Chapter 3

GETTING START WITH TISSUE CULTURE

Concepts
1. Introduction
2. Equipment and supplies
3. Water
4. The culture room
5. Characteristics of tissue culture media
6. Components of tissue culture medium
Inorganic mineral elements
Organic compounds
Plant growth regulators (PGRs)
Agar and alternative culture support systems
7. Preparation of tissue culture medium
Unit of concentration clarified
Making stock solutions of the mineral salts
Sterilizing equipment and media
Preparing the sterile transfer hood
Storage of culture media
8. Preparation of the explants for tissue culture
 Concepts

1. A tissue culture laboratory needs adequate physical space for work and storage,
and for equipments such as an autoclave, a distilled water source, balances,
refrigerators, various laboratory instruments, culture vessels, and flow hoods, to name a
few items

2. There are many growth media available, and type of basal culture medium selected
depend upon the species to be cultured. The growth of the plant in culture is also
affects by the selection of plant growth regulators (PGRs) and environmental (cultural)

3. There are about 20 different components in tissue culture medium. These include
inorganic mineral elements, various organic compounds, PGRs, and support
substances

4. PGRs are typically expressed in media as milligrams per liter (mg/l) or micromoles
(μM). When comparing the effects of several PGRs on tissue culture, prepare media
using micromolar concentrations because an equal of molecules of the various PGRs
will be present in each of the media
 1. Introduction

In a plant tissue culture laboratory has several

functional areas for teaching or for research and its
range from simple to complex

A laboratory has some components:

Operating room
Culture media preparing room
Sterilized room
Growth room
Microscope room
Cell culture room
Facilities and equipments room
Washing and cleaning room
2. Equipment and supplies
Operating room
Air conditions
Computer
Projector
Culture media preparing room
Air conditions
Refrigerators
Deep refrigerator
Magnetic stirer
Balances
pH meter
Media preparation
Sterilized room
Air conditions
UV light
Host laminar
Growth room
Air conditions
UV lights
Shelves (including lighting)
Microscope room
Air conditions
Microscopes
Cell culture room
Shaker
Bioreactor
Temporary culture system
Stable shaker incubator
Facilities and equipments room
HPLC / amino acid, oligosaccharide, bio-active products
HPLC / protein, nucleic acid
GC Gas chromatography
UV-VIS
UV-VIS protein, DNA
LC / MS
GC / MS
PCR
Electrophoresis
RAPD
AFLP
Gun bombardment
Bio-analyzer (protein, DNA)
DNA sequencer
DNA microarray
Washing and cleaning room
Deionization water 1 times
Deionization water 2 times
Distilled water 1 times
Distilled water 2 times
Superior clean water
Autoclave
 3. Water

Tap water contains cation, anions, particulates of various

kinds, resins, microorganism and gas…and it is not suitable
for tissue culture

Water was used in plant tissue culture must be deionization

or distilled water

Water was used in plant tissue culture:

Deionization water for preparing medium
Distilled water for preparing stock
Superior cleaning water for DNA techniques
 4. The culture room

Incubators or hoods were used as equipment to prevent

the infection of microorganism from the air

The culture room has UV light used after a day operating

to sterile the room

The culture room has refrigerators to keep stable of the

working temperature: 26-18oC

Every week, cultured room was steriled by formaldehyde

 5. Characteristics of tissue culture medium

Tissue culture medium is a medium contains the nutrition elements for

tissue and cell living

Tissue culture medium elements as:

Macro elements
Micro elements
Vitamin
Fe-EDTA
Plant growth regulators
Organic matter
Others
Agar
pH=5.8

There are about 20 main media; but they could classify to 3 groups of media
belonged to the concentration of ammonium salts
Rich nutritional media: MS, LS, N6, Nistch
Average nutritional media: B5
Poor nutritional media: WPM

Almost plants favor to MS medium in shoot tip culture and embryogenesis

culture; B5 medium in soybean culture; WPM medium in forest trees culture
 6. Components of tissue culture medium

6.1. Inorganic mineral elements

Macro elements
N NH4NO3 - KNO3
P NaH3PO4.7H2O - KH2PO4 – NaH2PO4 – NH4H2PO4
K KCl2 - KH2PO4 – KNO3
Ca CaCl2.6H2O - Ca(NO3)2.4H2O
Mg MgSO4.7H2O

