ABSTRACIThe effects ofgrowth temperature (2°C and 24C), abscisic acid (ABA) concentration, duration of exposure to ABA, and light were assessed for their ability to induce acclimation to freezing temperatures in callus cultures of Lotus corniculatus L. cv Leo, a perennial forage legume. The maximal expression of freezing tolerance was achieved on B% media containing 10 molar ABA, at 24°C for 7 or 14 days. Under these culture conditions, the freezing tolerance of the callus approximated that observed in field grown plants. In contrast, low temperatures (2°C) induced only a limited degree of freezing tolerance in these cultures. Viability was assessed by tetrazolium reduction and by regrowth of the callus. The two assays often differed in their estimates of absolute freezing tolerance. Regression analysis of the temperature profile suggested that there may be two or more distinct populations of cells differing in freezing tolerance, which may have contributed to the variability between viability assays.Winter annual and perennial plants have the ability to acclimate to freezing stress in response to environmental signals such as low temperature or short daylength (4). Since the acclimation process is associated with a period of slow growth and often dormancy, it has been proposed that the induction of freezing tolerance may be mediated by shifts in the hormonal balance, specifically in the levels of ABA (9). This hypothesis is based on three experimental observations. First, the endogenous levels of ABA increase when plants are exposed to low, hardening temperatures (17). Second, the exogenous application of ABA as a foliar spray or in hydroponic solution increases the hardiness of some plant species (1, 1 1), although others apparently do not respond (7). Third, the inclusion of ABA in the culture media induces freezing tolerance in both potato callus (1) Tomes. They were originally produced from the axillary buds of plants that were selected on the basis of their vigor and performance in culture. Callus cultures were grown according to the method of Swanson and Tomes (15) and 100 x 20 mm Petri dishes in a 16 h photoperiod at 35 ,uE m-2 s-' supplied by General Electric warm white fluorescent lights. The callus was subcultured every 3 weeks onto B5 media (5) containing 8 g L' agar, 1 mg L-' 2,4-D, and without kinetin. When included, ABA was added from a lOx stock solution prepared by initially dissolving ABA in 4 ml ethanol and diluting to 100 ml with distilled H20. The other nutrients were added from stock solution dissolved in distilled H20.Acclimation and Application of Freezing Stress. Approximately 0.5 g of callus from the stock culture was placed on a sheet of Whatman No. 50 fiber filter paper in a 60 x 20 mm Petri dish that contained a layer of B5 agar media, either with or without ABA. Following the required acclimation treatment, the callus and filter paper were transferred to a Petri dish that contained a layer of moistened sterile sand, covered by a second sheet of filter paper. The moistened layer of ...

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In vitro rooting and subsequent greenhouse survival of `Autumn Britten', `Boyne', `Comet',`Nova' and `Qualicum' raspberry (Rubus idaeus L.) plantlets were compared following four weeks on a rooting medium with and without activated charcoal, and with 0.1, 0.5, 1.0, 2.0 or 3.0 milligrams per litre IBA. The addition of charcoal significantly increased the percentage of plantlets that produced roots in vitro for the hard-to-root cultivars. Percent rooting in vitro was highest with the three lower levels of IBA. Root number was influenced only by the cultivar, while root diameter and length were affected by all the factors investigated. Greenhouse survival was affected by the cultivar, the presence or absence of charcoal and the IBA level in the in vitro rooting medium, with significant interactions. Provided charcoal was present in the rooting medium, the level of IBA didn`t alter survival. The addition of charcoal to the rooting medium improved greenhouse survival of the three hardest-to-root cultivars. Plug plant stem length; internode length and dry weight were increased by the presence of charcoal in the in vitro rooting medium for all but the easiest to establish cultivar. Chemical names used: 3-indolebutyric acid (IBA).