1. COLLEGE OF BIOTECHNOLOGY
Topic : Plant tissue culture ( History, sterilisation techniques, micro and macro
nutrients)
1
Submitted by : Shruti
Id No : 3364
PhD Plant molecular Biology and Biotechnology
2. Planttissueculture
A plant tissue culture laboratory, whether for research or for commercial purpose, should provide certain
basic facilities, such as
(i) Washing and storage of glassware, plasticware and other labwares.
(ii) Preparation, sterilization and storage of nutrient media.
(iii) Aseptic manipulation of plant material.
(iv) Maintenance of cultures under controlled conditions of temperature, light and humidity.
(v) Observation of cultures
(vi) Hardening of in vitro developed plants.
•
2
Plant Tissue culture involves the use of small pieces of plant
tissue (explants) which are cultured in a nutrient medium under
sterile conditions.
3. HISTORY
• Gottlieb Haberlandt, a German botanist, made the first attempts to
culture fully differentiated single cells isolated from the leaves in a
simple nutrient solution of Knop.
• The purpose of this experiment was to achieve divisions in these cells
and obtain complete plants from them to verify the concept of cellular
totipotency.
• 1902 Haberlandt presented the classic paper describing his pioneering
work, he is justifiably recognized as the father of plant tissue culture.
3
4. • A new line of investigation was initiated by Hannig (1904) that later emerged as an important applied
area of plant tissue culture. He excised nearly mature embryos of some crucifers and successfully
cultured them to maturity on mineral salts and sugar solution.
• In 1922, Kotte in Germany and Robbins in the USA suggested that the meristematic cells in shoot buds
and root tips could possibly be used to initiate in vitro cultures.
• 1925, 1929 Laibach demonstrated the practical application of embryo culture to produce interspecific
hybrids between sexually incompatible parents (Linum perenne x L. austriacum).
4
5. • In 1932, White started his famous work on isolated root
culture, White established continuously growing cultures
of tomato root tips.
• In 1937 White formulated the first synthetic plant tissue
culture medium (WM).
• 1939 Gautheret, Nobécourt and White, independently,
established continuously growing tissue cultures.
• These three scientists are credited for laying the foundation
for further work in the field of plant tissue culture.
5
6. • Van Overbeek et al. (1940) demonstrated for the first time,
the stimulatory effect of coconut milk on development of
young embryos of Datura.
• Almost the same time, Kranz and Lörz (1993) and Holm et
al. (1994) succeeded in in vitro cultivation of excised in
vitro and in vivo formed zygotes, respectively.
• 1947 Braun proposed the concept of tumor inducing
principal (TiP) of Agrobacterium tumefaciens responsible
for autonomous growth of crown gall tissue.
6
7. • 1952 Morel & Martin developed the technique of meristem culture.
• 1954 Muir et al. succeeded in inducing divisions in mechanically
isolated single cells cultured in the presence of a nurse tissue.
• 1955 Miller et al. discovered the first cytokinin (kinetin) from
autoclaved herring sperm DNA.
• 1957 Skoog and Miller put forth the concept of chemical control of
organogenesis (root and shoot differentiation) by manipulating the
relative concentrations of auxin and kinetin
• 1958 Steward (USA) and Reinert (Germany), independently, reported
the formation of embryos by the somatic cells of carrot (somatic
embryogenesis).
7
8. • In 1962, Murashige and Skoog formu- lated the now most
extensively used plant tissue culture medium, popularly called
MS medium.
• 1960 Morel described a method for rapid in vitro clonal
propagation of orchids (micropropagation).
• 1960 Cocking isolated plant protoplasts enzymatically.
• 1962 Kanta et al. developed the technique of in vitro pollination;
viable seed formation by in vitro pollination of naked ovules.
• 1962 Murashige & Skoog formulated the most widely used plant
tissue culture medium (MS).
• 1964 Guha and Maheshwari produced the first androgenic
haploid plants of Datura by anther culture.
• 1965 Johri and Bhojwani demonstrated the totipotency of
triploid endosperm cells.
