EFFECTS OF LIGHT AND GROWTH REGULATORS ON GERMINATION AND RADICLE GROWTH OF LETTUCE SEEDS HELD UNDER HIGH-TEMPERATURE STRESS AND WATER STRESS

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1 New Phytol. (1979) 82, EFFECTS OF LIGHT AND GROWTH REGULATORS ON GERMINATION AND RADICLE GROWTH OF LETTUCE SEEDS HELD UNDER HIGH-TEMPERATURE STRESS AND WATER STRESS BY T. W. HEGARTY AND HEATHER A. ROSS Scottish Horticultural Research Institute, Invergowrie, Dundee DD 2 5DA, Scotland {Accepted April 1978) SUMMARY Seeds of two cultivars of lettuce {Lactuca sativa L.), untreated or soaked in acetone containing kinetin and gibberellic acid (K + GAg), were germinated in light or dark at a range of (high) temperatures and at a range of water potentials. In addition, untreated and treated seeds were germinated at 2 C in the light and then transferred to the same high temperatures or to low water potentials. In both cvs light and K-I-GA3 promoted germination at high temperatures and at low water potentials, although the cvs differed quantitatively in their responses. The upper temperature limit of germination in the light, and of radicle growth after germination, were similar in treated and untreated seeds respectively, suggesting that light and K + GAg in combination may have completely removed the specific high-temperature block to germination. Although light and K + GA3 increased the tolerance of germination to water stress, germinated, untreated seeds were able to grow at even lower water potentials. The K + GA3 treatment, whilst promoting germination under high temperature or water stress, reduced radicle growth subsequent to germination, thus providing support for the possibility that, in germination, the initiation of cell elongation has a degree of metabolic independence of the other processes associated with growth. INTRODUCTION It is well established that light and some growth regulators (gibberellins, cytokinins, fusicoccin) can stimulate the germination of lettuce seeds (achenes) at high temperature, and in contact with osmotic or saline solutions (Kahn, 196; Odegbaro and Smith, 1969; Braun and Khan, 1976; Heydecker and Joshua, 1977). In many reports it has been shown simply that germination was increased by a particular treatment in a particular environment, but Reynolds and Thompson (1973) using the cv. Arctic King quantified the effect and found that kinetin increased the temperature at which there was 5% germination inhibition {GT^Q) from 26-5 to 28-5 C in the dark, and from 31- to c. 4 C in the light, whilst gibberellic acid was more effective in the dark {GT^Q = 31- C) than in the hght {GT^Q "C). In addition. Gray and Steckel (1977), using the cv. Hilde, found that kinetin increased the GT^Q in the light from 25-5 to 3-5 C. On the basis of the suggestion by Obroucheva (1975) that in roots the initiation of cell elongation and cell elongation itself are metabolically independent, and on the basis of our own results for calabrese {Brassica oleracea var. italica Plenck,) and cress {Lepidium sativwn L.) (Hegarty and Ross, 1978) that germination is more sensitive than radicle growth to water stress immediately after germination, we chose lettuce X/79/ fO2.OO/O 1979 The New Phytologist

