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Plant tissue culture

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Plant tissue culture is a practice used to propagate plants under sterile conditions, often to
produce clones of a plant. Different techniques in plant tissue culture may offer certain
advantages over traditional methods of propagation, including:
• The production of exact copies 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 that allows them to be moved with greatly
reduced chances of transmitting diseases, pests, and pathogens.
• The production of plants from seeds that otherwise have very low chances of germinating
and growing, i.e.: orchids and nepenthes.
• To clean particular plant of viral and other infections and to quickly multiply these plants
as 'cleaned stock' for horticulture and agriculture.
Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole
plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, or
(less commonly) roots can often be used to generate a new plant on culture media given the
required nutrients and plant hormones.

• 1 Techniques
• 2 Choice of explant
• 3 Applications
• 4 Laboratories

[edit] Techniques
Modern plant tissue culture is performed under aseptic conditions under filtered air. Living plant
materials from the environment are naturally contaminated on their surfaces (and sometimes
interiors) with microorganisms, so surface sterilization of starting materials (explants) in
chemical solutions (usually alcohol or bleach) is required. Mercuric chloride is seldom used as a
plant sterilant today, as it is dangerous to use, and is difficult to dispose of. Explants are then
usually placed on the surface of a solid culture medium, but are sometimes placed directly into a
liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are
generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones.
Solid media are prepared from liquid media with the addition of a gelling agent, usually purified

In-vitro tissue culture potato explants

The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate
versus ammonium salts or amino acids) have profound effects on the morphology of the tissues
that grow from the initial explant. For example, an excess of auxin will often result in a
proliferation of roots, while an excess of cytokinin may yield shoots. A balance of both auxin
and cytokinin will often produce an unorganised growth of cells, or callus, but the morphology
of the outgrowth will depend on the plant species as well as the medium composition. As
cultures grow, pieces are typically sliced off and transferred to new media (subcultured) to allow
for growth or to alter the morphology of the culture. The skill and experience of the tissue
culturist are important in judging which pieces to culture and which to discard.
As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce
plantlets which, when mature, can be transferred to potting soil for further growth in the
greenhouse as normal plants. Editing Plant tissue culture (section)
[edit] Choice of explant
The tissue which is obtained from the plant to culture is called an explant. Based on work with
certain model systems, particularly tobacco, it has often been claimed that a totipotent explant
can be grown from any part of the plant. However, this concept has been vitiated in practice. In
many species explants of various organs vary in their rates of growth and regeneration, while
some do not grow at all. The choice of explant material also determines if the plantlets developed
via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased
with inappropriate explants. Thus it is very important that an appropriate choice of explant be
made prior to tissue culture.
The specific differences in the regeneration potential of different organs and explants have
various explanations. The significant factors include differences in the stage of the cells in the
cell cycle, the availability of or ability to transport endogenous growth regulators, and the
metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic
ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of
cell division and either concentrate or produce required growth regulating substances including
auxins and cytokinins.
Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that
become problematic during the tissue culture process. Certain soil microflora can form tight
associations with the root systems, or even grow within the root. Soil particles bound to roots are
difficult to remove without injury to the roots that then allows microbial attack. These associated
microflora will generally overgrow the tissue culture medium before there is significant growth
of plant tissue.
Aerial (above soil) explants are also rich in undesirable microflora. However, they are more
easily removed from the explant by gentle rinsing, and the remainder usually can be killed by
surface sterilization. Most of the surface microflora do not form tight associations with the plant
tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization
or localized necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which
are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less
permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the
acceptable conditions of sterilization used for seeds can be much more stringent than for
vegetative tissues.
[edit] Applications
Plant tissue culture is used widely in plant science; it also has a number of commercial
applications. Applications include:
• Micropropagation is widely used in forestry and in floriculture. Micropropagation can
also be used to conserve rare or endangered plant species.
• A plant breeder may use tissue culture to screen cells rather than plants for advantageous
characters, e.g. herbicide resistance/tolerance.
• Large-scale growth of plant cells in liquid culture inside bioreactors as a source of
secondary products, like recombinant proteins used as biopharmaceuticals.
• To cross distantly related species by protoplast fusion and regeneration of the novel
• To cross-pollinate distantly related species and then tissue culture the resulting embryo
which would otherwise normally die (Embryo Rescue).
• For production of doubled monoploid plants from haploid cultures to achieve
homozygous lines more rapidly in breeding programmes, usually by treatment with
colchicine which causes doubling of the chromosome number.
• As a tissue for transformation, followed by either short-term testing of genetic constructs
or regeneration of transgenic plants.
• Certain techniques such as meristem tip culture may be employed that can be used to
produce clean plant material from virused stock, such as potatoes and many species of
soft fruit.
[edit] Laboratories
Although some growers and nurseries have their own labs for propagating plants via tissue
culture, a number of independent laboratories provide custom propagation services. The Plant
Tissue Culture Information Exchange lists many commercial tissue culture labs. Since plant
tissue culture is a very labour intensive process, this would be an important factor in determining
which plants would be commercially viable to propagate in a laboratory.
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1. What is biofertilizer?
Biofertilizers are ready to use live formulates of such beneficial microorganisms which on
application to seed, root or soil mobilize the availability of nutrients by their biological activity
in particular, and help build up the micro-flora and in turn the soil health in general.
2. Why should we use biofertilizers?
With the introduction of green revolution technologies the modern agriculture is getting more
and more dependent upon the steady supply of synthetic inputs (mainly fertilizers), which are
products of fossil fuel (coal+ petroleum). Adverse effects are being noticed due to the excessive
and imbalanced use of these synthetic inputs. This situation has lead to identifying harmless
inputs like biofertilizers. Use of such natural products like biofertilizers in crop cultivation will
help in safeguarding the soil health and also the quality of crop products.
3. What are the benefits from using biofertilizers?
• Increase crop yield by 20-30%.
• Replace chemical nitrogen and phosphorus by 25%.
• Stimulate plant growth.
• Activate the soil biologically.
• Restore natural soil fertility.
• Provide protection against drought and some soil borne

