This document discusses various types of plant tissue culture techniques. It defines explants as sterile pieces of plant material used to initiate cultures. Young, rapidly growing tissue is preferred for explants. Common types of in vitro culture discussed include callus culture, cell suspension culture, organ culture, and protoplast culture. Aseptic technique and sterilization methods like heating, filtration, and chemicals are used to prevent contamination during culture.
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
Introduction to organ culture in ptc and root cultureCollege
This presentation gives details about the organ culture in plant tissue culture and its basic applications, also this provide an detailed information about the technique of root culture and gives small view about its appilications.
Embryo culture is the culturing of embryos excised from the ovaries at earlier stages of their development. This technique helps to overcome problems associated with embryo development. Embryos are prevented from development by different factors like incompatibility with the female tissue, absence of endosperm etc. Hybrids produced by wide crosses usually fail to develop inside the ovaries of the mother plants. In such cases, the embryos can be rescued (the technique is called embryo rescue) and grown in culture media so as to produce viable progeny.
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
Introduction to organ culture in ptc and root cultureCollege
This presentation gives details about the organ culture in plant tissue culture and its basic applications, also this provide an detailed information about the technique of root culture and gives small view about its appilications.
Embryo culture is the culturing of embryos excised from the ovaries at earlier stages of their development. This technique helps to overcome problems associated with embryo development. Embryos are prevented from development by different factors like incompatibility with the female tissue, absence of endosperm etc. Hybrids produced by wide crosses usually fail to develop inside the ovaries of the mother plants. In such cases, the embryos can be rescued (the technique is called embryo rescue) and grown in culture media so as to produce viable progeny.
• Seed culture is an important technique when explants are taken from in vitro-derived plants and in propagation of orchids.
• Embryo culture represents the earliest technique to obtain viable offspring following interspecific and intergeneric hybridizations where routine fertilization failed to produce a well-defined and full-term embryo.
• Embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining haploid plants as well as for shortening the breeding cycle.
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
• Seed culture is an important technique when explants are taken from in vitro-derived plants and in propagation of orchids.
• Embryo culture represents the earliest technique to obtain viable offspring following interspecific and intergeneric hybridizations where routine fertilization failed to produce a well-defined and full-term embryo.
• Embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining haploid plants as well as for shortening the breeding cycle.
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
The types of tissue culture can be grouped by the structures formed in culture.
Plantlets
Seedlings
Callus
Somatic EmbryogenesisPlantlet formationThis is the most common form of micropropagation. Uses a portion of the stem with one to several nodes
1. Axillary shoot formation Meristem culture Shoot culture
2. Adventitious shoot formation Diploid plant regenerationPseudocorms
Pseudocorms are the structures initiated after seed germination in orchids Haploid and triploid regeneration
Embryo culture is a component of in vitro fertilisation where in resultant embryos are allowed to grow for some time in an artificial medium before being inserted into the uterus.
Callus cultures are initiated from a small part of an organ or tissue segment called the explants on a growth supporting solidified nutrient medium under sterile conditions. Any part of the plant organ or tissues may be used as the explants.Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation.Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants.Plant Tissue Culture products include Murashige and Skoog media, plant growth regulators, plant growth hormones, plant transformation systems,
Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds, or other plant parts in a sterile
environment on a nutrient medium.
Tissue culture had its origins at the beginning of the 20th century with the work of Gottlieb Haberlandt
(plants).
WHY PLANT TISSUE CULTURES ARE DONE ??
The production of clones of plants that produce particularly good flowers, fruits, or have other desirable traits.
To quickly produce mature plants.
The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
The regeneration of whole plants from plant cells that have been genetically modified.
The production of plants in sterile containers reduces disease transmission
Allows production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and Nepenthes.
To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.
***For PTC, the laboratory must have the following facilities:
Washing facility for glassware and ovens for drying glassware.
Medium preparation room with autoclave, electronic balance and pH meter.
Transfer area sterile room with laminar air-flow bench and a positive pressure ventilation unit called High Efficiency Particulate Air (HEPA) filter to maintain aseptic condition.
Culture facility: Growing the explant inoculated into culture tubes at 22-28° C with illumination of light 2400 lux, with a photoperiod of 8-16 hours and a relative humidity of about 60%.
*****Based on the explants some other plant tissue culture types are:
1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell culture.
Micropropagation and commercial exploitation in horticulture cropsDheeraj Sharma
Micro-propagation – principles and concepts, commercial exploitation in horticultural crops. Techniques - in vitro clonal propagation, direct organogenesis, embryogenesis, micrografting, meristem culture. Hardening, packing and transport of micro-propagules.
