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Let us make in-depth study of the meaning, principle, protocol and importance of the leaf culture.
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.
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.
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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.
It has 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.
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.
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(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 (Fig 2.2).
(6) Incubate the culture at 25° C under 16 hrs. light.
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 BAP or 2iPA. When shoot have grown to a height 3 cm, they may be removed and sub cultured onto MS medium devoid of growth hormones. Root formation is stimulated by this treatment. Therefore leaf culture of solanaceous species can be used as clonal micro-propagation.