8
9. Sterilization
• Whether it is labware or culture medium, plant material or environment in the laboratory, instruments
used for culture or the operator himself, all are sources of infection.
• It is, therefore, absolutely essential to maintain a completely aseptic environment inside culture vessels.
• Sterilization may be defined as the statistically complete destruction of all microorganisms including
the most resistant bacteria and spores.
9
10. 10
Disinfection: The destruction or removal of vegetative pathogens but not bacterial endospores. Usually used only on inanimate objects.
Sterilization: The complete removal or destruction of all viable microorganisms. Used on inanimate objects.
Antisepsis: Chemicals applied to body surfaces to destroy or inhibit vegetative pathogens.
Chemotherapy: Chemicals used internally to kill or inhibit growth of microorganisms within host tissues.
11. 11
Physical methods of microbial growth control are used in industry, medicine, and in the home to achieve
decontamination, disinfection, and sterilization.
• Heat
Moist heat where killing occurs by denaturation of proteins inside the cell,eg, autoclaving ,pasteurization,
intermittent tyndalization.
Dry heat where killing of microbes occurs through oxidation of the cell, eg, hot air oven, incerination,
flaming etc.
• Radiation
Ionizing radiation (UV) and non-ionizing radiation(X-rays) where microbial DNA, occurs as a result of
ionization and free radical production (gamma-rays and electrons) or excitation (UV light).
Physical methods
12. 12
Chemicals are routinely used to control microbial growth, and an antimicrobial agent is a natural or
synthetic chemical that kills or inhibits the growth of microorganisms.
•Oxidizing Agents High-level disinfectants and antiseptics such as peroxides, ozone, and per acetic acid
kill by oxidation of microbial enzymes.
•Phenolic and alcohols are popular disinfectants that act by denaturing proteins and disrupting cell
membranes
•Halogens (iodine and chlorine) kill by oxidizing cellular constituents; cell proteins may also be
iodinated. Iodine is applied as a tincture or iodophore. Chlorine may be added to water as a gas,
hypochlorite, or an organic chlorine derivative.
•Heavy metals tend to be bacteriostatic agents. Their use can be such as the use of silver nitrate in the
eyes of newborn infants and copper sulfate in lakes and pools.
• Cationic detergents and surfactant are often used as disinfectants and antiseptics; they disrupt
membranes and denature proteins.
•Aldehydes derivates such as formaldehyde and glutaraldehyde can sterilize as well as disinfect because
they kill spores.
•Ethylene oxide (Gaseous agent) gas penetrates plastic wrapping material and destroys all life forms by
reacting with proteins. It is used to sterilize packaged, heat-sensitive materials.
Chemical methods
13. 13
Filtration is an excellent way to reduce the microbial population in solutions of heat-labile material by use
of a variety of filters. Filters are used to sterilize these heat-labile solutions. There are two types of filter:
(a) depth filters and (b) membrane filters.
• Depth filters:
Depth filters are of the following types: (a) Candle filters, (b)Asbestos filters and (c)Sintered glass filters
• Membrane filters: Membrane filters are made up of various types
(a) cellulose acetate, (b) cellulose nitrate, (c) polycarbonate, (d) polyvinylidene fluoride, or (e) other
synthetic materials; nucleopore filters.
Mechanical methods
14. • Glassware and Plasticware
The glass culture vials may be dry sterilized before pouring the medium to kill such bacteria, which
might withstand autoclaving.
For pre sterilized medium the culture vials with proper closure may be sterilized by autoclaving or dry
heating in an oven at 160–180 °C for 3 h.
Not all types of plastic labware can be heat sterilized. Only polypropylene, polymethylpentene,
polyallomer, Tefzel ETFE and Teflon FEP may be repeatedly autoclaved at 121 °C.
Methodsappliedforsterilisationinplanttissue
culture
14
15. • Culture Medium. Mostly, the culture medium is sterilized by autoclaving. Autoclave is an apparatus
in which sterilization is done by steam heating under pressure. The culture vials containing medium
and closed with a suitable bacteria proof closure are autoclaved at 15 psi and 121 °C for 15–40 min
from the time the medium reaches the required temperature and pressure.