2 5 T. W. HEGARTY AND H. A. Ross in which to investigate the germination and radicle growth responses to germination stimuli (light and growth regulators) given to seeds held under either high-temperature or water stress. MATERIALS AND METHODS The effects of growth regulators have been studied by incorporating them into the germination medium (Reynolds and Thompson, 1973), by introducing them into the seed via a non-aqueous solvent such as acetone or dichloromethane before evaporating off the solvent (Meyer and Mayer, 1971; Tao and Khan, 1974), or by soaking seeds in an aqueous solution of the chemical (Odegbaro and Smith, 1969; Gray and Steckel, 1977). We have used a single treatment following the method of Khan, Tao and Roe (1973) involving a h soak in acetone solution containing kinetin (-5 mm) and gibberellic acid (GA3, -25 mm). After treatment the seeds were drained of acetone and placed in a vacuum desiccator for -5 h. Commercial seeds of cvs Hilde and Avondefiance were compared in all the experiments because the results of Gray (1975) suggested that the former had a low, and the latter a somewhat higher GT^Q in the light. In the germination experiments seeds were sown, 5 to a Petri dish (plastic, 1 mm square), on to a single thickness of germination paper (Bridger T-IO-D) saturated with water or the appropriate osmotic solution of polyethylene glycol 6 (PEG). The covered dishes were, if required, immediately wrapped in two thicknesses of aluminium foil (dark treatment) before being placed in polyethylene bags to help prevent evaporation. The Petri dishes were then placed in lit incubators running at the desired temperatures. In the growth experiments seeds were sown, 25 to a Petri dish, on to germination papers saturated with water and left for h at 2 C in the light. Fifteen germinated seeds with radicle lengths approximately 1 mm or greater were then selected at random from each dish, radicle lengths were measured to the nearest mm, and the germinated seeds were transferred to a fresh Petri dish containing a germination paper saturated with water or the appropriate osmotic solution. The Petri dishes, in polyethylene bags, were then placed in the lit incubators at the appropriate temperature and radicle lengths were measured after h and h. There were four experiments, and in all experiments seeds of both cvs were used untreated or treated with kinetin+ga3 (K + GA3). In Experiment I, seeds were germinated in the light or dark at 23-, 25-5, 28-, 3-5, 33- and 35-5 C. Final germination was counted after 5 days. In Experiment II the temperature range was extended and seeds were germinated only in the light at 26-, 29-, 32-, 35-, 38' and 41- C. Final germination was also counted after 5 days. In addition, seeds pre-germinated in the light at 2 C were grown on at these elevated temperatures and radicle growth was measured over h. In Experiment III, seeds were germinated in the light and dark at 2 C in water ( bar) or in PEG solutions of 2, 4, -6, 8, 1, 12, 14 or 16 bar. The final germination count was made after 9 d In Experiment IV, seeds pre-germinated in the light at 2 '^C were transferred to fresh Petri dishes with water ( bar) or PEG solutions of - 6, - 8, - 1, - 12, -14, - 16 or - 18 bar for h at 2 C. There was no dark treatment. In all experiments there were three replications. The temperature or water potential at which 5% of the seeds failed to germinate and GW^^y respectively), and at which 5% of the germinated seeds failed to

3 Germination, light and grozvth regulators 51 show an increment in radicle length of at least 2 mm in the period to h after transfer {RT^^^ and RW^^ respectively), were calculated using the logit link function of the GLIM (generalized linear interactive modelling) program available at the Edinburgh Regional Computing Centre. The convention was adopted of restricting calculation of the line of best fit to data showing successive reductions in percentage germination or percentage seedlings showing radicle grow^th with increasing temperature or with decreasing water potential. RESULTS The germination values obtained in Experiments I and II have been combined; the fitted curves and the derived GT-^Q values are shown in Figure 1 and Table 1 respectively. The cv. Hilde, with a low GT^Q for untreated seeds in the dark of 25-4 C, showed a substantial reponse to both K+GA3 and light. On average, light IOO a) 75 * 5 25 I 1 b) 75 ^ Temperature ( C) Fig. 1. Temperature-response curves for germination (%) and radicle growth (percentage germinated seeds showing at least 2 mm growth in the period to h after transfer from, 2 "'C) for two lettuce cvs, cv. Hilde (a) and cv. Avondefiance (b). Closed symbols: untreated seeds. Open symbols: seeds treated with ldnetin4-gibberellic acid. #, O, Experiment 1;, Q, Experiment 2; percentage germination in the light (continuous lines). A, A, Experiment 1, percentage germination in the dark (dotted lines). T. V> Experiment 2, radicle growth (hroken lines).

4 T. W. HEGARTY AND H. A. Ross increased the G2\Q by almost 4 C and K + GAg by 7-5 ^C. The effects of both stimuli were less in the cv, Avondefiance, which had a higher GT^^^ in the dark of 29-2 ^C. Light had less effect than K+ GA3 in this cultivar. In the presence of both stimuli cv. Hilde had a slightly higher GT^^ than cv. Avondefiance. Table 1. Tetnperature ( C) at which there was a 5% reduction in germination or a 5% reduction in the number of germinated seeds with radicles grozving by at least 2 mm (RTQQ). Data are for two lettuce cvs with seeds either untreated or treated with kinetin + gibberellic acid {K-i- GA^). Standard errors are in parentheses cv. Hilde cv. Avondefiance Untreated Untreated GAfor germination in dark for germination in light 25-4 (-15) 29-3 (-1) 37-2 (-15) 33-1 (-13) 37- (-14) 33-7 (-18) 29-2 (-15) 31-5 ( 9) 35-4 (-14) 35-2 (-17) 36-1 (-11) 32-9 (-18) Table 2. Radicle lengths {mm) of seeds germinated at 2 C, transferred to one of five temperatures a?idgrowing by at least 2 mm between and h after transfer. Data are for two lettuce cvs with seeds either untreated or treated with kinetin-\-gibberellic acid {K-\- GA^) measured at transfer {O h) and and h after transfer. Standard errors* in separate table below cv. Hilde cv,, Avondefiance A Tennp. (^C) Untreated * K + GA3 K Untreated K + GA M 1-3 M Standard errors * Because of the different magnitudes of, and different numbers of seedlings contributing to, each mean, standard errors have been calculated for each individual mean. The root growth data are given in Table 2, with fitted curves show^n in Figure 1 and RT^QS in Table 1, It is clear from Figure 1 and Table 1 that whilst K + GA3 increased the GT^Q, the RT^Q was reduced by this treatment. In both cvs it was found that the RT^Q of the untreated seeds, which represents the physiological hightemperature limit for radicle growth, was close in value to the GT^^ for treated seeds in the light. Ungerminated and germinated seeds were killed at 41 X, but germinated seeds showing no growth at 38 C grew when transferred to 26 ''C. Radicle growth was generally reduced as the temperature was raised though this was more evident in cv. Hilde than in cv. Avondefiance (Table 2). Germinated seeds of cv. Hilde had

