Carrot Callus Formation

Initiating Callus Formation in an in vitro setup

Methodology

Surface Sterilization

• Chlorate(I) bleach solution containing a wetting agent (detergent).

Methodology

2,4-Dichlorophenoxya

cetic acid

Precautions

• Only fresh, undamaged carrots which sink in water should be used.

• Use a very sharp scalpel– Injured tissues cause release of compounds that

are air oxidized darkening of tissue and medium tissue death

Results

Role of 2-4D

• Synthetic stable form of auxin• Stimulates cell division in the cambium in

combination with cytokinins in tissue culture • Stimulates growth • Stimulates root initiation on stem cuttings and

lateral root development in tissue culture• Callus induction

Callus Tissue

• an amorphous aggregate of loose parenchyma cells, which proliferate from the mother cells– Initiated by wounding

• Factors affecting callus growth: – Chemical (mineral and PGRs)– Environmental (Genetic composition; light,

humidity, and etc.)

Media Requirements

• Inorganic Salts (nitrate, potassium, ammonium) • PGRs (hormones)• Vitamins (enzyme cofactors, nicotinic acid, thiamine,

pyridoxine)• Carbon Source (sucrose)• Gelling Agent (agar of TC grade or Difco-bacto agar)• Amino Acids and Amides• Antibiotics *• Natural Complexes *• Antioxidants * (reduce excessive browning of explants)

Growth patterns leading to organized development

• Induction of growth • Division Phase• Differentiation

Induction of growth

• Transfer to fresh medium induces differentiated cells (quiescent) to enter an active cell cycle

• Cells are in G1 phase but begin S (DNA/RNA synthesis) and proceed through a short G2 phase prior to mitosis.

Division Phase

• rapid increase in cell number through periclinal divisions subjacent to the periphery of the callus, and followed by anticlinal (perpendicular) divisions

Differentiation Phase

• cell division slows, during this period differentiation occurs which is then followed by cell expansion resulting in the development of an organized structure

• Formation of SOMATIC EMBRYO (EMBRYOID)