4. What are the advantages of bio-fertilizers?

1. Cost effective.
2. Suppliment to fertilizers.
3. Eco-friendly (Friendly with nature).
4. Reduces the costs towards fertilizers use, especially
regarding nitrogen and phosphorus.

5. What types of biofertilizers are available?

1. For Nitrogen
○ Rhizobium for legume
○ Azotobacter/Azospirillu
m for non legume
○ Acetobacter for
sugarcane only.
○ Blue –Green Algae
(BGA) and Azolla for
low land paddy.
2. For Phosphorous
○ Phosphatika for all
crops to be applied with
Azospirillum and
3. For enriched compost
○ Cellulolytic fungal
○ Phosphotika and
Azotobacter culture


6. What biofertilizers are recommended for crops?

• Rhizobium + Phosphotika at 200 gm each per 10 kg of seed
as seed treatment are recommended for pulses such as
pigeonpea, green gram, black gram, cowpea etc, groundnut
and soybean.
• Azotobacter + Phosphotika at 200 gm each per 10 kg of
seed as seed treatment are useful for wheat, sorghum,
maize, cotton, mustard etc.
• For transplanted rice, the recommendation is to dip the
roots of seedlings for 8 to 10 hours in a solution of
Azospirillum + Phosphotika at 5 kg each per ha.

7. How biofertilizers are applied to crops?

1. Seed treatment:
200 g of nitrogenous biofertilizer and 200 g of Phosphotika
are suspended in 300-400 ml of water and mixed
thoroughly. Ten kg seeds are treated with this paste and
dried in shade. The treated seeds have to be sown as soon
as possible.
2. Seedling root dip:
For rice crop, a bed is made in the field and filled with
water. Recommended biofertilizers are mixed in this water
and the roots of seedlings are dipped for 8-10 hrs.
3. Soil treatment:
4 kg each of the recommended biofertilizers are mixed in
200 kg of compost and kept overnight. This mixture is
incorporated in the soil at the time of sowing or planting.

