1. Plant tissue culture Chapter -3-
By : Saib Al owini
Text book : plant propagation by tissue culture 3rd Edition
2008©

2. 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

3. Macro and micro nutrients Inorganic
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

4. Stimulates growth but not essential FOR ALL ;
I, Na, AL, Si, Co, Se

5. Uptake of inorganic nutrients
An ions or cat ions
As salts and dissociate in cells
Nitrogen : nitrate (NO3 -) or ammonium (NH4 +)
phosphorus : ( HPO4 -2) ( H2PO4 -1)
Sulfur: sulfate ion ( SO4 -2)
Uptake occur by :
Active or passive or passive

6. Uptake depend on PH Element concentration Temperature
Biochemical and physical status.

7. Uptake depend on cont---
Mg 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 ….

8. 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

9. mmol/kg

10. 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

11. • Appear in the old foliage since N is mobile in the plant • Symptoms include an overall chlorosis of theolder leaves

12. Calcium nitrate Ca(NO3)2 Basic
Ammonium nitrate NH4NO Acidic
Potassium nitrate KNO Basic
Urea CO(NH2) Acidic

14. Nitrate ( oxidized form)
N2 from atmosphere very rare
Nitrate in mor acidic ( )
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

15. 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

17. Prefer ph more than 5.4
In un buffered media after some times ammonia uptake will stopped.

18. 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

19. Ammonium is toxic why ! (PH) : (C ) : hyperhydric
(PEP) : high but without plastids action

20. Ammonium pathways

21. :Detoxification ( if the ammonium is the sol source )
Add alkaline :
In carrot
Ph will decrease by each 8 hours nearly
Must mentain about 5.4 .
TCA acids / with buffer
Malate , succinate , fumaric
AMMONIUM MALATE AMMONIUM CITRATE
Need to ( GDH)
SOME ENZYMES

22. UREA
BY UREASE TO AMMONIUM
If NO3 with urea many plant prefer NO3

23. Nitrate and ammonium NH4 with NO3 is the most favort
NH % PROTEIN
TOGETHER TWICE NO3
With different relative variation
For embryo culture : 1.9g/l kno3

24. Shoot culture / strawberry shoot culture
-10.9m M NO3 + 6mM NH Phytotoxicity
Without NH chlorotic
** N mM SHOOT
30 Mm shoot

25. More n will give undifferentiated tissue
Without n cells go to differentioantion

26. 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, L-asparagine, and adenine.
When amino acids are added alone, they can be inhibitory to cell growth. .

27. Beneficial effects of amino acids
Rapid grwoth
Protoplast cell division
Conservation of ATP
AS chelating agent
Enhanced nitrogen assimilation
Not toxic as ammonium
As buffer

28. 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

29. 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 !!

30. 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

31. Phosphate Plant media contain 1.3-1.7 mM.
Phosphate deficiency lead to interrupt PH.

32. Potassium (K+) Major cat ion
( chloroplast and cytoplasm 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

33. 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

34. Calcium (Ca++) • 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

35. 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

36. Magnesium (Mg++) 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

37. Sulfur Sulfur : SO4-2 Uptake coupled with N assimilation
a.a, cystein metionin
Lipid
Activates enzymes
Detoxification of radical ( glutathione)
1-2.5 mM