5 Germination, light and grozvth regulators 53 longer radicles throughout the experiment compared with those of cv. Avondefiance, but relative growth was similar at the lower temperatures. Results from Experiment III are shown in Figure 2 and Table 3. For cv. Hilde the GW^Q of untreated seeds was very high at 3-1 bar. This was substantially altered by K+GA3 and by light which, when combined, gave a GW^Q of 9-7 bar, although Water potential (bar) Fig. 2. Water potential (polyethylene glycol solution) response curves for germination (%) and radicle growth (percentage of germinated seeds showing at least 2 mm growth in the period to h after transfer from water) for two lettuce cvs, cv. Hilde (a) and cv. Avondefiance (b). Closed symbols: untreated seeds. Open symbols: seed treated with kinetin + gibberellic acid., O, Percentage germination in the Hght (continuous lines). A, A, Percentage germination in the dark (dotted lines). T, Vj Radicle growth (broken lines). this was little different from the effect of K + GA3 alone. The effects of light and K+ GA3 on cv. Avondefiance were relatively small, and in this cv. seeds treated with K+GA3 had a marginally lower GW^Q when germinated in the light than when germinated in the dark. Radicle growth measurements from Experiment IV are summarized in Table 4, the fitted curves are shown in Figure 2 and the derived data are shown in Table 3. In both cvs K + GA3 gave higher values of RW^Q (i.e. less tolerance of water stress) than untreated seeds, and in both cvs the RW^Q values of untreated seeds were

6 - 54 T- W. HEGARTY AND H. A. Ross substantially lower than the GW^^ values of treated, light-germinated seeds. There was a marked contrast between the cvs in the range of GPF^jj and7?pfg,^ values obtained, with cv, Hilde having a GWr,Q of untreated seeds in the dark of 3*1 bar yet a /^PF^g of untreated seeds of 16- bar, compared with 8-3 bar and 12-6 bar respective in cv. Avondefiance. Table 3. Water potentials {bar) at which there was a 5% reduction in germination (GW5Q) or a 5% reduction in the numbers of germinated seeds with radicles growing by at least 2 mm (RWSQ). Data are for two lettuce cvs with seeds either untreated or treated with kinetin -\- gibberellic acid {K -\- GA^). Standard errors are in parentheses cv. Hilde cv. Avondefiance Untreated K + GA, Untreated K + GAfor germination in dark -3-1 (-12) -9-3 (-9) -7-8 (-9) -9-7 (-7) for germination in light -8-3 (-11) - (-11) -91 (-11) -9-6 (-11) -16- (-25) -9-7 (-2) (-25) (-23) Table 4. Radicle lengths {mm) of seeds germinated at 2 ^C, transferred to one of eight water potentials {WP, bar) and growing by at least 2 mm between and h after transfer. Data are for two lettuce cvs with seeds either untreated or treated with kinetin -{-gibberellic acid {K-\- GA^, measured at transfer {O h) and and h after transfer Standard errors* in separate table below WP Untreated A cv Hilde K + GA A Standard c M errors Untreated cv. Avondefiance A. 1-1 M K + GA A * Because of the different magnitudes of, and different numbers of seedlings contributing to, each mean, standard errors have been calculated for each individual mean. As in Experiment II, K + GA3 reduced radicle growth, and below - 6 bar, decreasing water potential also decreased radicle growth in untreated seeds. At bar, growth was less than at - 6 bar but this may have been due to the very different patterns of development of the radicle in water and PEG solutions. In water the radicles were

