Tissue culture is a technique used to grow plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. It has various applications in horticulture including micropropagation, germplasm conservation, haploid and dihaploid production, embryo rescue and synthetic seed production. The process involves selecting an explant from a mother plant, inducing callus formation, initiating shoot and root development, and acclimatizing the plantlets. Factors like the growth medium, environmental conditions and explant source influence the outcome. Tissue culture has advantages like rapid mass propagation of disease-free clones and conservation of endangered species, but also risks of genetic variation and infection if not performed carefully.
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,
Hello ever one i hope its useful for preparation of notes regarding plant tissue culture for Pharmacognosy .. B.pharm II yr IV sem.. plz give comments it may useful for me and i can rectify the things.
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,
Hello ever one i hope its useful for preparation of notes regarding plant tissue culture for Pharmacognosy .. B.pharm II yr IV sem.. plz give comments it may useful for me and i can rectify the things.
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
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
The presentation gives overview of production of secondary metabolites using callus culture as well as tissue culture techniques. Various batch and continuous culturing process are described on the basis of secondary metabolite to be synthesised.
Plant tissue culture,its methods, advantages,disadvantages and applications.Komal Jalan
Plant tissue culture is the most widely used technique for growing very large number of plant using a very small part of the main plant(explant). Tissue culturing is very common for many popular and demanding crops.Few of them discussed here are Potato,Papaya,Pinepple,Banana,Gerbera,Sunflower,Orchids
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
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
The presentation gives overview of production of secondary metabolites using callus culture as well as tissue culture techniques. Various batch and continuous culturing process are described on the basis of secondary metabolite to be synthesised.
Plant tissue culture,its methods, advantages,disadvantages and applications.Komal Jalan
Plant tissue culture is the most widely used technique for growing very large number of plant using a very small part of the main plant(explant). Tissue culturing is very common for many popular and demanding crops.Few of them discussed here are Potato,Papaya,Pinepple,Banana,Gerbera,Sunflower,Orchids
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.
INVITRO CULTURE: TECHNIQUES, APPLICATIOSNS & ACHIEVEMENTS.
INVITRO TECHNIQUES AND BIOTECHNOLOGY USE IN AGRICULTURE AND CROP IMPROVEMENT. APPLICATIONS OF VARIOUS BIOTECHNOLOGICAL TECHNIQUES AND METHODS. TISSUE CULTURE, MICROPROPAGATION, EMBRYO CULTURE, ANTHER CULTURE, POLLEN CULTURE, ENDOSPERM CULTURE, OVULE CULTURE, OVARY CULTURE, ETC.
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.
Micropropagation (tissue culture or invitro culture) refers to the multiplication of plants, in an aseptic condition and in artificial growth medium from plant parts like meristem tip, callus, embryos anthers, axillary buds etc. It is a method by which a true to type and disease free entire plant can be regenerated from a miniature piece of plant in aseptic condition in artificial growing medium rapidly throughout the year.
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1. TISSUE CULTURE AS A PLANT
PRODUCTION TECHNIQUE FOR
HORTICULTURE CROPS
2. WHAT IS TISSUE CULTURE?
Tissue culture is the term used for “the process of growing
cells artificially in the laboratory.
PLANT TISSUE CULTURE-Technique of growing plant cells,
tissues, and organs in an artificially prepared nutrient
medium under aseptic condition.
REQUIREMENT OF TISSUE CULTURE:
• Appropriate tissue (explant)
• A suitable growth medium (Some of the common media
are MS, LS, Gamborg B5, White’s, Hellar for haploids etc)
• Growth regulators
• Aseptic (sterile) conditions
• Frequent subculturing :
Plant cells are totipotent.
Origin : Early
20th century with
the work of
Gottleib
Haberlandt
(plants).
4. PROCESS OF PLANT TISSUE CULTURE
Selection of explant from
mother plant
Parent plant
Inoculation Light &warmth for shoot
initiation
Shoots are transferred to
rooting media(higher
auxin conc)
Shoot formation Callus induction
Plants are kept for
hardening in green
house ,sold to a
nursery and then
potted up.
5. Tissue culture plants sold to
a nursery & then potted up
The rooted shoots are
potted up (deflasked)
and ‘hardened off’
This is necessary as
many young tissue
culture plants have no
waxy cuticle to prevent
water loss.
6. FACTORS AFFECTING TISSUE CULTURE
• Growth Media
– Minerals, Growth factors, Carbon source, Hormones
• Environmental Factors
– Light, Temperature, Photoperiod, Sterility, Media
• Explant Source
– Usually, the younger, less differentiated explant, the better for tissue
culture
– Different species show differences in amenability to tissue culture
– In many cases, different genotypes within a species will have variable
responses to tissue culture; response to somatic embryogenesis has
been transferred between melon cultivars through sexual hybridization
8. MICRO-PROPAGATION
Large scale production of genetically identical clones in vitro is called
Micro-propagation.