Fe-solution
Fe Fe-EDTA

Micro elements
Concentration (M)
Mn MnSO4.4H2O 15-100
B H3BO4 6-100
Zn ZN(SO4).7H2O 15-30
Cu CuSO4.5H2O 0.04-0.08
Co CoCl2.6H2O 0.1-0.4
I KI 2.5-20
Mo (NH4)6Mo7O24.4H2O 0.007-1
NaMoO4.2H2O 0.007-1
Vitamins
Concentration (mg/l)
Myo-inositol 100
Acid nicotinic (niacin) 0.5-1
Pyridoxin HCl (B6) 0.05-1
Thiamin HCl (B1) 1-5
Riboflavin (B2) 1-10
Panthothenate calci (B5) 0.5-2.5
Biotin (H) 0.01-1
Acid folic (M) 0.1-0.5
Tocopherol (E) 1-50

Organic compounds
Concentration
Coconut water 10-15%
Yeast extract 1-2 g/l
Casein hydrolysate 1-2 g/l
Bee milk 1-5 g/l
Malt 1-10 g/l
Potato extract 3-5%
Corn extract 3-5%
Banana 3-5%
Plant growth regulators (PGRs)
Concentration (mg/l)
2.4D 0.2-5
α-NAA 0.1-5
β-IAA 5-20
β-IBA 1-5
Kinetin 0.1-2
BA 0.1-2
2iP 0.1-2
GA3 0.1-2

Agar and alternative culture support systems

Sigma agar
Difco Bacto agar
HaiPhong Agar

Antioxidants Concentration
Ascorbic acid 50-150 mg/l
Citric acid 50-150 mg/l
PVP 5-50 mg/l
Activated charcoal 1-3 g/l

pH
5.8 (by KOH or NaOH)
 7. Preparation of tissue culture medium

7.1. Unit of concentration clarified

Percentage based upon volume ( v/v ): 5% (v/v) coconut water

means 50ml CW / liter medium

Percentage based upon weight ( w/v ): 6% (w/v) agar solution

means 6g agar / liter medium

Molar solution: a mole ( M ) is the same number of grams as the

molecular weight (Avogadro’s number of molecules)

Milligram per liter ( mg/l ): this is simple to calculate and use

because it is direct weight and commonly used for macro nutrients
and PGRs. 1mg = 10-3grams

Micrograms per liter ( μg/l ):commonly used for micro nutrients.

1μg = 10-3mg = 10-6g

Parts per million (ppm ): sometimes media components are

expressed in ppm. 1ppm = 1mg/liter
7.2. Making stock solutions of the mineral salts

Mineral salts can be prepared as stock solutions 10 to 1000 times (10x to

1000x) the concentration in the medium

Preparing the stock must be written the labels:

Type of stock Macro (20x)
Type of medium MS
Instructions 10mg/l medium or 1mg=1ml
Date made 1/12/2006
Who made the stock Ms. NT Ngoc Lieu

Example for preparing stock MS (1962)

Stock Macro MS 20x
Stock Micro MS 1000x
Stock Vitamin MS 1000x
Stock Fe-EDTA 200x
Stock BA 1mg = 1ml
Stock NAA 1mg = 10ml
7.3. Sterilizing equipments and media

Tissue culture media, in addition to providing an ideal for the growth of plant cells, are
also ideal substratum for the growth of microorganism (bacteria and fungi)

The significance in sterile is microorganism that has the speed growth is more than
the cells or tissues, and the microorganism will be contaminated the cell and tissue
before they growth.