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
Definition of hairy root culture ,multiple shoot culture ,Production of hairy root and multiple shoot , advantages an disadvantages of hairy root and multiple shoot culture, Sterilization and sterilizing agents wit concentration and exposure time
Plant biotechnology also known as green biotechnology is the use of biotechnology in plant or crop production. There are several techniques used such as ell culturing. Organ culture, explant culture, cell suspension culture are some culture types. This is a very useful technology in which have several applications like synthetic seed production, somaclonal variation, cybridization, hybridization.
Much faster rates of growth can be induced in vitro than by traditional means.
Multiplication of plants which are very difficult to propagate by cuttings or other traditional methods.
Production of large numbers of genetically identical clones in a short time
Seeds can be germinated with no risk of damping off/ predation.
Under certain conditions, plant material can be stored in vitro for considerable periods of time with little or no maintenance
Tissue culture techniques are used for virus eradication, genetic manipulation, somatic hybridization and other procedures that benefit propagation, crop improvement, and basic research.
By means of tissue culture it is possible to produce pathogen free plantlets by mass multiplication in a very limited amount of area from a very small sterile part of a mother plant. This method is also used to produce/ multiply plants that are to be transported across national border and so for their faster multiplication.But the establishment of a tissue culturing unit needs huge financial investments, skilled labors/technicians and required areas for its establishment are major constraints. Plant tissues grow and multiply in the labs only when there is an uncompetitive, growing condition with uninterrupted supply of nutrients.
Medium:
It contains all the elements that contribute the required nutrients that aid to the growth of the tissues; it is in liquid state or semi-solid in nature. The tissues are grown on the media. It consists of 95% of water, major and minor nutrients, plant growth hormones, vitamins, sugar rich compounds and chelating agents.
Totipotency:
It is the ability of a tissue or an organ of a plant to produce the whole plant, under the optional laboratory conditions and this is called as Totipotency. This is the baseline over which plant tissue culture relies upon.
Callus Culture:
When the cells divide into an undifferentiated mass it is called as callus. Any part of a plant can be used to produce the calli. It may be a stem, leaf, meristem or any other part. It is used to produce variations among the plantlets.
Suspension culture:
The callus produced from the explants are grown on nutrient solutions (that are semi solid) for a period of time and they are induced to produce plants with new traits.
Embryo Culture:
The method of culturing mature and immature embryos in media is called embryo culture. By this method, it is possible to produce plants from dormant seeds and seeds with metabolites that inhibit germination. This method is very important in crop improvement programs.
Somatic Embryogenesis:
When the plants are grown on nutrient media, calli are formed. When these calli are subjected to growth in cytokinin medium, somatic embryos are formed. They are circular, elongated,
Single cell culture
• As stated earlier, cells derived from a single cell through mitosis constitute a clone and the process of obtaining clones is called cloning (asexual progeny of a single individual make up.
1. Sterile pieces of a whole plant from which cultures
are generally initiated
Explants
• The smaller the explant the better the
chances to overcome specific
phytopathological problems (virus,
microplasm, bacteria), but it decreases the
survival rate
2. Inoculum
A subculture of plant material which is already in culture
Generally all plant cells can be used as an explant,
however young and rapidly growing tissue (or tissue at
an early stage of development) are preferred.
Types of explant
4. Types of In vitro culture
(explant based)
Culture of intact plants (seed and seedling culture)
Embryo culture (immature embryo culture)
Organ culture
Callus culture
Cell suspension culture
Protoplast culture
5. Seed culture
Growing seed aseptically in
vitro on artificial media
Increasing efficiency of
germination of seeds that
are difficult to germinate in
vivo
it is possible to
independent on asymbiotic
germination. Production of
clean seedlings for explants
or meristem culture
6. Embryo culture
Growing embryo aseptically in
vitro on artificial nutrient media
Overcoming seed dormancy and
self-sterility of seeds
Study embryo development
7. Organ culture
Any plant organ can serve as an explant to initiate
cultures
No. Organ Culture types
1. Shoot Shoot tip culture
2. Root Root culture
3. Leaf Leaf culture
4. Flower Anther/ovary culture
8. Shoot apical meristem culture
Production of virus free
germplasm
Mass production of
desirable genotypes
Facilitation of exchange
between locations
(production of clean
material)
Cryopreservation (cold
storage) or in vitro
conservation of
germplasm
10. Ovary or ovule culture
Production of haploid plants
A common explant for the initiation of somatic
embryogenic cultures
Overcoming abortion of embryos of wide hybrids at
very early stages of development due to incompatibility
barriers
In vitro fertilization for the production of distant hybrids
avoiding style and stigmatic incompatibility that inhibits
pollen germination and pollen tube growth
11. Anther and microspore culture
Production of haploid plants
Production of homozygous diploid lines
through chromosome doubling, thus reducing
the time required to produce inbred lines
Uncovering mutations or recessive phenotypes
12. Sterilization
Killing or excluding microorganisms or their spores with
heat, filters, chemicals or other sterilants
Tissue culture is an aseptic technique
Aseptic technique:
-Sterile
-Free of pathogenic microorganisms
-Free from the living germs of disease and fermentation
-Conditions established to exclude contaminants
13. Axenic culture
Germfree
Uncontaminated
Free from germs or pathogenic organisms
Free from other microorganism
Containing only 1 organism
A culture of an organism that is entirely free from all
other contaminating organisms
Pure cultures that are completely free of the presence of
other organisms
14. Source of contamination
The explant or culture
The vessels
The media
The instruments
The environment where handling is taking place
15. Aseptic Techniques
Chemical treatments
• disinfectants,
• antibiotics,
• sublimat
Physical treatments
• heating: the most important disinfection method
• electromagnetic radiation,
• filtration
• ultrasonic waves.
