Download Nitrate and ammonium

Plant tissue culture
Chapter -3By : Saib Al owini
Text book : plant propagation by tissue culture 3rd Edition 65-113
©2008
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Functions of medium
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Access to atmosphere for gas exchange
Removal of plant metabolite waste
Amino acids
Sugars
Buffers
Macro and micro nutrients
Inorganic
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1. It is required to complete the life cycle
2. Its function cannot be replaced by another
nutrient
3. If it is directly involved in plant metabolism
Stimulates growth but not essential FOR ALL ;
I, Na, AL, Si, Co, Se
Uptake of inorganic nutrients
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An ions or cat ions
As salts and dissociate in cells
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Nitrogen : nitrate (NO3 ) or ammonium (NH4 )
-2
-1
phosphorus : ( HPO4 ) ( H2PO4 )
Sulfur: sulfate ion ( SO4 -2)
• Uptake occur by :
• Active or passive or passive
Uptake depend on
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PH
Element concentration
Temperature
Biochemical and physical status.
Uptake depend on cont--• Mg+2 competes with other ions uptake .
• ca++ , k+
• Active uptake of phosphate falloff with
alkaline
• Ammonium or nitrate /
Ammonium more than nitrate with lower temperature
* ca++ : Not absorbed efficiently so ….
Ion transport
• In mother plant by :
• Root (xylem)
• Leaf stoma
Explants : cut edges
1-2 day Mg, K, ca, Na
- 2end day HPO4 and ammonium in 15 day media
will be free .
- Na, k, SO4 after 15 day decrease to 1/5
- Agar and gelrite increase inorganic. Cont
mmol/kg
Nitrogen
• Is a component of amino acids, proteins,
enzymes, nucleic acids and high energy
compounds
• Nitrogen is taken up by plants either as the
• ammonium ion NH4+
• or the nitrate ion NO3• Some plants have a preference for one form
over another but most plants don't
• Appear in the old foliage since N is mobile in
the plant
• Symptoms include an overall chlorosis of
theolder leaves
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Calcium nitrate Ca(NO3)2
Basic
Ammonium nitrate NH4NO3
Acidic
Potassium nitrate KNO3
Basic
Urea
CO(NH2) 2
Acidic
Nitrate ( oxidized form)
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N2 from atmosphere very rare
Nitrate in mor acidic ( 4.2- 4.6)
Nitrate
nitrate reductase (2) nitrite
nitrite
nitrite reductase ammonium
These enzymes substrate conc dependent
Need ATP
Don’t use nitrate as a sol source
NH4 IS low-priced
Ammonium ( reduced form)
• Amines R-NH2 and amides: R-CO-NH-R• Amines (NH4+), seldom a.a
• Amides :
- NH2-CO-NH2 (urea)
- ureides, which include allantoin and allantoic
• Prefer ph more than 5.4
• In un buffered media after some times
ammonia uptake will stopped.
Importance of NH4
• Cell division /
formation of cell wall
. growth /
For activity of regulator
With legumes ( ammonium nitrate, ammonium
chloride) not ammonium sulphate
All sharing with NO3
Ammonium is toxic why !
• (PH) :
• (C ) : hyperhydric
• (PEP) : high but without plastids action
Ammonium pathways
Detoxification :
( if the ammonium is the sol source )
• Add alkaline :
In carrot
Ph will decrease by 0.9 -0 .7 each 8 hours nearly
Must mentain about 5.4 .
• TCA acids / with buffer
• Malate , succinate , fumaric
• AMMONIUM MALATE
• Need to ( GDH)
• SOME ENZYMES
AMMONIUM CITRATE
UREA
• BY UREASE TO AMMONIUM
• If NO3 with urea many plant prefer NO3
Nitrate and ammonium
• NH4 with NO3 is the most favort
• NH4 --- 10% PROTEIN
• TOGETHER TWICE NO3
• With different relative variation
For embryo culture : 1.9g/l kno3
• Shoot culture / strawberry shoot culture
-10.9m M NO3 + 6mM NH4
Phytotoxicity
Without NH4
chlorotic
** N
75mM
SHOOT
30 Mm
shoot
• More n will give undifferentiated tissue
• Without n cells go to differentioantion
Amino Acids
• The most common sources of organic nitrogen used
in culture media are amino acid mixtures.
• Its uptake more rapidlly than in organic
. a.a(e.g., casein hydrolysate), L-glutamine, Lasparagine, and adenine.
When amino acids are added alone, they can be
inhibitory to cell growth.
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Beneficial effects of amino acids
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Rapid grwoth
Protoplast cell division
Conservation of ATP
AS chelating agent
Enhanced nitrogen assimilation
Not toxic as ammonium
As buffer
• Tyrosine has been used to stimulate
morphogenesis in cell cultures but should only
be used in an agar medium.
• Supplementation of the culture medium with
adenine sulfate can stimulate cell growth and
greatly enhance shoot formation. L-tyrosine stimulates shoot formation
Phosphate
• Constituent of:
• Sugar Phosphates, ATP, nucleic acids, enzymes,
phospholipids baitlayers.
• Forms taken up as: H2PO4-, HPO4-2, (PO43- )
also organic forms
• Mobility within plant: High
• USED IN OXIDIZED FORM (PO43- ) NOT AS !!
Phosphate
• H2PO4- OR HPO4-2 convertible ACCORDING
TO ph
• HPO4 has more affinity to cell membrane sites
• In cytoplasm 5-10 mM
• NOT exceed about 25mM in cytoplasm
• If exceeded will be stored as phytic acid
Phosphate
• Plant media contain 1.3-1.7 mM.
• Phosphate deficiency lead to interrupt PH.
Potassium (K+)
• Major cat ion
( chloroplast and cytoplasm 100- 200 mM. )
- K is not metabolized but
Osmotic potential
PH regulation
transport quickly
pH regulation
Enzyme activation by changing conformation of
proteins (ATPase)
Influencing protein
• interacting with IAA and GA
• Cofactor
• KNO3 …. To ammonia then by phloem to root
To enter anion
-K deficiency in plant culture : hyperhydricity
-Decrease in the rate of phosphate absorption .
- K UPTAKE reduced in ca absence
Calcium (Ca++)
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• Structural Functions:
Stabilizing cell wall (together with Mg++)
Not mobile
Metabolic Functions:
Ca++ is toxic, so in cytosol as bound proteins
-Interaction with IAA in elongation of cells
• Activator of membrane-bound enzymes
• Is cofactor for ATP Hydrolysis
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Cellulose don’t formed unless ca exist
APTases of ion channels
Osmoregulation
Ca++ functions in the mitochondria (in
contrast to Mg++ functioning in chloroplast)
• Common intracellular messenger
• Calcification
• Form : Kcl2
Magnesium (Mg++)
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Structural Functions:
In structure of chlorophyll
In cross-connections of cellulose fibrils in cell wall
Metabolic Functions:
Stabilizing enzymes,
Regulates H+ gradient in stroma of chloroplasts in ATP
synthesis, and thus determines pH of chloroplast
• Osmoregulation an pH regulation in cell, as antagonist
to Ca++ and K+
• Form : Mgso4
Sulfur
• Sulfur : SO4-2
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Uptake coupled with N assimilation
a.a, cystein metionin
Lipid
Activates enzymes
Detoxification of radical ( glutathione)
1-2.5 mM