For this reason it is necessary to sterilize the media, culture vessels, tools, and
instruments, and to surface disinfect the explants as well

Sterilizing in autoclave at temperature used as 121oC at 15psi for 15minutes

7.4. Preparing the sterile transfer hood

The explants were prepared for sterilizing by using chemicals or antibodies

By the chemical as: Ca-Hypochlorite or Na-Hypochlorite (5-10% in 20-10 minutes)

HgCl2 (0.1-1o/oo in 7-2 minutes)
Javelin (50%,v/v, in 15-30 minutes)
By antibodies: Streptomycin, penicillin

The hood (or incubator) was sterilized by surface the glass with alcohol (70%) before
working, and open UV-light after a day working
7.5. Storage of culture media
Stocks of Macro and Micro were prepared for use in 4 weeks
Stock of Vitamin was prepared for using in 3 weeks
Stocks of PGRs were prepared for using in 2 weeks
Refrigerator (10oC) was used to storage the stock
Deep refrigerator (2-4oC) was used to store the powder form of vitamin and
PGRs
 8. Preparation of the explants for tissue culture

8.1. Physiological status of the explants

The explants were young and juvenile physiology
Explants were sourced from shoot tip and lateral bud

8.2. Sterilization method

Step1 Washing by soap and tap water
Step2 Primary sterile by alcohol 70% in 1 minutes (washed by 3 times of steriled water)
Step3 Sterilizing by chemical Ca-Hypochlorite (washed by 3 times of steriled water)
Step4 Prepare the explants to the favored size and culture in test tube
Step5 Transfer test tube to growth room

8.3. The size of explants in tissue culture

Meristem 0.1-1 mm
Shoot tip 5-10 mm
Anther 0.1-1 mm
Leaves 5-10 mm
Root 1-20 mm
Stem 5-20 mm
Chemicals
 Distilled Water

Ion exchange Distilled water system

 Balance - pH meter

Analyzed balance pH meter

Media Preparation
 Container type

Container Petri dish Rouge Bottle

Test tube PP box Nylon bag Nylon bag

 Autoclave

Bench autoclave

Stand autoclave
Industrial Autoclave
Incubator (30-200oC)
Hood
Plant Tissue Culture in Hood
Culture Room
Mother plant (donor plant)
Explants
Juvenility located in trees
Juvenility gradients in trees

B: Enough mature
F: Youngest position
 Juvenility and Propagation

(A) embryo (B) seedling (A-B) Micropropagation

(C) Cutting with root development (D) Cutting with poor root
Sterilization
Infected by fungi
Infected by bacteria
 BROWNING - Remedies

Explants were browned

-Release the agents produce phenol

-Supply the antioxidants
-Inhibit the activities of phenolase
-Decrease the activities of phenolase
-Admen the agents prevent browning
of activated charcoal or PVP

Inhibition the browning

 BROWNING - Remedies

+ activated charcoal

+ PVP
Charcoal
Growth Room
GROWTH ROOM – Infection checking
GROWTH ROOM – Industrial production
AUXIN - Natural
 AUXIN - Rooting

Yucca aloifolia Mungbean

AUXIN – 2.4D and NAA
CYTOKININ
CYTOKIN / AUXIN
Interactions
GIBBERELLIN
ABA - Compound
 CYCOCEL (CCC)

Inhibition of shoot growing

ETHYLENE
Relative humidity (RH %)

RH in bottle is 100%
Refrigerator
Freezer ( -80oC )
PHYTOTRON - Environmental chamber
Phytotron
Nitrogen Tank

- 196oC
 Shaker

Platform

Rotate

Plagiotrop
SHAKER – for micropropagation
Bioreactor
 Bioreactor

30 L
100 L
Embryo Culture System
Embryo Culture Shaker
Microscope
 Centrifuge

Stand

Bench
Cell Collection System
Tissue Cutting
Microtome
ELISA
UV / VIS - Spectrophotometer
HPLC
Analytica - HPLC
MS – Mass Spectrophotometer
Analytica - GC
PEPTIDE - Synthesizer
DNA / RNA - Synthesizer
PROTEIN - Crytalography
DNA - Spectrophotometer
DNA - Analyzer
DNA - Electrophoresis
DNA - PCR
DNA - Sequencer
DNA - Purifier
DNA - Hybridization
DNA – Scan Array System
DNA – Gel Dryer
DNA – Film Processor
DNA – Gendoc Camera System
DNA – Freeze dryer
DNA – Evaporation dryer
DNA - AFLP
DNA – Western Blot
DNA – Southern-Northern Blot
Reflectrometer

Colormeter
Luminometer
RH - Food Sun-photometer