Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L.

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Abstract
A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10−5 molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N2 after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10−2 molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10−4 molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT50 of −5°C to an LT50 of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.