Plant tissue culture Chapter -3- By : Saib Al owini Text book : plant propagation by tissue culture 3rd Edition 65-113 2008©

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 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

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 nearly Must 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 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, 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