This document discusses the process of initiating callus formation from carrot tissue in an in vitro setup. It describes how to surface sterilize and injure the carrot tissue to induce callus growth. The plant growth regulator 2,4-D is used to stimulate cell division and callus induction. Callus tissue is an amorphous aggregate of loose parenchyma cells that proliferate from wounded mother cells. The document outlines the media requirements and growth patterns, including induction of growth, division phase, and differentiation phase, that lead to organized development and potential formation of somatic embryos.

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)