ADVANTAGES:
•Millions of shoot tips from a small piece of plant tissue in a short
period of time and space.
•Maintain Genetically uniform progeny.
•Can produce exact copies of plants required that have desirable
traits.
•Supplies plant throughout the year irrespective of seasonal variation
COMMERCIAL APPLICATION:
•Micro propagation of Orchids demonstrated profit all over the
world.
•Many other crops like banana, papaya, potato, gerbera, carnation,
Anthurium, apple, pear,cherry etc have been grown through micro
propagation are commercially exploited.
•Development of disease resistant crops .Some valuable clones
recovered from virus are
Tobacco- TMV, Sugarcane-Mosaic virus etc.
•Among the innovative techniques of micro propagation, the
concept of somatic embryogenesis with synthetic seed
production or artificial seed technology is very promising,
9. GERMPLASM CONSERVATION
• Maintenance of germplasm of plants vulnerable to pathogen, insects and various climatic
hazards and plants with early embryo degeneration, are difficult to maintain
• Plant species which are endangered, rare threatened with extinction are needed to be
conserved by ex-citu method of germplasm conservation.
• Provides cost effective alternative to growing plants under field condition , nurseries or
green houses.
• Cryopreservation of germplasm is really effective in conservation biotechnology.
EMBRYO CULTURE
• Using embryo culture seed dormancy can be overcome.
• Embryo rescue which can circumvent post zygotic barriers.
• To raise the rare hybrids by rescuing embryo from incompatible crosses is the most popular
application. The production of embryos from somatic or “non-germ” cells.
• Usually involves a callus intermediate stage which can result in variation among seedlings
10. HAPLOID&DIHAPLOID PRODUCTION
• Anther or Ovary/ovule culture
• Production of pure homozygous line in leser time period.
• Androgenic haploids are used to produce different lines of aneuploids like monosomic,nullisomic,trisomic
etc.
• Induction of mutagenesis
• Recessive traits(e.g low glucosinolate in Brassica ) can be made expressed.
• Use in hybrid development and early release of varieties.
11. ARTIFICIAL SEED PRODUCTION
. Synthetic or Artificial Seeds (living seed-like structure) are any totipotent cells(somatic
embryos or meristem tips etc )which is artificially encapsulated by chemicals (Hydrogels)
which behave like true seeds if grown in soil and can be used as substitutes of true seeds and
possess the ability to convert into a plant ex vitro or invitro.
SOMATIC EMBRYO
ARTIFICIAL SEED
COAT
ARTIFICIAL
ENDOSPERM
GERMINATED SYNTHETIC SEEDS
12. PROTOPLAST FUSION
• Production of somatic hybrids.
• Production of CMS line.
• Somatic Hybrids with specific alien gene.
• Production of Unique Nuclear Cytoplasmic
Combination.
• Production of Autotetraploid or Allotetraploid.
• Production of Heterozygous line.
SOMACLONAL VARIATION
• Play an important role in
polyploidisation.
• Genetic variability can be raised which
include resistance to a particular disease,
herbicide resistance,stress tolerance etc
13. ADVANTAGES OF PLANT TISSUE CULTURE
• A single explant can be multiplied into several thousand plants in less than a year - this allows fast
commercial propagation of new cultivars. Very useful solution for the prevention of starvation in third
world countries since the process is highly efficient i.e. by using only one plant it is possible to produce
more than one thousand copies of the same plant with higher productivity if its genome is changed.
• Taking an explant does not usually destroy the mother plant, so rare and endangered plants can
be cloned safely.
• Once established, a plant tissue culture line can give a continuous supply of young plants
throughout the year.
• The production of clones of plants that produce particularly good flowers, fruits, or have other desirable
traits.
• The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
• Allows production of plants from seeds that otherwise have very low chances of germinating and growing,
i.e.: orchids and Nepenthes.
• The regeneration of whole plants from plant cells that have been genetically modified.
• Tissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability.
• The production of plants in sterile containers reduces disease transmission. To clean particular plants of
viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and
agriculture.
• Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture.
• Tissue culture allows fast selection for crop improvement - explants are chosen from superior
plants, then cloned.
14. DISADVANTAGES OF PLANT TISSUE CULTURE
• Chromosomal abnormalities appear as cultures age: undesirable changes.
• Long term culture lose regenerative potential.
• Expensive,labour intensive especially if less than 1000 plants are needed.
• The procedure is quite variable and also it depends on the type of the species
so sometimes it needs trial-and-error type of experiments if there is no
available review about that species.
The procedure needs special attention and diligently done observation.
There may be error in the identity of the organisms after culture.
Infection may continue through generations easily if possible precautions are not
taken
Decrease genetic variability.
15. ADVANTAGES OF TISSUE CULTURE
CROP IMPROVEMENT
• Tissue culture has been exploited to create genetic variability from which
crop plants can be improved.
• to improve the state of health of the planted material and to increase the
number of desirable germplasms available to the plant breeder
• The culture of single cells and meristems can be effectively used to
eradicate pathogens from planting material and thereby dramatically
improve the yield of established cultivars.