8. How could one get good response to biofertilizer

• Biofertilizer product must contain good effective strain in
appropriate population and should be free from
contaminating microorganisms.
• Select right combination of biofertilizers and use before
expiry date.
• Use suggested method of application and apply at
appropriate time as per the information provided on the
• For seed treatment adequate adhesive should be used for
better results.
• For problematic soils use corrective methods like lime or
gypsum pelleting of seeds or correction of soil pH by use of
• Ensure the supply of phosphorus and other nutrients.

9. What would be probable reasons for not getting response

from the application of biofertilizers?
1. On account of quality of product
○ Use of ineffective strain.
○ Insufficient population
of microorganisms.
○ High level of
2. On account of inadequate storage facilities
○ May have been
exposed to high
○ May have been stored
in hostile conditions.
3. On account of usage
○ Not used by
recommended method
in appropriate doses.
○ Poor quality adhesive.
○ Used with strong doses
of plant protection
4. On account of soil and environment
○ High soil temperature or
low soil moisture.
○ Acidity or alkalinity in
○ Poor availability of
phosphorous and
○ Presence of high native
population or presence
of bacteriophages.

10. What precautions one should take for using

• Biofertilizer packets need to be stored in cool and dry place
away from direct sunlight and heat.
• Right combinations of biofertilizers have to be used.
• As Rhizobium is crop specific, one should use for the
specified crop only.
• Other chemicals should not be mixed with the biofertilizers.
• While purchasing one should ensure that each packet is
provided with necessary information like name of the
product, name of the crop for which intended, name and
address of the manufacturer, date of manufacture, date of
expiry, batch number and instructions for use.
• The packet has to be used before its expiry, only for the
specified crop and by the recommended method of
• Biofertilizers are live product and require care in the storage
• Both nitrogenous and phosphatic biofertilizers are to be
used to get the best results.
• It is important to use biofertilizers along with chemical
fertilizers and organic manures.
• Biofertilizers are not replacement of fertilizers but can
supplement plant nutrient requirements.
11. Where can I get further information on biofertilizers?
You may visit the following Internet sites:,

Biofertilizers - Biofertilizers are defined as biologically active products or microbial

inoculants of bacteria, algae and fungi (separately or in combination), which may
help biological nitrogen fixation for the benefit of plants.
Biofertilizers also include organic fertilizers (manure, etc.), which are rendered in
an available form due to the interaction of micro-organisms or due to their
association with plants. Biofertilizers thus include the following:
(i) symbiotic nitrogen fixers Rhizobium spp.;
(ii) asymbiotic free nitrogen fixers (Azotobacter, Azospirillum, etc.);

(iii) algae biofertilizers (blue green algae or BGA in association with Azolla);
(iv) phosphate solubilising bacteria;
(v) mycorrhizae;
(vi) organic fertilizers.
The need for the use of biofertilizers has arisen, primarily for two reasons.
First, because increase in the use of fertilizers leads to increased crop productivity,
second, because increased usage of chemical fertilizer leads to damage in soil texture
and raises other environmental problems. Therefore, the use of biofertilizers is both
economical and environment friendly. The pragmatic approach will be to develop the
integrated nutrient supply system involving a combination of the use of chemical
fertilizers and biofertilizers. India is not self sufficient in fertilizer production.

An estimated capital investment of Rs. 7,000 crores was needed by the end of
Seventh Five Year Plan period to achieve self sufficiency. Realizing the importance
of biofertilizers in supplementing the use of chemical fertilizers, the Government of
India had launched the 'National Project on Development and use of Biofertilizers’
during the Sixth Five Year Plan.

Under this 7 project, one national centre and six regional centres and 40 BGA
production centres have been established. These centres will produce 800 tonnes
of Rhizobia and 600 tonnes of BGA annually.