• Thermolabile compounds cannot be autoclaved along with rest of the nutrient medium. These are,
instead, filter sterilized.
15
A horizontal autoclave (a) and a small vertical autoclave (b)
16. • Instruments. The instruments for aseptic manipulation, such as forceps, scalpels, needles, and
spatula, should be sterilized before use by wrapping in aluminium foil and autoclaving. Again,
during aseptic manipulation the instruments are sterilized several times by dipping in 95 %
ethanol and flaming and used after cooling.
• Several labs use glass bead sterilizers (Steripot), in which temperature rises to 250 °C within 5–
20 min. Embedding the instruments in the heated beads for 5–7 min is adequate to sterilize
them. Infrared sterilizers are also available for sterilizing instruments in the hood.
•
16
17. • Plant Material Plant surfaces harbour a wide range of microbial contaminants. This source of
infection can be avoided by thorough surface sterilization of the plant material before planting it on
the nutrient medium.
• Plant tissues can be surface sterilized using various sterilants. The sterilant type, its concentration
and the duration of treatment have to be determined empirically.
•
17
Sterilizing agent Duration (min) Effectiveness
Calcium hypochlorite 5–30 Very good
Sodium hypochlorite 5–30 Very good
Hydrogen peroxide 5–15 Good
Bromine water 2–10 Very good
Silver nitrate 5–30 Good
Mercuric chloride 2–10 Satisfactory
Antibiotics 30–60 Fairly good
Table : Effectiveness of some surface sterilizing agents
18. Transfer Area and Growth room.
• The chances of the cultures getting infected exist whenever the culture vials are opened to inoculate the
sterilized plant tissue on the medium (inoculation) or for subculturing. To avoid this, all transfer operations
are carried out under strictly aseptic conditions.
• Most laboratories use laminar air- flow cabinets to carry out aseptic manipulations. These are very
convenient, as work can be stared within 10–15 min of switching on the airflow and can be continued for
long hours.
• A laminar airflow cabinet basically has drum type fans rotating at high speed to suck air from outside
through a coarse filter which removes large particles, and the semi-clean air is thrown in the opposite
direction. The dust-free air, which is under pressure, gets pushed through a fine filter, known as the ‘‘High
Efficiency Particulate Air (HEPA)’’ filter.
• The HEPA filter prevents the entry of particles larger than 0.3 lm. The ultra clean air, free of fungal and
bacterial contaminants, flows through the working area in the direction of the operator.
18
19. Macronutrients and micronutrients
Plant tissues and organs are grown in vitro on artificial media, which supply the nutrients necessary for growth.
Macronutrients.
• the macronutrients are required in millimolar (mM) quantities.
• Calcium (Ca2+), potassium (K+), magnesium (Mg2+), nitrogen (NO3-), sulphur (SO42-) and phosphorous (PO43-) are the
macroelements and the essential ingredients of plant tissue culture media.
• These are added to the medium as calcium nitrate, potassium dihydrogen phosphate and magnesium sulphate.
• Alternately, potassium and calcium may be provided as KCl or KNO3 and CaCl2.2H2O, respectively.
• Nitrogen is one of the vital elements required for the growth of plants in cultures as also in nature. Inorganic nitrogen is usually
supplied in the form of ammonium (NH4+) and/or nitrate (NO3-) ions. Sometimes, organic form of nitrogen, such as urea,
amino acids (glutamine) and/or casein hydrolysate (a complex mixture of amino acids and ammonium), is also included in the
medium.
19
Micronutrients.
•Some micronutrients, although required in small quantities, are essential for tissue growth in cultures.
They act as cofactors of enzymes.
•boron (B), zinc (Zn), manganese (Mn), iron (Fe), copper (Cu), molybdenum (Mo), chlorine (Cl).
20. Table : Role of culture media constituents Bhojwani, S. S., & Dantu, P. K. (2013)