• Large-scale micropropagation laboratories are providing millions of plants
for the commercial ornamental market and the agricultural, clonally-
propagated crop market. With selected laboratory material typically taking
one or two decades to reach the commercial market through plant
breeding, this technology can be expected to have an ever increasing
impact on crop improvement as we approach the new millenium.
16. Research on some horticulture crops
1.POTATO (Solanum tuberosum)
Tissue culture plants are when grown
In insect proof net houses produces
mini tubers.
Tissue culture of mini tubers give 20-
30% higher yield.
Protected against bacterial disease
aphids(bacterial disease-crop failure).
Uniform evenly shaped potatoes.
17. BANANA
• Disease & pest free
planting material.
• Little space needed for
multiplying large
number of plants
• Plantlets more
vigourous , uniform
shorter harvest period,
higher bunch weights.
• Allows for faster
distribution of superior
germplasm.
PLUMUlE SHOOT TIP
EMBRYO
GERMINATION
WELL DEVELOPED ROOT
& SHOOT
COMPLETE
PLANTLET
ACCLIMITIZA
-TION STATE
SECONDARY
HARDENING
EMBRYO CULTURE OF WILD Musa SP
SYNTHETIC SEED OF BANANA
EXPLANT-SUCKER
5CM PLUG PLANTS ARE GROWN IN
GREEN HOUSE
18. FRUITS
PINEAPPLE
• Resistance to mealy
bugs,nematodes,wilt
disease,heart and root rot.
• Uniform yield,uniform fruit
weighing.
• Faster growth and spineless
darker leaves
• Sweeter pulp
• Ripens evenly and naturally
PAPAYA
•Rapid and mass propagation of homogenous
and uniform plants for both commercial and
research purposes.
•As the sex of the plant is known,the required
nummber of male to female plant species can
be produced.
•To decrease mortality% and to increase
gaermination%.
•To increase yield.
•To avoid papaya viral disease.
•To obtain export quality food.
19. FLOWERS
SUNFLOWER
• IMMATURE EMBRYO CULTURE-For the shortening of the
entire lifecylce
• To overcome seed dormancy.
• In vitro tissue culture methods in sunflower accelerates
the breeding process.
• Gene from sunflower known as HAHB4, which helps the
plant endure water shortages. That gene is introduced
into wheat, soybean and maize.
GERBERA
Gerbera – hybrid seed
germination followed by axillary
bud proliferation and separation
20. ORCHIDS
• Seeds are exceedingly small with small
reserves,loose their viability very fast so they
need to be germinated in a nutrient medium.
• Higher seed germination % even in rare to
germinate species
• Exact clones are produced .
• Mass and rapid propagation.
• Diseased free plant
• Embryo rescue and culture in hybrid plant
production.
CAPSULE OF ORCHID MATURE CAPSULE BEARING SEEDS
CALLUS (OR PLB)OF ORCHID INVITRO GERMINATION BY SEEDS
(a) Clump formation at the base of leaf segments on
week three, (b) PLB initiation, (c) PLB development
into shoots and roots, (d) direct shoot formation (e)
shoot induced roots on week six and (f) 3-month-
old plantlets
21. Conclusion
• Plant tissue culture represents most promising areas of
application and giving an outlook into the future.
• While tissue-culture offers tremendous benefits, it
could be a bane if undertaken improperly. It could be
the fastest means of spreading disease in any crop. If
the source of the material to be tissue-cultured is
disease-infected, the resulting tissue-cultured plantlets
will carry the disease. Since tissue culture could
produce thousands if not millions of plantlets
tremendously fast, the disease could spread super fast.
Thus conducting tissue culture we must be sure that
the parent plant is free from infection or disease.
planted in a sterile medium, and placed in a greenhouse
Care must be taken during this transition to acclimatize the plant to their new environment
Weak, sterile plant
Usually want to double the chromosomes, creating a dihaploid plant with normal growth & fertility
Chromosomes can be doubled by
Colchicine treatment
Embryo rescue of interspecific crosses
Creation of alloploids (e.g. triticale)
Bulbosum method
Anther culture/Microspore culture
Culturing of Anthers or Pollen grains (microspores)
Derive a mature plant from a single microspore
Ovule culture
Culturing of unfertilized ovules (macrospores)
Sometimes “trick” ovule into thinking it has been
Spontaneous doubling
Tends to occur in all haploids at varying levels
Many systems rely on it, using visual observation to detect spontaneous dihaploids
Can be confirmed using flow cytometry
that can be used for sowing as a seed and that possess the ability to convert into a plant under in vitro or ex vitro conditions and that retain this potential also after storage. It is within an artificial medium that supplies nutrients and is encased in an artificial seed covering. which functionally mimic seeds and can develop into seedling under suitable conditions
The technology designed to combine the advantages of clonal propagation with those of seed propagation and storage. Also cab be used as channel for new plant lines produced through biotechnology advances.