1) Tissue culture medium is a medium contains the nutrition elements for tissue and cell living Tissue culture

medium elements as: Macro elements Micro elements Vitamin Fe-EDTA Plant growth regulators Organic matter Others Agar pH=5.8 There are about 20 main media; but they could classify to 3 groups of media belonged to the concentration of ammonium salts Rich nutritional media: MS, LS, N6, Nistch B5

Average nutritional media: Poor nutritional media: WPM

Almost plants favor to MS medium in shoot tip culture and embryogenesis culture; B5 medium in soybean culture; WPM medium in forest trees culture

Macro elements N P K Ca Mg Fe NH4NO3 - KNO3 NaH3PO4.7H2O - KH2PO4 NaH2PO4 NH4H2PO4 KCl2 - KH2PO4 KNO3 CaCl2.6H2O - Ca(NO3)2.4H2O MgSO4.7H2O Fe-EDTA

Micro elements Concentration (M) Mn B Zn Cu Co I Mo MnSO4.4H2O H3BO4 ZN(SO4).7H2O CuSO4.5H2O CoCl2.6H2O KI (NH4)6Mo7O24.4H2O NaMoO4.2H2O Vitamins Concentration (mg/l) Myo-inositol 100 0.5-1 0.05-1 1-5 1-10 15-100 6-100 15-30 0.04-0.08 0.1-0.4 2.5-20 0.007-1 0.007-1

Acid nicotinic (niacin) Pyridoxin HCl (B6) Thiamin HCl (B1) Riboflavin (B2)

Panthothenate calci (B5) 0.5-2.5 Biotin (H) Acid folic (M) Tocopherol (E) Organic compounds Concentration Coconut water Yeast extract Casein hydrolysate 1-2 g/l 1-2 g/l 10-15% 0.01-1 0.1-0.5 1-50

Bee milk Malt Potato extract Corn extract Banana 2) 2.1. Auxins 3-5%

1-5 g/l 1-10 g/l 3-5%

3-5%

Auxin stimulates the production of callus and roots Auxin causes extension of stem Auxin stimulates of cell elongation, cell division in cambium tissue Auxin + cytokinin stimulate differentiation of phloem and xylem High concentration of auxin can induce somatic embryogenesis The essential function of auxins and cytokinin is to reprogram somatic cells in a determine state of differentiation Reprogramming causes dedifferentiation and then redifferentiation into a new pathway. Thus, a cell that had been destined to develop into part of a leaf might become embryogenic, producing somatic embryo The mechanism that auxin causes dedifferentiation is not understood High concentration of exogenous auxin can be toxic because they stimulate production of concentrations of ethylene, which can cause growth inhibition

2.2. Cytokinin Cytokinin cause cell division, as the name suggest; such cell division can lead to shoot regeneration in vitro by stimulating the formation of shoot apical meristems and shoot buds The cell division caused by cytokinins can produce undifferentiated callus A high concentration of cytokinin will block root development

Cytokinins can cause release of shoot apical dominance, thereby stimulating growth of lateral buds and resulting in multiple shoot formation (fig8.3) 2.3. Gibberellins GA3 stimulates the shoot elongation or the conversion of buds into shoots (fig 8.4) GA3 interferes with bud initiation at a very early stages of meristem formation and thereby may reduce shoot production in vitro if given to plant tissue cultures at the shoot bud initiation stage It is necessary to optimize the stage-specific effects of GA3 2.4. Abscicis acid ABA facilitates somatic embryo maturation In certain species, embryos may not be mature adequately for lack of ABA Endogenous ABA functions similarly during seed development in monocotyledonous and dicotyledonous plants and conifers ABA induces the formation of LEA proteins found at late stage of embryogenesis in both somatic and sexual embryos ABA might stimulate some regeneration processes and, rarely, may also reduce the production of somatic embryos 3) Physiological status of the explants The explants were young and juvenile physiology Explants were sourced from shoot tip and lateral bud The size of explants in tissue culture Meristem Shoot tip Anther Leaves 0.1-1mm 5-10mm 0.1-1mm 5-10mm

Root 1-20mm Stem 5-20mm