• Crude Agar Contains Lots of Impurities
  Minerals, Organic Compounds, which may interfere
  with tissue culture

• Phytoagar is Purified (Lacking Most Impurities)
  and Has a Melting Point of About 65C and a
  Gelling Point Between 40-50C

• Agarose is A Purified Fraction of Agar and
  Typically Has Low Melting and Gelling Points.
  More Expensive and Use for Protoplasts

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Agar and Alternatives

• Gellan Gums Gelrite and Phytagel Require
  Additional Ca Ions to Gel. Care Must be Taken to
  Assure That Tissues Have Sufficient Calcium for
  Growth

• Mechanical Supports Filter Paper Bridges,
  Rafts, Rock Wool (Fiberglass), Foam and Glass or
  Polyurethane Beads

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Autoclave
15 lbs/sq. in yields 121 C or 250 F
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• From one to many propagules rapidly
• Potential for disease-free propagules
• Rapid build up of stock of new, superior variety

Micropropagation
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Tissue Culture of Roses
Bob Trigiano and
Bonnie Ownley
The University of Tennessee
Photo by Mark Windham
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Stage 0 Selection of Materials
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Stage I Placing Your Tissue in Culture

• Surface sterilization with household bleach
  (20) and dishwashing liquid (two drops)
• Sterile water
• Sterile waste beaker

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Preparing the Tissue
Cut Stem Above and
Below a Node (Leaf)
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Trim Most of the Leaf From the Stem -- Leave a
Small Piece of the Petiole
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Surface Sterilization with 20 Clorox and two
drops of detergent for 10 Minutes
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Rinse with Sterile Water Three Times for Five
Minutes Each
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Sterilize Forceps and Scalpels
Flame and Cool
Burn-Off Alcohol
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Preparing the Tissue for Culture
Trim the Petiole
Cut the Ends
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Stage II MultiplicationThree Weeks After Culture
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Shoot Elongation
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Callus Growth
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Contamination
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Stage III Rooting (IBA Medium)Rose Shoots After
Four Weeks
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Rooted Shoots After Four Weeks
Remove from Test Tube and Wash with Water
Keep Moist at All Times!
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Stage IV Acclimatization of Plantlets
Rooted Shoot in Soilless Medium
Plastic Bag to Maintain High Humidity
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Acclimatization by misting at Twyford
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Meristem Tip Culture for Virus Elimination
1
2
4
3
0.5 mm
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0.5 mm
5
7
Note quick browning of meristems
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Types of Cultures

•  
• Seed culture
• Organ culture
• Callus culture
• Cell culture
• Protoplast culture

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Different Techniques of Plant Tissue Culture

• Callus and Cell culture
• Somatic embryogenesis
• Haploid culture
• Protoplast culture
• Micropropagation
• Organogenesis
• Production of virus-free plants
• Somaclonal variation
• In vitro Mutagenesis

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Somatic Embryogenesis from Grape Callus
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Somatic Embryogenesis Stimulation of callus or
suspension cells to undergo a developmental
pathway that mimics the development of the
zygotic embryo.
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