Introduction: Biofertilizers are culture of microorganisms used for inoculating seed or soil or both under ideal
conditions to increase the availability of plant nutrients.

Classification of Bio-fertilizers -
How to use the Bio-fertilizers
Biofertilizers are mixed with little water for preparing slurry. In that slurry small quantity of sugar or jaggary or
gum is added so that the inoculant may get energy for their prolonged survival. The slurry is poured over the
seeds which should be kept in a container. The seed is mixed well with the slurry by pouring the mixture into
another container. Thus by pouring fourth and back into both containers the seed is nicely mixed with inoculant.
Now the treated seed should be dried in cool and dry shady place and sown immediately in the field.
Application of Azolla Culture: There are two methods.
• Azolla can be used as green manure in rice field. In this case Azolla culture is inoculated in the main field
about 15-20 days before transplanting of rice seedling so that it can be incorporated in the field at the
time of puddling.

• The nicely prepared field is divided into smaller plots of 100 sq.m. by raising bunds and 5-10 cm deep
• water level is maintained upto about 20-25 days after inoculation so that a thick azolla mat may be
formed. After about 25 days of inoculation when thick mat is formed, the water level is reduced and
azolla mat is mixed into the soil while weeding.

Water Resources | Soil Conservation & Forestry | Package of Practices

Plant tissue culture

Most methods of plant transformation applied to GM crops require that a
whole plant is regenerated from isolated plant cells or tissue which have been
genetically transformed. This regeneration is conducted in vitro so that the en-
vironment and growth medium can be manipulated to ensure a high fre-
quency of regeneration. In addition to a high frequency of regeneration, the
regenerable cells must be accessible to gene transfer by whatever technique is
chosen (gene transfer methods are described in Chapter 3). The primary aim
is therefore to produce, as easily and as quickly as possible, a large number of
regenerable cells that are accessible to gene transfer. The subsequent regenera-
tion step is often the most difficult step in plant transformation studies. How-
ever, it is important to remember that a high frequency of regeneration does
not necessarily correlate with high transformation efficiency.
This chapter will consider some basic issues concerned with plant tissue cul-
ture in vitro, particularly as applied to plant transformation. It will also look
at the basic culture types used for plant transformation and cover some of the
techniques that can be used to regenerate whole transformed plants from
transformed cells or tissue.
Plant tissue culture
Practically any plant transformation experiment relies at some point on tissue
culture. There are some exceptions to this generalisation (Chapter 3 will look
at some), but the ability to regenerate plants from isolated cells or tissues in
vitro underpins most plant transformation systems.
Plasticity and totipotency
Two concepts, plasticity and totipotency, are central to understanding plant
cell culture and regeneration.
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Plants, due to their sessile nature and long life span, have developed a
greater ability to endure extreme conditions and predation than have animals.
Many of the processes involved in plant growth and development adapt to en-
vironmental conditions. This plasticity allows plants to alter their metabo-
lism, growth and development to best suit their environment. Particularly
important aspects of this adaptation, as far as plant tissue culture and regen-
eration are concerned, are the abilities to initiate cell division from almost any
tissue of the plant and to regenerate lost organs or undergo different develop-
mental pathways in response to particular stimuli. When plant cells and tis-
sues are cultured in vitro they generally exhibit a very high degree of plasticity,
which allows one type of tissue or organ to be initiated from another type. In
this way, whole plants can be subsequently regenerated.
This regeneration of whole organisms depends upon the concept that all
plant cells can, given the correct stimuli, express the total genetic potential of
the parent plant. This maintenance of genetic potential is called ‘totipotency’.
Plant cell culture and regeneration do, in fact, provide the most compelling
evidence for totipotency.
In practical terms though, identifying the culture conditions and stimuli re-
