1. Carrot Callus Formation
Initiating Callus Formation in an in
vitro setup

2. Methodology

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

4. Methodology
2,4-
Dichlorophenoxya
cetic acid

5. 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

6. Results

9. 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

10. 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.)

11. 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)

12. Growth patterns leading to organized
development
• Induction of growth
• Division Phase
• Differentiation

13. 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.

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

15. 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)