Establishment of plants: Seeds of cauliflower (Brassica oleracea var. botrytis cv. Medailon) and barley (Hordeum vulgare cv. Pastoral) were sown in plastic module trays (10 ml plug volume) filled with sieved John Innes seed compost. Trays were placed in a glasshouse with a minimum temperature of 15 0C with automatic irrigation. Plants were fertilized weekly with ‘Baby Bio’ (NPK fertilizer, 23 ml per 18 litres of water). Plants were grown up to 4 weeks old when the cauliflower had 3 leaves and barley had 4 leaves. Frost Resistance Test: A pilot test was carried out to determine the critical parameters for conducting the frost test. Three hundred sixty plants of each crop species were transferred from the glasshouse to a cold chamber (4 0C and 12h photoperiod) for acclimation (Fuller, 1993). On 0th day, 60 plants were placed into labelled boiling tubes. Fifteen plants (Controls) were left in the cold chamber and 8 plants were taken for proline analysis. The remaining 45 plants were placed in a Sanyo incubator and subjected to the following regime: 4 0C for 1 hour -3 0C for 2 hours - 5 0C for 2 hours -7 0C for 2 hours -9 0C for 2 hours (for barley only). At -3 0C, a small amount of crushed ice was added to each tube to nucleate freezing of each plant and prevent supercooling (Fuller et al., 1994). Ice was also added to the Control tubes. At the end of each test temperature [-3 0C, -5 0C and -70C (and -9 0C for barley)] 15 tubes were removed from the incubator and returned to the cold chamber to thaw overnight (Fuller, 1993). Both frost tested and control plants were then potted into compost in seed trays and placed in a net tunnel to recover for 14 days before scoring for survival. Frost tests were repeated with 2, 7, 10, 13 and 17 day acclimated plants.
Salt Resistance Test: A pilot test was undertaken to determine the critical parameters for conducting the salt acclimation level and 50 mM NaCl was chosen. Eight hundred plants of each crop species, with roots washed free of soil, were placed in a hydroponic system through polystyrene tiles into nutrient solution Twenty two plastic tanks (87 litres volume) were filled with 40 litres of water followed by 140 ml each of A and B standard hydroponic solutions. Eight of the tanks were designated acclimation solutions and salt (NaCl) added to achieve 50 mM equivalent. Similarly, 0 mM, 100 mM, 200 mM and 300 mM of salt solutions (2 replicate tanks per treatment) were designated the test solutions and prepared. Initially, all plants were placed in the acclimation tanks in polystyrene tiles (16 cm2, 16 plants per tile). On 0th day, 8 tiles from each of the two crop species were selected randomly from acclimation tanks and transferred to each treatment solution (1 tile per replicate). Three plants were taken for Proline analysis. This procedure was repeated on 2nd, 7th, 10th, 14th and 17th day of acclimation. After 2 weeks on the test solutions, plants were scored for survival. In all trays, an aerator to oxygenate the solution was connected and the solution surface completely covered by tiles to reduce evaporation loss. The electric conductivity of each solution was measured every week and necessary measures were taken to maintain the required nutrient concentrations.
Proline Analysis The proline level in the plants was determined for both the pilot and actual tests. Selected plants were immediately frozen (-85 0C for 1 day) to stop all biochemical activities and then freeze dried (-35 0C, until constant weight) (Eed, 2001). Dried leaf (0.04 g), with 2 ml ultra clean water, was finely ground in a pestle, taken into an eppendorf tube and centrifuged at 15000 rpm for 15 minutes. Three aliquots of the resultant supernatant were diluted to 1 ml in 3 crimptop sample vials. The HPLC method was used to determine proline level in plants using a Dionex AAA-Direct Amino Acid Analyser System. A series of proline standards analysed by the Acid Ninhydrin Method (Bates et al., 1973) and the Dionex system, found a good linear fit between the two methods (Fuller and Paisey – unpublished) and concluded that the Dionex system is reliable, versatile, convenient and a suitable replacement for the acid ninhydrin method. At first, 2 vials containing water were run by Dionex to clean the column and stabilize the detector’s reading. Then, 5 standard samples were run to obtain a calibration curve for all analytes. Afterward, vials containing prepared samples were placed in the autosampler and run overnight. The Peaknet software gave output curves for all analytes from which the area of proline was integrated.
Statistical Analysis: Damage scores due to frost and salinity were analyzed with ‘Anova’ using Minitab v. 13.