Culture media

[aayush_005]

CULTURE
Objective
When cultured in vitro, all the needs, both chemical and
physical, of the plant cells have to be 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 supplements 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.
Now, you must have understood that growth and morphogenesis
of plant tissue in vitro largely depend on the composition
of the culture media. No single medium can be suggested for
all types of plants and organs. So, the details of culture
medium have to be worked out for each plant material separately.
Several media have been developed which are commonly
used e.g.,
i. Murashige & Skoog’s medium (MS medium)
ii. Gamborg et al. (B5 medium).
iii. White’s medium.
The most important of these media is MS medium.
Media Composition
The major constituents of most plant tissue culture media are:
i. Inorganic nutrients (macro - and micro-nutrients)
ii. Carbon source (sugar)
iii. Organic supplements
iv. Growth regulators
v. A gelling / solidifying agent (agar).
The concentration of inorganic and organic constituents in
culture media is expressed either in mass value (mg/l or ppm)
or mole values (1M or 1 mol/l).
Let us now discuss each constituent of the media in detail.
Inorganic Nutrients
A variety of mineral elements (salts) supply the needed macro -
and micronutrients required in the life of a plant. Elements
required in concentrations greater than 0.5 m mol L-1 are referred
to as Macronutrients and those less than 0.05 mM/L as
Micronutrients.
Macronutrients
They include six major elements:

Nitrogen(N), Phosphorus(P), Potassium(K), Calcium (Ca),
Magnesium (Mg) and Sulphur (S), present as salts in the media.
All are essential for plant cell & tissue growth. Culture media
should contain at least 25 mmolL-1 nitrate and potassium.
Better results are obtained if the source of ‘N’ in media is
contributed by both nitrates and ammonium (2-20mmol L-1) or
any other reduced nitrogen source. Other major elements Ca, P,
S and Mg, at concentrations in the range of 1-3 mmolL-1 appear
adequate (see Table 1). The physiological role of various
elements is summarized in Table 2.
Micronutrients:
These are Iron (Fe), Manganese (Mn), Zinc (Zn), Boron(B),
Copper (Cu) and Molybdenum (Mo). Chelated forms of Fe and
Zn are commonly used in preparing culture media eg. for
embryo induction, EDTA-iron chelate is used in place of iron
citrate. Generally, 0.1 u mol L-1 Cu and Co, 1 umol L-1 Fe and
Mo, 5 u mol L-1 I, 5- 30u mol L-1 Zn, 20- 90 umol L-1 Mn and 2-
5100 u mol L-1 B are added to culture media, depending upon
the requirement of the experiment.

Carbon and Energy Source
The most preferred carbon source in plant tissue culture is
‘Sucrose’. Glucose supports equally good growth, while fructose
is less efficient. Sucrose, while autoclaving the medium is
converted to glucose and fructose. In the process, first glucose is
used and then, fructose.
Plant cells and tissues in the culture medium lack autotrophic
ability and therefore, need external carbon for energy. Even
tissues which are initially green or acquire green pigments under
special conditions during the culture period are not autotrophs
for carbon. The addition of an external carbon source to the
medium enhances proliferation of cells and regeneration of
green shoots.

