Professional Documents
Culture Documents
Contents
[hide]
• 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
agar.
Biofertili
zer
product
s
(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.
Essential element
Concentration in
Concentration
stock solution (mg/l)
in medium (mg/l)
Macroelements
b
NH
4
NO
3
33 000
1 650
KNO
3
38 000
1 900
CaCl
2
.2H
2
O
8 800
440
MgSO
4
.7H
2
O
7 400
370
KH
2
PO
4
3 400
170
Microelements
c
KI
166
0.83
H
3
BO
3
1 240
6.2
MnSO
4
.4H
2
O
4 460
22.3
ZnSO
4
.7H
2
O
1 720
8.6
Na
2
MoO
4
.2H
2
O
50
0.25
CuSO
4
.5H
2
O
5
0.025
CoCl
2
.6H
2
O
5
0.025
Iron source
c
FeSO
4
.7H
2
O
5 560
27.8
Na
2
EDTA.2H
2
O
7 460
37.3
Organic supplement
c
Myoinositol
20 000
100
Nicotinic acid
100
0.5
Pyridoxine-HCl
100
0.5
Thiamine-HCl
100
0.5
Glycine
400
2
Carbon source
d
Sucrose
Added as solid
30 000
a
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.
b
Added as solid.
SPB2 2/27/2003 4:06 PM Page 38
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.
Microelements
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
1
) 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
reasons.
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.
Summary
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,
namely:
(1) auxins;
(2) cytokinins;
(3) gibberellins;
(4) abscisic acid;
(5) ethylene.
Each class of plant growth regulator will be briefly looked at.
Auxins
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-
L
-alanine and
indole-acetyl-
L
-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.
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