7 Germination, light and growth regulators 55 relatively thick, with root hairs densely distributed along almost the whole length of the radicle. However, at - 6 bar and lower water potentials the radicles were characteristically very thin and had very few and diminutive root hairs which were confined to the proximal end of the radicle. DISCUSSION Haber and Luippold (196a) considered that in lettuce, germination (rootlet protrusion) was a result of cell elongation and not cell division. They found that at 26 C the onset of cell division and cell elongation coincided. However, at 3 C most seeds underwent cell division but did not germinate, and in seeds held in mannitol solution at 26 C cell division preceded cell expansion by many days. Haber and Luippold (196b) also considered that dormancy of this type, which is not generally related either to a significant depression of overall metabolism or to prevention of cell division, is a subtle block that specifically prevents the initiation of cellular expansion. Light and growth regulators have been shown before to stimulate germination of lettuce seeds under high-temperature stress and at low water potential (Kahn, 196; Reynolds and Thompson, 1973; Braun and Khan, 1976), and kinetin has also been shown to inhibit root growth in lettuce seedlings (Ikuma and Thimann, 1963a; Heydecker and Joshua, 1977). Our results have shown that in both cvs the RT^^^ of treated seeds was lower than the GT^fy of light-germinated, treated seeds. This suggests that light and K+ GA3 in combination stimulated the elongation of existing cells in the embryo, resulting in rootlet protrusion. However, they impeded further growth, which would have been dependent both upon cell division and upon expansion, once germination had been initiated. There is thus an apparent similarity with the situation described by Obroucheva (1975) for roots, in which the initiation of cell elongation has a degree of metabolic independence of the processes of cell elongation itself as well as of cell division. It may be relevant that in embryonic axes isolated from dormant hazel {Corylus avellana L.) seeds, GA3 was found to stimulate early growth of the axis but in the longer term led to reduced root growth (Jarvis, Wilson and Fowler, 1978), We have also quantified the extent to which the block to germination at high temperature and low water potential can be displaced using light and one particular combination of kinetin and GA3. Indeed, the similarity of the values of the RT^^ of untreated seeds to the GT^^ of light-germinated, treated seeds in both cvs. could indicate that the block to germination induced at high temperature was completely overcome by this particular combination of treatments. It may also indicate that the response shown by the treated seeds no longer represented a response specific to germination, but simply that the physiological high-temperature limit for root growth in these seed populations had been reached. In contrast, although in the cv. Hilde the GW^Q was reduced from 3-1 bar for untreated seeds in the dark to 9*7 bar for K + GA3 treated seeds in the light, the RWr^^ of the untreated seeds, at 16- bar, was still substantially lower. This does not rule out the possibility, however, that the use of other combinations of these growth regulators, or of different growth regulators, might be more effective than the present treatment in reducing further the value and hence overcoming the block to germination induced by water stress.

