PREVENTING THERMOINHIBITION IN A THERMOSENSITIVE LETTUCE GENOTYPE BY SEED IMBIBITION AT LOW TEMPERATURE

Lettuce (Lactuca sativa L.) seed germination is strongly temperature dependent and under high temperatures, germination of most of genotypes can be erratic or completely inhibited. Lettuce seeds of Dark Green Boston (DGB) were incubated at temperatures ranging from 15° to 35°C at light and dark conditions. Other seeds were imbibed in dark at 20°; 25°; 30°; and 35°C for 8 and 16 hours and then transferred to 20 or 35°C, in dark. Seeds were also incubated at constant temperature of 20° and 35 °C, in the dark, as control. In another treatment, seeds were primed for 3 days at 15°C with constant light. DGB lettuce seeds required light to germinate adequately at temperatures above 25°C. Seeds incubated at 20°C had 97% germination, whereas seeds incubated at 35°C did not germinate. Seeds imbibed at 20°C for 8 and 16 hours had germination. At 35°C, seeds imbibed initially at 20°C for 8 and 16 hours, had 89 and 97% germination, respectively. Seeds imbibed at 25°C for 16 hours, germinated satisfactory at 35°C. High temperatures of imbibition led to no germination. Primed and non-primed seeds had 100% germination at 20°C. Primed seeds had 100% germination at 35°C, whereas non-primed seeds germinate only 4%. The first hours of imbibition are very critical for lettuce seed germination at high temperatures.


INTRODUCTION
Optimum germination temperature for most lettuce genotype seeds is around 20°C (AOSA, 1993), although some germinate at temperatures ranging from 5 to 33°C (Gray, 1975).Different cultivars vary in their response to germination at high temperatures (Thompson et al., 1979).In general, temperatures above 30°C ad-versely affect lettuce seed germination.High temperatures may decrease either germination rate or germination percentage (Cantliffe et al., 2000).
Several methods to circunvent the effects of high temperature have been proposed including resistant germplasm, adjusting the seed production environment, and the use of growth hormones (Nascimento & Cantliffe, 1999).Seed priming has also been used to allow germination of lettuce seed at higher temperatures (Cantliffe et al., 1981;Guedes & Cantliffe, 1980;Khan et al., 1980/ 81;Valdes et al., 1985;Wurr & Fellows, 1984).The objectives of the present study were to determine the effects of different temperatures and light on seed germination for a thermosensitive lettuce genotype, and to evaluate the effects of low temperature imbibition for seed germination at high temperature.
Experiment 2: Seeds were imbibed in the dark at 20°; 25°; 30°; and 35°C for 8 and 16 hours and then transferred to 20° or 35°C, in the dark.Other seeds incubated at constant temperature of 20° and 35°C in the dark were used as controls.Germination procedures were used as described for Experiment 1.
Experiment 3: Priming was used as a control procedure.Seeds were primed in 200 mm test tubes for 3 days at 15°C with constant light (~26 µmol m -2 s -1 ) in an aerated solution of polyethylene glycol (PEG), at an osmotic potential of -1.2 MPa (30 mL of solution g -1 of seed).Aeration was provided by flow pump.The air was pre-hydrated by bubbling through water to minimize evaporation of the soaking solution.Afterwards, seeds were placed in a Buchner funnel, rinsed three times with 100 mL of distilled water and redried in an incubator at 15°C and 45% RH for two days.Radicle emergence was recorded daily for seven days.Total percent germination (normal seedlings) was evaluated at seven days (AOSA, 1993).
Four replications of 50 seeds were used in these experiments.Analysis of variance (ANOVA) using a randomized factorial design was performed.Treatments means were compared by the Tukey and F-tests (P = 0.05).

