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Title: Plant growth regulators in thermodormancy and germination of fruits of Lactuca sativa L. cv. 'Grand Rapids'
Author: Robertson, James
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1975
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The aim of this research project has been to investigate the hormonal control of germination in light sensitive lettuce seeds. We have used the classic lettuce seed variety, Grand Rapids which, given suitable moisture conditions, will readily germinate below a critical temperature and not above that temperature. This 'thermo-dormancy' is known to be overcome by red light, mediated through phytochrome, and by a number of exogenously applied chemicals. Little information was available in the literature regarding the endogenous hormonal levels in lettuce seeds. It was decided to examine the involvement of endogenous hormones. Principally we have examined the levels of indole acetic acid and abscisic acid during imbibition at 20°C below the 'critical temperature* and at 30°C above the 'critical temperature'. Abscisic acid and indole acetic acid (hereafter ABA and IAA) have both been identified in fruits of Grand Rapids by combined gas liquid chromatography-mass spectrometry (GCMS). In the mass spectrum (MS) of any chemical the fragment occurring in the largest amounts is known as the most 'abundant ion'. It is possible to 'focus' on one ion instead of taking a full MS and this technique allows the measurement of smaller amounts of material. Using this technique of single ion monitoring the levels of both ABA and IAA have been measured. With ABA the levels have also been measured using an electron capture detector in a gas liquid chromatogram. This detector is several orders of magnitude more sensitive for ABA than the more commonly used flame ionisation detector. Using these techniques we have shown that the levels of both 'free' and 'bound* (released by base hydrolysis) ABA vary during imbibition at 20°C or 30°C. However, it was not possible to correlate these changes with germination behaviour. Furthermore, there were differences between different seed batches. With respect to IAA the data obtained were difficult to interpret. The major problem in working with IAA is that IAA was present in low levels and this necessitated the use of large quantities of seeds for extraction. Changes do take place in the endogenous levels of IAA but as with ABA these could not be correlated with changes in germination. Initially, it was hoped to look at gibberellin levels in lettuce fruits. It is possible to erect a hypothesis that the control of photosensitive seeds is under the dual control of promoters and inhibitors and that seeds cannot germinate above the critical temperature because of the presence of inhibitors or the lack of promoters. Control might reside in either reducing the level of inhibitors which would allow the promotive forces present to bring about germination, or in increasing the promotive forces. The latter could entail either the new formation or release of promoters to active sites. Of course, the two are not mutually exclusive. Consequently, we wished to examine the role of gibberellins in lettuce seed germination. Preliminary studies using bioassays indicated that there were major difficulties in studying the levels of gibberellin-like substances during germination. Metabolism of applied radioactive gibberellins might yield meaningful results but in the absence of any information about the gibberellins present in lettuce this is not feasible. An indirect approach to this problem was carried out using exogenous growth retardants. The theory behind this work is that these growth retardants specifically inhibit gibberellin biosynthesis. If this is the case then applied gibberellins should completely overcome the inhibitory effects of growth retardants, provided saturating levels of gibberellins are added. However, any statistical interaction would be evidence against the proposed mode of action of growth retardants. In fact it was found that such an interaction occurred and that cytokinins and red light both overcame the inhibitory effects of the growth retardants. Consequently, it was not possible to say whether or not red light caused the production of gibberellins. From this work it also became apparent that lettuce seed germination was promoted by exogenous cytokinins. There is some controversy in the literature about whether cytokinins alone will break thermodormancy or whether the seeds require small amounts of radiant energy in the red region to respond. Furthermore, no comprehensive study of the effect of exogenous cytokinins, abscisic acid, indole acetic acid, gibberellins and red and far-red light on Grand Rapids seeds has been reported. In order to clarify this situation and it was hoped to throw some 'light' on the control of seed germination, this study was undertaken. We have found that cytokinins promote germination in complete darkness at 30°C. Abscisic acid induced dormancy of lettuce seed can be overcome by red light, by cytokinins and by gibberellins. The fact that gibberellins and red light have been reported as incapable of overcoming ABA would appear to be a function of the concentrations of exogenous chemical added and the time scale of counting germination. In my experiments germination, as assessed by radicle emergence, was counted after 24 hrs and subsequently at 24 hr intervals. This allows analysis in terms of both rate and final germination. Red light was also found to overcome IAA inhibition of germination both at 20°C and 30°C. The inhibition of lettuce seed germination by IAA was lost with time as was ABA inhibition at 20°C. For IAA a 'reverse' escape experiment was conducted where seed was transferred to IAA from water after 1, 2, 3, ----- 24 hrs in water at 20°C. It was found that there was an increasing escape from the inhibitory effect of IAA after 12 hrs imbibition. This was interpreted as showing IAA to have a possible role in germination and not merely radicle growth. A similar experiment with ABA provided more conclusive evidence in favour of ABA inhibiting germination. Finally, in an attempt to establish what role, if any, either ABA or IAA had in germination, the metabolism of [¹⁴C] ABA and [³H] IAA by Grand Rapids lettuce seeds was studied. The results showed that seeds carried out metabolic conversions of ABA but that these changes were not associated with the release from thermodormancy. Changes in labelled IAA indicated that the conversions although requiring the presence of seeds, could not be considered to result from metabolic processes. The results from the IAA metabolism studies do not show a role for IAA. In summary, we have shown the presence of ABA and IAA for the first time in Grand Rapids lettuce seeds, but have been unable to support the hypothesis that ABA and IAA are controlling compounds in the release or imposition of thermodormancy. If one or both of these compounds imposes ,dormancy, it can only be the result of its presence and not at a more fundamental level.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available