8 56 T. W. HEGARTY AND H. A. Ross Our results also emphasize how important seed lot differences can be in contributing to different germination responses in lettuce cultivars. The GT^Q values of light-germinated, untreated seeds in this experiment were 31-5 C for cv. Avondefiance and 29-3 C for cv. Hilde, and those can be compared with the values 28-5 and 25-7 C respectively found by Gray (1975). In our experiments untreated seeds of cv. Hilde had the least tolerance to both high-temperature stress and water stress when germinated in the dark, but the greatest tolerance of these stresses once germinated. There is, however, insufficient evidence toshow whether any of these responses are necessarily linked. In addition, the results show that the responses of seeds to light and K + GA3, separately and in combination, differed with the applied stress, being additive under temperature stress but interacting under moisture stress. Further investigation might yield valuable information on the underlying mechanisms involved in the responses to high-temperature and water stresses. There are many theories on the precise mode of action of light and growth regulators in breaking seed dormancy, especially in lettuce (Haber and Luippold, 196b; Ikuma and Thimann, 1963b; Scheibe and Lang, 1965; Lewak and Khan, 1977). The mechanism of phytochrome action is rapid and involves changes at the biophysical level of the cell, such as changes in the properties of membranes (Kendrick, 1976), and in lettuce seeds held in PEG solution phytochrome stimulation has been shown to result in a decrease of solute potential within the axes (Carpita and Nabors, 1977). Growth regulators may also act on osmoregulation either directly or indirectly via the cell membrane (Marre, 1977) and it is possibly at this level that a link may be found between the actions of, and responses of stimuli to, high-temperature stress and water stress on germination (Hegarty, 1978). REFERENCES BRAUN, J. W. & KHAN, A. A. (1976). Alleviation of salinity and high temperature stress by plant regulators permeated in lettuce seeds via acetone. Jottrnal of the American Soeietv of Horticultural Science, 11, 716. CARPITA, N. C. & NABOBS, M. W. (1977). Phytochrome-induced changes in osmotic and pressure potentials of photodormant lettuce seeds. Plant Physiology, 59, Suppl., 34, Abstract 188. GRAY, D. (1975). Effects of temperature on the germination and emergence of lettuce {Lactiica sativa, L.) varieties. Journal of Horticultural Science, 5, 349. GRAY, D. & STECKEL, J. R. A. (1977). Pre-sowing seed treatment with cytokinin to prevent high temperature dormancy in lettuce {Lactuca sativa) seeds. Seed Science and Technology, 5, 473. HABER, A. H. & LUIPPOLD, H. J. (196a). Separation of mechanisms initiating cell division and cell expansion in lettuce seed germination. Plant Physiology^ 35, 168. HABER, A. H. & LUIPPOLD, H. J. (196b). Effectsof gibberellin, kinetin, thiourea, and photomorphogenic radiation on mitotie activity in dormant lettuce seed. Plant Physiology, 35, 6. HEGARTY, T. W. (1978). The physiology of seed hydration and dehydration, and the relation between water stress and the control of germination: a review. Plant, Cell and Environment, 1, 11. HEGARTY, T. W. & Ross, H. A. (1978). Differential sensitivity to moisture stress of seed germination and seedling radicale growth. Annals of Botany, 42, 13. HEYDECKER, W. & JOSHUA, A. (1977). Alleviation of the thermodormancy of lettuce {Lactuca sativa L.) seeds. Journal of Horticultural Science, 52, 87. IKUMA, H. & THIMANN, K. V. (1963a). Action of kinetin on photosensitive germination of lettuce seed as compared with that of gibberellic acid. Plant and CeU Physiology, 4, 113. IKUMA, H. & THIMANN, K. V. (1963b). The role of the seed-coats in gemiination of photosensitive lettuce seeds. Plant and Cell Physiology, 4, 169. JAHVIS, B. C, WILSON, D. A. & FOWLER, M. W. (1978). Growth of isolated embryonic axes from dormant seeds of hazel {Corylus avellana L.). Nerv Phytologist, 8, 117. KAHN, A. (196). An analysis of 'dark-osmotic inhibition' of germination of lettuce seeds. Plant Physiology, 35, L KENDRICK, R. E. (1976). Photocontrol of seed germination. Science Progress, 63, 347.

9 Germinatioriy light and growth regulators 57 KH.\N, A. A., TAO, K. L. & ROE, C. H. (1973). Application of chemicals in organic solvents to dry seeds. Plant Physiology, 52, 79. LEWAK, S. &: KHAN, A. A. (1977). Mode of action of gibberellic acid and light on lettuce seed germination. Plant Physiology, 6, 575. MARRE, E. (1977). Effects of fusicoccin and hormones on plant cell membrane activities: observations and hypotheses. In: Regulation of Cell Membrane Activities in Plants (Ed. by E. Marre & O. Ciferri), pp Elsevier/North Holland Biomedical Press, Amsterdam. MEYER, H. & MAYER, A. M. (1971). Permeation of dry seeds with chemicals: use of dichlormethane. Science, 171, 583. OBROUCHEVA, N. V. (1975). Physiology of growing root cells. In: The Development and Function of Roots (Ed. by J. G. Torrey & D. T. Clarkson), pp Academic Press, London. ODEGBARO, O. A. & SMITH, O. E. (1969). Effects of kinetin, salt concentration, and temperature on germination and early seedling growth of Lactuca sativa L. Jottrnal of the American Society for Horticultural Science, 94, 167. REYNOLDS, T. SC THOMPSON, P. A. (1973). Effects of kinetin, gibberellins and (±) abscisic acid on the gemiination of lettuce {Lactuca sativa). Physiologia Plantarujn, 28, 516. SCHEIBE, J. & LANG, A. (1965). Lettuce seed germination: evidence for a light-induced increase in growth potential and for phytochrome mediation of the low temperature effect. Plant Physiology, 4, 4S5. TAO, K. L. & KHAN, A. A. (1974). Penetration of dry seeds with chemicals applied in acetone. Plant Physiology, 54, 956.

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