RESULTS AND DISCUSSION
DGB seeds germinated satisfactorily at temperatures below 22.5°C in the dark (Table 1).Early studies reported that light affects lettuce seed germination (Borthwick et al., 1952;Bewley & Black, 1994).For example, seed dormancy in lettuce may be induced when seeds are imbibed for a prolonged period in the dark (Toole et al., 1956).This effect may be alleviated if red light is applied to the imbibing seed early in the germination process (Scheibe & Lang, 1965).An interaction between imbibition conditions and temperature was observed in this study (Table 1).
Seed imbibition under light increased the threshold for germination at higher temperatures; under light conditions, seeds germinated adequately at 27.5°C.These results agree with several reports which have shown that lettuce seeds imbibed at high temperatures are more dependent on red light for germination (Evenari et al., 1953;Georghiou & Thanos, 1983).Nascimento & Cantliffe (2000) reported that germination of 'Everglades', a thermotolerant lettuce genotype, exceeded 90% at temperatures up to 35°C under light conditions.However, germination of 'Everglades' decreased when seeds were imbibed under high temperature in the dark.However, DGB seeds did not germinate satisfactorily at temperatures above 27.5°C,even under light conditions (Table 1).
Table 1 -Percentage of germination of 'Dark Green Boston' lettuce seeds at different temperatures in light and dark conditions.
In each column, means with the same upper-case letter and, in each row, means with the same lower-case letter are not different, by Tukey Test (P ≤ 0.01).There was interaction between conditions and temperature (P ≤ 0.01).
Scientia Agricola, v.60, n.3, p.477-480, Jul./Sept.2003 In another study, seeds imbibed at constant temperature of 20°C in the dark, had 97% germination; at 35°C, seeds did not germinate (Table 2).However, when seeds were previously imbibed at low temperature (20°C) for 8 hours, germination at 35°C was 89%.Interactions among the treatments were observed in this study (Table 2).Lettuce seeds may germinate at high temperatures when the seeds are previously imbibed for a period of time at low temperatures.When seeds were imbibed for 16 hours at 20°C, germination at 35°C was 97%.The longer the seeds are imbibed at low temperatures, the higher their germination percentage after they were transferred to high temperatures.A prolonged period of imbibition at low temperatures might promote production of enzymes needed for lettuce endosperm loosening and consequent seed germination at high temperature (Nascimento, 1998).In addition, the length of phase II is temperature dependent.However, germination and endo-β-mannanase synthesis in lettuce at 15°C during the priming proceeded at adequate rates to circumvent thermodormancy later, at 35°C.At 25°C, seeds required 16 hours of imbibition to germinate adequately.Seeds imbibed at this temperature for 8 hours had only 43% germination (Table 2).Higher temperatures of imbibition affected seed germination at both temperatures of germination (Table 2).
Primed and non-primed seeds had 100% germination at 20°C (Table 3).At 35°C, non-primed seeds had only 4% germination, whereas primed seeds had 100%.An interaction between priming and germination temperature was observed for total germination but not for germination rate (Table 3).Primed seeds germinated faster than non-primed seeds, even at 20°C.Benefic effects of seed priming at high temperature were previously reported for lettuce (Cantliffe et al., 1981;Guedes & Cantliffe, 1980;Khan et al., 1980/81;Valdes et al., 1985;Wurr & Fellows, 1984, Eira, 1988), as confirmed herein.In general, lettuce seeds are primed in an osmotic solution under low temperatures, e.g., 15°C (Nascimento & Cantliffe, 1998).Seed priming extends the lag phase of water uptake by restricting radicle emergence, and in this phase major metabolic events occur preparing the seed for phase III and radicle protrusion (Bewley & Black, 1994).Therefore, a prolonged phase II during priming might promote a specific mechanism needed for seed germination at high temperatures (Nascimento et al., 2001).
The mechanism by which seed priming overcomes thermoinhibition or thermodormancy for lettuce is unknown.Guedes et al. (1981) observed a progressive loosening of the endosperm membrane during priming, a possible indicative of endosperm weakening, thus allowing lettuce seed germination to proceed at high temperatures.Nascimento et al. (2001) concluded that the activity of endo-β-mannanase, an enzyme which hydrolyses the mannan-polymers, and is produced and secreted by the lettuce endosperm, increased markedly in primed seeds before radicle protrusion, and this may be a crucial mechanism for endosperm weakening and consequently lettuce seed germination, especially under high temperatures.
Temperatures during lettuce seed imbibition above the optimum for a specific cultivar may lead to two different phenomena: thermoinhibition, a reversible process, where the seeds do not germinate above a certain temperature, but will germinate after transfer to a lower temperature.Prolonged exposure to supraoptimal temperatures may induce a true dormancy, a phenomenon known as thermodormancy or secondary dormancy (Khan, 1980/81).In this case, the seeds will not germinate even when the temperature is reduced to a favorable range.

Table 2 -
Percentage of germination of 'Dark Green Boston' lettuce seeds imbibed for 8 and 16 hours at different temperatures and incubated at 20 and 35°C in the dark.In each column, means with the same lower-case letter and, in each row, means with the same upper-case letter are not different by Tukey Test (P ≤ 0.01).There were interactions among treatments (P ≤ 0.01).

Table 3 -
Total germination (TG) and time to first germination (FG) of 'Dark Green Boston' lettuce primed and non-primed seeds at 20 and 35°C in the light.