16. Disinfectans
They penetrate into bacteria,
They will denature bacterial protein,
They decrease the activity of bacterial enzyme,
They inhibit bacterial growth and metabolism,
They damage the structure of cell membrane,
They change membrane permeability.
17. Disinfectans
– Liquid laundry bleach (NaOCl at 5-6% by vol)
• Rinse thoroughly after treatment
• Usually diluted 5-20% v/v in water; 10% is most common
– Calcium hypochlorite – Ca(OCl)2
• a powder; must be mixed up fresh each time
– Ethanol (EtOH)
• 95% used for disinfesting plant tissues
• Kills by dehydration
• Usually used at short time intervals (10 sec – 1 min)
• 70% used to disinfest work surfaces, worker hands
– Isopropyl alcohol (rubbing alcohol) is sometimes
recommended
18. Antibiotics
Used only when necessary or when disinfestants are
ineffective or impractical
Its use by incorporating in the media
Common antibiotics are carbenicillin, cefotaxime,
rifampicin, tetracycline, streptomycin
Problems with antibiotics
• tend to be selective
• resistance acquisition
• Make unclear, the presence of microbes
• cell/tissue growth inhibition
19. An ideal antibiotics
Broad-spectrum
Did not induce resistance
Selective toxicity, low side effects
Preserve normal microbial flora
19
BC Yang
20. Modes of action
Inhibitors of cell wall synthesis.
Penicillins, cephalosporin, bacitracin,
carbapenems and vancomycin.
Inhibitors of Cell Membrane.
Polyenes - Amphotericin B, nystatin, and
condicidin.
Imidazole - Miconazole, ketoconazole and
clotrimazole.
Polymixin E and B.
Inhibitors of Protein Synthesis.
Aminoglycosides - Streptomycin, gentamicin,
neomycin and kanamycin.
Tetracyclines - Chlortetracycline, oxytetracycline,
doxycycline and minocycline.
Erythromycin, lincomycin, chloramphenicol and
clindamycin.
20
Amphotericin
Tetracyclines
Aminoglycosides
vancomycin
BC Yang
21. UV radiation
Ultraviolet is light with
very high energy levels
and a wavelength of
200-400 nm.
One of the most
effective wavelengths for
disinfection is that of
254 nm.
21
BC Yang
22. Heating
• Oven (dry heat)
Suitable for tools, containers a 160°-180° C for 3 h
• Microwaves (off the shelf)
Useful for melting agar (but not gellan gum types of solidifying agents)
Special pressurized containers are required for sterilizing in a microwave
• Flaming or heating of tools
Flaming – e.g., 95% EtOH in an alcohol burner is useful for sterilizing metal
instruments
Bacticinerators – heats metal tools in a hot ceramic core
Heated glass beads
23. Heating
• Autoclave
Steam heat under pressure (It typically generates 15 lbs/in2
and 250°
F (1.1 kg/cm2
and 121° C))
It is faster and more effective
For liquids (such as water, medium), autoclave time depends on
liquid volume
Recommended autoclaving times (sterilization time only):
250 ml requires 15 min
500 ml requires 20 min
1000 ml requires 25 min
Excessive autoclaving can break down organics – a typical symptom
is caramelized sucrose
24. Heating
• Flaming or heating of tools
Flaming – e.g., 95% EtOH in an alcohol burner is useful for
sterilizing metal instruments
Bacticinerators – heats metal tools in a hot ceramic core
Heated glass beads
25. Filtration
– Filtration of culture medium
• Some medium ingredients are heat labile, e.g., GA, IAA, all proteins,
antibiotics
• Most devices use a paper cellulose filter with small pore spaces (0.22
µm)
• Syringes used for small volumes, vacuum filtration for large volumes
– Filtration of air
• Transfer hoods generate wind at 27-30 linear m per min (or 90-100 ft
per min)
• Too slow and air drops contaminants onto your work surface; too
fast causes turbulence and excess filter wear
• air "corridors" must be kept free of barriers to be effective
29. Callus Culture
Callus:
An un-organised mass of cells, produced when explants are
cultured on the appropriate solid medium, with both an auxin and a
cytokinin and correct conditions.