Organic Supplements
1. Vitamins: Plants synthesise vitamins endogenously and
these are used as catalysts in various metabolic processes.
When plant cells and tissues are grown in vitro, some
essential vitamins are synthesized but only in sub- optimal
quantities. Hence, it is necessary to supplement the medium
with required vitamins and amino acids to achieve the best
growth of the tissue. Thiamine (B1), nicotinic acid (B3),
pyridoxine (B6), calcium pantothenate (B5) and myo inositol
are used more often, in the range of 0.1- 10.0 mg/l.
2. Amino Acids: Cultured tissues are normally capable of
synthesizing the amino acids necessary for various metabolic
processes. In spite of this, the addition of amino acids to
the media is important for stimulating cell growth in
protoplast cultures and for establishing cell lines. Casein
14 2.621
© Copy Right: Rai University
PLANT TISSUE CULTURE
hydrolysate (0.05- 0.1%), L- glutamine (8 mmol/l), Lasparagines
(100 mmol/l), L- glycine (2mmolL-1), L-arginine
and L-cysteine (10mmolL-1) are common sources of organic
nitrogen used in culture media.
3. Other Organic Supplements: These include organic extracts
eg. protein (Casein) hydrolysates, coconut milk, yeast and
malt extracts, ground banana, orange juice and tomato juice.
They are of undefined nature. In tissue culture, success
achieved with coconut milk (5 to 20%) and protein (casein)
hydrolysate (0.05 to 1.0%) has been significant. Potato extract
has been found a suitable medium for anther culture.
4. Activated Charcoal: The addition of activated charcoal (AC)
to culture media stimulates growth and differentiation in
orchids, carrot, ivy and tomato. AC adsorbs inhibitory
compounds and darkening of the medium occurs. AC is
generally acid-washed and neutralised before its addition at
concentrations of 0.5- 3% to the culture medium. It also
helps to reduce toxicity by removing toxic compounds (eg.
Phenols) produced during the culture and permits
unhindered cell growth.
5. Antibiotics: Some plant calls have a systemic infection of
microorganisms. To prevent the growth of these microbes,
it is essential to enrich the media with antibiotics, eg.
Streptomycin or kanamycin at low concentration effectively
controls systemic infection and do not inhibit the growth of
cell cultures.
Growth Regulators
These include: i) auxins, ii) cytokinins, iii) Gibberellins, iv)
Abscisic acid and, (v) Ethylene. The growth, differentiation and
organogenesis of tissues occurs only on the addition of one or
more of these hormones to the medium.
Auxins
Commonly used auxins are : ( Table 3)
i. Indole-3 - acetic acid (IAA).
ii. Indole –3- butyric acid (IBA)
iii. 2,4-dichlorophenoxyacetic acid (2,4-D)
iv. Naphthoxyacetic acid (NOA)
IAA occurs naturally in the plant tissues. Auxins have the
property of inducing cell division. In nature, hormones of this
group are involved in elongation of stem, internodes, tropism,
apical dominance, abscission and rooting. 2, 4-D is mostly
employed to induce callus production. Auxins are generally
dissolved either in ethanol or dilute NaOH.
Cytokinins
These are adenine derivatives which are mainly concerned with
cell division, modification of apical dominance and shoot
differentiation, in the tissue culture. Commonly used cytokinins
include: (Table 3)
i. 6-Benzylaminopurine (BAP)
ii. Isopentyl-adenine (2,ip)
iii. Furfurylamino purine (Kinetin).
iv. Zeatin.
v. 6- Benzyladenine (BA).
Zeatin and 2,ip are naturally occurring cytokinins. Cytokinins are
generally dissolved in dilute HCL or NaOH.
The ratio of auxins and cytokinins is important with respect to
morphogenesis in the culture system. For embryogenesis, callus
initiation and root initiation, the ratio of auxins to cytokinin is
high, while the reverse leads to axillary and shoot proliferation.
Equally important is concentration of the two hormones. Eg.
2,4-D and BA at a concentration of 5.0 mgL-1 promote callus
formation, but if used at 0.1 mg L-1 concentration, they
promote shoot formation. Cytokinins have been shown to
activate RNA synthesis and to stimulate protein and enzyme
activity in certain tissues.

[NilmfemaSkemi]

amish sheds duramax sheds how to build a shed. cool b.o.b. ringtones bob magic ringtone buy b.o.b. ringtones. big boi ringtone shutterbugg ringtone big boi sony ericsson ringtones. lady gaga verizon ringtones alejandro ringtone lady gaga cell phone ringtones. send kesha ringtone blah blah blah ringtone kesha mp3 ringtone. which battery last longer superman stamina how can i last longer. buy kris allen ringtones live like were dying ringtone buy kris allen ringtones. download eminem mp3 ringtone not afraid ringtone cheap eminem ringtones. justin bieber ringtone baby ringtone justin bieber samsung ringtones. buy glee cast ringtones glee cast forget you ringtone glee cast mp3 ringtone. b.o.b. nokia ringtones nothin on you ringtone b.o.b. mp3 ringtone. drake cell phone ringtones find your love ringtone drake sony ericsson ringtones. send black eyed peas ringtone imma be ringtone buy black eyed peas ringtones. 3oh!3 us cellular ringtones my first kiss ringtone 3oh!3 latest ringtones. kelly clarkson top ringtones already gone ringtone kelly clarkson popular ringtones. lady gaga samsung ringtones telephone ringtone lady gaga verizon wireless ringtones. jay-z virgin mobile ringtones young forever ringtone jay-z ringtones. lil wayne boost ringtones drop the world ringtone cool lil wayne ringtones. lady gaga virgin ringtones paparazzi ringtone lady gaga real ringtones. drake mobile ringtones over ringtone drake t mobile ringtones. glee cast popular ringtones glee cast teenage dream ringtone download glee cast ringtones. download ludacris ringtones to computer how low ringtone ludacris at&t ringtones. cheap jason derulo ringtones whatcha say ringtone cool jason derulo ringtones. black eyed peas nextel ringtones rock that body ringtone black eyed peas virgin mobile ringtones. buy jason derulo ringtones in my head ringtone download jason derulo mp3 ringtone. male enhancer maxoderm cream natural male enhancement pill. wood shed Arrow Sheds shed designs. la roux new ringtones la roux bulletproof ringtone la roux mp3 ringtone. iyaz nokia ringtones replay ringtone iyaz at&t ringtones. download jason derulo mp3 ringtone ridin solo ringtone download jason derulo ringtones to your phone. hgh dosage Genf20 hgh benefits. kanye west polyphonic ringtones kanye west monster ringtone kanye west ringtone. lady gaga new ringtones lady gaga bad romance ringtone lady gaga ringtone. buy jack johnson ringtones online you and your heart ringtone jack johnson verizon wireless ringtones. jay-z ringtones empire state of mind ringtone jay-z new ringtones. glee cast nokia ringtones glee cast total eclipse of the heart ringtone glee cast polyphonic ringtones. artists for haiti cell phone ringtones we are the world ringtone artists for haiti t mobile ringtones. adam lambert latest ringtones whataya want from me ringtone adam lambert mp3 ringtone. lady antebellum boost ringtones need you now ringtone lady antebellum mp3 ringtone. drake nokia ringtones miss me ringtone send drake ringtones. beyonce latest ringtones sweet dreams ringtone beyonce sony ericsson ringtones. justin bieber boost ringtones somebody to love ringtone justin bieber sprint ringtones. download justin bieber ringtones one time ringtone justin bieber latest ringtones. glee cast free ringtones glee cast poker face ringtone buy glee cast ringtones online. kesha alltel ringtones your love is my drug ringtone kesha virgin ringtones. kenny chesney at&t ringtones the boys of fall ringtone kenny chesney mobile ringtones. boys like girls free ringtones two is better than one ringtone send boys like girls ringtones.

