A method for the induction of a high rate of cell division and embryogenesis of Nicotiana rustica pollen was developed. Binucleate pollen grains were fractionated by Percoll density gradient (35/45%) centrifugation and cultured in 0.4 molar mannitol at 30°C (the first culture). After 3 days in culture pollen was recollected by a second Percoll fractionation (0/30%) and transferred to and cultured in a medium containing the Murashige-Skoog macro-elements, 0.4 molar mannitol, 40 millimolar galactose, 3 millimolar glutamine, and 5 micromolar ABA for 10 days (the second culture). The cell population consisting of about 80% dividing pollen was transferred to a Murashige-Skoog medium containing 0.4 molar mannitol, 3 millimolar glutamine, and no phytohormone (the third culture), where about 40% of dividing pollen developed into embryos or embryogenic calli.
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