sterile, lidded jar and cover with a chlorate(I) bleach solution ( 1.4% available chlorine) containing a wetting agent (detergent). Reseal the jar and shake for five seconds every five minutes for 20 minutes.
Kelangan minimum (5mm) lower di na nag iinduceng callus formation; more than ok lng
Not sterile with air spaces (possible habitat for microbes)Cut on all sides (stimulate wounding) to promote callus formationDark: devlopmentchloroplastids differentiation na stops meristematic activity (undifferentiated state)
5 weeks: start development secondary metabolic products
2,4 D : dual role, acts like cytokinin and auxin promoting rich callus growth
Amorphous – shape; no organized meristemPotentiality to produce embyroidsPGR – Plant Growth Regulators
Precautions• Only fresh, undamaged carrots which sink inwater should be used.• Use a very sharp scalpel– Injured tissues cause release of compounds thatare air oxidized darkening of tissue andmedium tissue death
Role of 2-4D• Synthetic stable form of auxin• Stimulates cell division in the cambium incombination with cytokinins in tissue culture• Stimulates growth• Stimulates root initiation on stem cuttings andlateral root development in tissue culture• Callus induction
Callus Tissue• an amorphous aggregate of loose parenchymacells, 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, nicotinicacid, 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 organizeddevelopment• Induction of growth• Division Phase• Differentiation
Induction of growth• Transfer to fresh medium inducesdifferentiated cells (quiescent) to enter anactive cell cycle• Cells are in G1 phase but begin S (DNA/RNAsynthesis) and proceed through a short G2phase prior to mitosis.
Division Phase• rapid increase in cell number throughpericlinal divisions subjacent to the peripheryof the callus, and followed by anticlinal(perpendicular) divisions
Differentiation Phase• cell division slows, during this perioddifferentiation occurs which is then followedby cell expansion resulting in the developmentof an organized structure• Formation of SOMATIC EMBRYO (EMBRYOID)