A tissue that develops in response to injury caused by physical or
chemical means
Most cells of which are differentiated although may be and are
often highly unorganized within the tissue
31. Stimuli :
In vivo : wound, microorganisms, insect feeding
In vitro : Phytohormones
1. Auxin
2. Cytokinin
3. Auxin and cytokinin
4. Complex natural extracts
Callus formation
32. Callus
• During callus formation there is some degree of
dedifferentiation both in morphology and metabolism,
resulting in the lose the ability to photosynthesis.
• Callus cultures may be compact or friable.
Compact callus shows densely aggregated cells
Friable callus shows loosely associated cells and the callus
becomes soft and breaks apart easily.
• Habituation:
The lose of the requirement for auxin and/or cytokinin by
the culture during long-term culture.
•
33. When friable callus is placed into the appropriate liquid
medium and agitated, single cells and/or small clumps of cells
are released into the medium and continue to grow and divide,
producing a cell-suspension culture.
The inoculum used to initiate cell suspension culture should
neither be too small to affect cells numbers nor too large too
allow the build up of toxic products or stressed cells to lethal
levels.
When callus pieces are agitated in a liquid medium, they tend
to break up.
Cell-suspension cultures
34. Cell suspension culture
Suspensions are much
easier to bulk up than
callus since there is no
manual transfer or solid
support
Cell suspension culture
techniques are very
important for plant
biotransformation and
plant genetic
engineering.
36. Protoplast
The living material of a plant or bacterial cell, including the
protoplasm and plasma membrane after the cell wall has been
removed.
37. Plant Regeneration Pathways
Existing Meristems (Microcutting)
Uses meristematic cells to regenerate whole plant.
Organogenesis
Relies on the production of organs either directly from an
explant or callus structure
Somatic Embryogenesis
Embryo-like structures which can develop into whole plants in a
way that is similar to zygotic embryos are formed from somatic
cells
38. Cell Differentiation
The process by which cells become specialized in form
and function. These cells undergo changes that organize
them into tissues and organs.
Morphogenesis
As the dividing cells begin to take form, they are
undergoing morphogenesis which means the “creation of
form.”
Morphogenetic events lay out the development very early
on development as cell division, cell differentiation and
morphogenesis overlap
39. Morphogenesis
• These morphogenetic events “tell” the organism
where the head and tail are, which is the front
and back, and what is left and right.
• As time progresses, later morphogenetic events
will give instructions as to where certain
appendages will be located.
41. Organogenesis
• The ability of non-
meristematic plant tissues to
form various organs de novo.
• The formation of
adventitious organs
• The production of roots,
shoots or leaves
• These organs may arise out
of pre-existing meristems or
out of differentiated cells
• This may involve a callus
intermediate but often occurs
without callus.
45. Somatic Embryogenesis
• The formation of
adventitious embryos
• The production of
embryos from somatic or
“non-germ” cells.
• It usually involves a callus
intermediate stage which
can result in variation
among seedlings
46. Various terms for non-zygotic
embryos
Adventious embryos
Somatic embryos arising directly from other organs or
embryos.
Parthenogenetic embryos (apomixis)
Somatic embryos are formed by the unfertilized egg.
Androgenetic embryos
Somatic embryos are formed by the male gametophyte.
47. Two routes to somatic
embryogenesis
(Sharp et al., 1980)
• Direct embryogenesis
– Embryos initiate directly from explant in the absence
of callus formation.
• Indirect embryogenesis
– Callus from explant takes place from which embryos
are developed.
50. Somatic embryogenesis as a
means of propagation is
seldom used
High probability of mutations
The method is usually rather difficult.
Losing regenerative capacity become greater with
repeated subculture
Induction of embryogenesis is very difficult with many
plant species.
A deep dormancy often occurs with somatic
embryogenesis
52. Steps of Micropropagation
• Stage 0 – Selection & preparation of the mother plant
– sterilization of the plant tissue takes place
• Stage I - Initiation of culture
– explant placed into growth media
• Stage II - Multiplication
– explant transferred to shoot media; shoots can be constantly
divided
• Stage III - Rooting
– explant transferred to root media
• Stage IV - Transfer to soil
– explant returned to soil; hardened off