quired to manifest this totipotency can be extremely difficult and it is still a
largely empirical process.
The culture environment
When cultured in vitro, all the needs, both chemical (see Table 2.1) and physi-
cal, of the plant cells have to met by the culture vessel, the growth medium and
the external environment (light, temperature, etc.). The growth medium has to
supply all the essential mineral ions required for growth and development. In
many cases (as the biosynthetic capability of cells cultured in vitro may not
replicate that of the parent plant), it must also supply additional organic sup-
plements such as amino acids and vitamins. Many plant cell cultures, as they
are not photosynthetic, also require the addition of a fixed carbon source in the
form of a sugar (most often sucrose). One other vital component that must also
be supplied is water, the principal biological solvent. Physical factors, such as
temperature, pH, the gaseous environment, light (quality and duration) and
osmotic pressure, also have to be maintained within acceptable limits.
Plant cell culture media
Culture media used for the in vitro cultivation of plant cells are composed of
three basic components:
(1) essential elements, or mineral ions, supplied as a complex mixture of salts;
(2) an organic supplement supplying vitamins and/or amino acids; and
(3) a source of fixed carbon; usually supplied as the sugar sucrose.
2 : Plant tissue culture
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For practical purposes, the essential elements are further divided into the
following categories:
(1) macroelements (or macronutrients);
(2) microelements (or micronutrients); and
(3) an iron source.
Complete, plant cell culture medium is usually made by combining several
different components, as outlined in Table 2.2.
Media components
It is useful to briefly consider some of the individual components of the stock
As is implied by the name, the stock solution supplies those elements required
in large amounts for plant growth and development. Nitrogen, phosphorus,
potassium, magnesium, calcium and sulphur (and carbon, which is added sep-
arately) are usually regarded as macroelements. These elements usually com-
prise at least 0.1% of the dry weight of plants.
Plant tissue culture
Table 2.1
Some of the elements important for plant nutrition and their physiological function.
These elements have to supplied by the culture medium in order to support the growth of
healthy cultures in vitro
Component of proteins, nucleic acids and some coenzymes
Element required in greatest amount
Regulates osmotic potential, principal inorganic cation
Cell wall synthesis, membrane function, cell signalling
Enzyme cofactor, component of chlorophyll
Component of nucleic acids, energy transfer, component of
intermediates in respiration and photosynthesis
Component of some amino acids (methionine, cysteine) and some
Required for photosynthesis
Electron transfer as a component of cytochromes
Enzyme cofactor
Component of some vitamins
Enzyme cofactor, electron-transfer reactions
Enzyme cofactor, chlorophyll biosynthesis
Enzyme cofactor, component of nitrate reductase
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Nitrogen is most commonly supplied as a mixture of nitrate ions (from the
) and ammonium ions (from the NH
). Theoretically, there is an
advantage in supplying nitrogen in the form of ammonium ions, as nitrogen
must be in the reduced form to be incorporated into macromolecules. Nitrate
ions therefore need to be reduced before incorporation. However, at high con-
centrations, ammonium ions can be toxic to plant cell cultures and uptake of
ammonium ions from the medium causes acidification of the medium. In
order to use ammonium ions as the sole nitrogen source, the medium needs to
be buffered. High concentrations of ammonium ions can also cause culture
2 : Plant tissue culture
Table 2.2
Composition of a typical plant culture medium. The medium described here is that of
Murashige and Skoog (MS)

Essential element
Concentration in
stock solution (mg/l)
in medium (mg/l)



33 000
1 650

38 000
1 900


8 800


7 400


3 400



1 240


4 460


1 720







Iron source



5 560


7 460
Organic supplement

20 000
Nicotinic acid
Carbon source

Added as solid
30 000

Many other commonly used plant culture media (such as Gamborg’s B5 and Schenk and
Hildebrandt (SH) medium) are similar in composition to MS medium and can be thought of as
‘high-salt’ media. MS is an extremely widely used medium and forms the basis for many other
media formulations.

50 ml of stock solution used per litre of medium.


5 ml of stock solution used per litre of medium.


