FOR B.PHARM 3RD YEAR

1. Leaf Culture

2. What is the Meaning of Leaf Culture?
• Leaf culture is the culture of excised
young leaf primordia or immature
young leaf of the shoot apex in a
chemically defined medium where
they grow and follow the
developmental sequences under
controlled conditions.

3. Principle:
• Leaf primordia or very young leaves are excised,
surface sterilized and inoculated on an agar
solidified medium.
• In culture leaf remains in healthy condition for a
long period.
• Leaves can be taken from aseptically grown plants
for culture.
• Since leaves have a limited growth potential, so in
culture the amount of leaf growth depends upon the
stage of maturity at the time of excision.
• Leaf primordia or very young leaf have more growth
potential than nearly mature leaves.

4. • Most of the work on leaf culture has been done
with lower plants, particularly fern (Osmunda),
although higher plant species, such as tobacco
and sunflower, have been used.
• In culture, the fern leaf primordia (1.2 mm),
excised from underground buds, develop into
leaves having a normal morphology except that
they are much reduced in size than in vivo
leaves due to a reduced number of cells rather
than a decrease in cell size.
• The growth of cultured leaf primordia is also
completed earlier than intact leaf.

5. • This also been found that there is a correlation
between leaf primordia size and its mode of
development in culture.
• In Osmunda cinnamomea, smallest leaf
primordia (300 µm in length) give rise to shoots
instead of leaf in culture. However, with
increasing size of primordia, there is an
increased tendency to form leaves.
• These results indicate that some unidentified
leaf forming substances gradually accumulate as
the primordia develop.

6. Protocol:
(1) Detach vegetative bud or very young leaf from shoot
apex at the vegetative phase of the plant. Wash the
explants thoroughly with running tap water.
(2) Immerse the leaf buds or young leaves in 5% Teepol for
10 minutes. Wash the explants to remove Teepol.
(3) Leaf buds or young leaves are surface sterilized by
immersion in 70% v/v Ethanol for 30 seconds. This
treatment is followed by 10-15 minutes incubation in
sodium hypochlorite solution with 0.8% available
chlorine. Rinse the explants 3-4 times in sterile distilled
water.
(4) Excise the leaf primordia from the leaf bud with the help
of surgical scalpel.
(5) Inoculate the leaf primordia or young leaf onto 20 ml of
solidified medium in a culture tube.

8. Importance of Leaf Culture:
(1) Culture of excised leaf primordia is valuable to study the
effects of various nutrients, growth factors and changing
environmental conditions on leaf development under
conditions divorced from the complexities of the intact
plant.
(2) In case of fern, leaf primordia cultures are used to study
the formation of sporangia and the size at which a
primordium is destined to become a leaf.
(3) Young leaves of most of the solanaceous species form
numerous shoot buds instead of callus formation when they
are cultured in solidified MS medium supplemented with
1-5 µm kinetin or 6-BAP or 2iPA,(6-(3-methyl-2-
butenylamino)-9-β-d-ribofuranosylpurine).

9. THANK YOU