(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. (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. (6) Incubate the culture at 25° C under 16 hrs. (5) Inoculate the leaf primordia or young leaf onto 20 ml of solidified medium in a culture tube (Fig 2.2). (4) Excise the leaf primordia from the leaf bud with the help of surgical scalpel. Rinse the explants 3-4 times in sterile distilled water. This treatment is followed by 10-15 minutes incubation in sodium hypochlorite solution with 0.8% available chlorine. (3) Leaf buds or young leaves are surface sterilized by immersion in 70% v/v Ethanol for 30 seconds. (2) Immerse the leaf buds or young leaves in 5% Teepol for 10 minutes. Wash the explants thoroughly with running tap water. (1) Detach vegetative bud or very young leaf from shoot apex at the vegetative phase of the plant. These results indicate that some unidentified leaf forming substances gradually accumulate as the primordia develop. However, with increasing size of primordia, there is an increased tendency to form leaves. In Osmunda cinnamomea, smallest leaf primordia (300 µm in length) give rise to shoots instead of leaf in culture. It has also been found that there is a correlation between leaf primordia size and its mode of development in culture. The growth of cultured leaf primordia is also completed earlier than intact leaf. 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. 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.
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