Added as solid.
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problems by increasing the frequency of vitrification (the culture appears pale
and ‘glassy’ and is usually unsuitable for further culture). Using a mixture of
nitrate and ammonium ions has the advantage of weakly buffering the
medium as the uptake of nitrate ions causes OH

ions to be excreted.
Phosphorus is usually supplied as the phosphate ion of ammonium, sodium
or potassium salts. High concentrations of phosphate can lead to the precipi-
tation of medium elements as insoluble phosphates.
These elements are required in trace amounts for plant growth and develop-
ment, and have many and diverse roles. Manganese, iodine, copper, cobalt,
boron, molybdenum, iron and zinc usually comprise the microelements, al-
though other elements such as nickel and aluminium are frequently found in
some formulations.
Iron is usually added as iron sulphate, although iron citrate can also be
used. Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction
with the iron sulphate. The EDTA complexes with the iron so as to allow the
slow and continuous release of iron into the medium. Uncomplexed iron can
precipitate out of the medium as ferric oxide.
Organic supplements
Only two vitamins, thiamine (vitamin B
) and myoinositol (considered a B
vitamin) are considered essential for the culture of plant cells in vitro. How-
ever, other vitamins are often added to plant cell culture media for historical
Amino acids are also commonly included in the organic supplement. The
most frequently used is glycine (arginine, asparagine, aspartic acid, alanine,
glutamic acid, glutamine and proline are also used), but in many cases its in-
clusion is not essential. Amino acids provide a source of reduced nitrogen and,
like ammonium ions, uptake causes acidification of the medium. Casein
hydrolysate can be used as a relatively cheap source of a mix of amino acids.
Carbon source
Sucrose is cheap, easily available, readily assimilated and relatively stable and
is therefore the most commonly used carbon source. Other carbohydrates
(such as glucose, maltose, galactose and sorbitol) can also be used (see Chap-
ter 3), and in specialised circumstances may prove superior to sucrose.
Gelling agents
Media for plant cell culture in vitro can be used in either liquid or ‘solid’
forms, depending on the type of culture being grown. For any culture types
that require the plant cells or tissues to be grown on the surface of the
medium, it must be solidified (more correctly termed ‘gelled’). Agar, produced
from seaweed, is the most common type of gelling agent, and is ideal for rou-
tine applications. However, because it is a natural product, the agar quality
can vary from supplier to supplier and from batch to batch. For more
Plant tissue culture
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demanding applications (see, for instance, the section on microspore culture
below and Chapter 3), a range of purer (and in some cases, considerably more
expensive) gelling agents are available. Purified agar or agarose can be used,
as can a variety of gellan gums.
These components, then, are the basic ‘chemical’ necessities for plant cell cul-
ture media. However, other additions are made in order to manipulate the pat-
tern of growth and development of the plant cell culture.
Plant growth regulators
We have already briefly considered the concepts of plasticity and totipotency.
The essential point as far as plant cell culture is concerned is that, due to this
plasticity and totipotency, specific media manipulations can be used to direct
the development of plant cells in culture.
Plant growth regulators are the critical media components in determining
the developmental pathway of the plant cells. The plant growth regulators
used most commonly are plant hormones or their synthetic analogues.
Classes of plant growth regulators
There are five main classes of plant growth regulator used in plant cell culture,
(1) auxins;
(2) cytokinins;
(3) gibberellins;
(4) abscisic acid;
(5) ethylene.
Each class of plant growth regulator will be briefly looked at.
Auxins promote both cell division and cell growth The most important
naturally occurring auxin is IAA (indole-3-acetic acid), but its use in plant cell
culture media is limited because it is unstable to both heat and light. Occa-
sionally, amino acid conjugates of IAA (such as indole-acetyl-
-alanine and
-glycine), which are more stable, are used to partially alleviate
the problems associated with the use of IAA. It is more common, though, to
use stable chemical analogues of IAA as a source of auxin in plant cell culture
media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used
auxin and is extremely effective in most circumstances. Other auxins are
available (see Table 2.3), and some may be more effective or ‘potent’ than 2,4-
D in some instances.
2 : Plant tissue culture
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