Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either 12H4JIAA (m/z 194, 134) or I2HS5IAA (m/z 195, 135).Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of rH31ABA (m/z 264, 250 The method to be described here, for simultaneous quantitation by GC-MS-SIM of free IAA and free ABA in small samples (0.1-0.5 g dry weight), arose from research on morphogenesis in tissue cultures and intact plants of apple (9, 13), prune (1), and 'Abbreviations: GC-MS-SIM, gas chromatography-mass spectrometry-selection ion monitoring; Me-IAA, methyl ester of IAA; Me-ABA, methyl ester of ABA; MH+, protonated molecular ion; m/z, mass to charge ratio. citrus (10). Of the few methods available for simultaneous measurement of IAA and ABA (2, 4-6, 8, 11) none was suitable for our purposes. Those based on chromatographic procedures alone (2, 5, 8, 11) require extensive purification of extracts and lack the specificity of GC-MS. Published methods involving GC-MS (4, 6) are primarily qualitative in nature and do not include use of deuterated internal standards and calibration curves. These methods lack the necessary precision for quantitative work. Accordingly, it was decided to modify a currently used quantitative GC-MS assay for ABA (12) to enable IAA to be measured in the same extract. MATERIALS AND METHODSPlant Materials, Extraction, and Derivatization. The plant materials were leafy microcuttings from aseptic proliferating shoot cultures of apple (cv Jonathan) and prune (cv D'Ente), 1-year-old extension shoots of Jonathan apple, and fully expanded mature leaves from adult cropping orange trees (cv Valencia).All glassware was soaked in a 5% solution of Surfasil (Pierce Chemical Co., Rockford, IL) in hexane for 5 min, rinsed with methanol and oven-dried for 1 h at 100°C. All the sample manipulations were carried out under dim light to minimize isomerization and photodecomposition. Samples and extracts were stored in the dark at -20°C when not in use. All solvents were of analytical reagent grade and were distilled before use.The tissues were harvested, frozen by immersion in liquid N2, and freeze-dried. Although the possibility of losses of IAA and ABA during freeze drying cannot be completely ruled out, care was taken to ensure that the same technique and conditions were used for the freeze drying of all tissues. Samples of the freezedried material (100-500 mg) were ground in a mortar and extracted overnight (4°C) with 70% (v/v) IAA (purchased from MSD Isotopes, Montreal, Canada). The slurry was centrifuged (2 min) and the supernatant was retained.The pellet was reextracted with 70% acetone and centrifuged and the combined supernatants were then evaporated to the aqueous phase under a ...
Proliferating axillary shoots of the difficult-to-root apple cultivar `Jonathan' acquired an enhanced ability to form adventitious roots with increasing number of subcultures in vitro . The transition between the difficult-to-root and the easy-to-root condition occurred at the fourth subculture .Endogenous levels of free IAA and ABA in shoot tissues were analysed by gas chromatography/mass spectrometry/single ion monitoring (GC/MS/SIM) using negative ion chemical ionisation . Tissues from the mother plants grown in the glasshouse contained more IAA and ABA than those from tissue-culture material . After establishment in vitro there was no variation in the IAA content throughout the subcultures but a decrease in ABA content was observed after the fourth transfer . The IAA/ABA ratio increased from 0 .2 in difficult-to-root shoots from the initial culture up to 0 .7 in easy-to-root shoots from the long-term subculture. 1 . IntroductionThe propagation of apples from cuttings is difficult because most apple cultivars and rootstocks do not readily form roots under conventional nursery conditions [23] . However, the rooting ability of apple cuttings can be improved by `conditioning' the mother plants using treatments such as heavy pruning and blanching [11], application of growth retardants [6,9] or exposure to low light intensity [7] . Success in the striking of cuttings is determined as much by the management of the stock plants prior to severance as by the conditions under which root formation occurred [18] . Most of the research done on root formation is concerned with cuttings after excision from the stock plants .Recent reviews on adventitious root formation [10,13] have drawn attention to the importance of the physiological state of the mother plants at time of collection of the cuttings . Investigations on the hormonal balance in stock and the results are conflicting . In Dahlia [3] and in Rhododendron [24] the capacity of cuttings to form adventitious roots is not correlated with the endogenous level of IAA in the mother plants . In stock plant of Bougainvillea, Hibiscus [5] and Vitis [2], however, enhanced root formation of cuttings is associated with high IAA content . Similarly very little is known about the occurrence of ABA in mother plants . In Rhododendron the rooting ability of cuttings is positively correlated with a high ABA content in stock plants [24] but high ABA levels are found to be inhibitory in Sequoia sempervirens [8] .Difficult-and easy-to-root microcuttings of the same apple cultivar can be produced in vitro by repeated subculture under specific conditions [19] .
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