[NilmfemaSkemi]

drake mp3 ringtone find your love ringtone drake real ringtones. kris allen t mobile ringtones live like were dying ringtone kris allen samsung ringtones. lean to shed arrow sheds building a shed. lady gaga popular ringtones lady gaga alejandro ringtone lady gaga mp3 ringtones. download lady gaga ringtones to computer paparazzi ringtone download lady gaga mp3 ringtones. download glee cast ringtone glee cast forget you ringtone glee cast boost ringtones. black eyed peas polyphonic ringtones rock that body ringtone black eyed peas nextel ringtones. la roux mp3 ringtones la roux bulletproof ringtone la roux at&t ringtones. backyard shed Duramax Sheds tool shed. lil wayne virgin mobile ringtones drop the world ringtone download lil wayne ringtone. download big boi mp3 ringtones shutterbugg ringtone download big boi ringtones. kesha sprint ringtones your love is my drug ringtone kesha top ringtones. glee cast virgin mobile ringtones glee cast teenage dream ringtone glee cast ringtone. 3oh!3 verizon wireless ringtones my first kiss ringtone download 3oh!3 ringtone. how can you last longer in bed superman stamina how to prevent premature ejaculation. cool eminem ringtones not afraid ringtone eminem real ringtones. jason derulo t mobile ringtones what you say ringtone cheap jason derulo ringtones. justin bieber t mobile ringtones one time ringtone buy justin bieber ringtones. best hgh on the market genf20 how to get hgh. cool lady gaga ringtones lady gaga telephone ringtone lady gaga new ringtones. send b.o.b. ringtone nothing on you ringtone b.o.b. samsung ringtones. kelly clarkson virgin mobile ringtones already gone ringtone cheap kelly clarkson ringtones. safe male enhancement maxoderm cream the best male enhancement products. buy boys like girls ringtones online two is better than one ringtone download boys like girls ringtones to your phone. artists for haiti latest ringtones 25 for haiti ringtone download artists for haiti ringtones. cheap jay-z ringtones young forever ringtone jay-z free ringtones. download jason derulo mp3 ringtones in my head ringtone jason derulo nokia ringtones. kenny chesney ringtones the boys of fall ringtone kenny chesney verizon ringtones. glee cast ringtone glee cast total eclipse of the heart ringtone glee cast alltel ringtones. kanye west sony ericsson ringtones kanye west monster ringtone download kanye west ringtones to computer. download jay-z ringtones to your phone empire state of mind ringtone jay-z mp3 ringtone. jack johnson mobile ringtones you and your heart ringtone cheap jack johnson ringtones. beyonce new ringtones sweet dreams ringtone beyonce virgin mobile ringtones. drake at&t ringtones miss me ringtone drake latest ringtones. glee cast verizon ringtones glee cast poker face ringtone glee cast nokia ringtones. justin bieber nokia ringtones justin bieber somebody to love ringtone download justin bieber ringtone. ludacris new ringtones how low ringtone cool ludacris ringtones. download adam lambert ringtones to computer whataya want from me ringtone cheap adam lambert ringtones. black eyed peas alltel ringtones imma be ringtone black eyed peas latest ringtones. drake mp3 ringtone drake over ringtone drake sprint ringtones. jason derulo real ringtones ridin solo ringtone cheap jason derulo ringtones. download justin bieber mp3 ringtone justin bieber baby ringtone justin bieber t mobile ringtones. buy iyaz ringtones replay ringtone iyaz boost ringtones. lady gaga popular ringtones lady gaga bad romance ringtone lady gaga cell phone ringtones. cheap b.o.b. ringtones magic ringtone buy b.o.b. ringtones online. kesha at&t ringtones blah blah blah ringtone download kesha ringtone. lady antebellum latest ringtones need you now ringtone lady antebellum mp3 ringtone.