Plant tissue culture Chapter -3-

By : Saib Al owini

Text book : plant propagation by tissue culture 3rd Edition 65-1132008©

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

• 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

• 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

Uptake depend on

• 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 stomaExplants : cut edges1-2 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

• Calcium nitrate Ca(NO3)2 Basic• Ammonium nitrate NH4NO3 Acidic• Potassium nitrate KNO3 Basic• Urea CO(NH2) 2 Acidic

Nitrate ( oxidized form)

• 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 nearlyMust mentain about 5.4 .

• TCA acids / with buffer • Malate , succinate , fumaric

• AMMONIUM MALATE AMMONIUM CITRATE

• Need to ( GDH)• SOME ENZYMES

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 PhytotoxicityWithout 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, L-

asparagine, and adenine. When amino acids are added alone, they can be

inhibitory to cell growth. .

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

• 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++)

• • 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

• 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++)

• 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

• Uptake coupled with N assimilation • a.a, cystein metionin • Lipid • Activates enzymes • Detoxification of radical ( glutathione) • 1-2.5 mM