occurre in both water and 1% K3PO4 4to 6 h prior to cell division. Both proc c d sooner in water ... Florida Agricultural Experiment Station Journal Series No. 4935. .... mounts after various time durations (O to 44 h) using the tech- niques of Dyer ...
Plant Physiol. (1984) 75, 290-294 0032-0889/84/75/0290/05/$01.00/0
Mechanism of Seed Priming in Circumventing Thermodormancy in Lettuce' Received for publication June 27, 1983 and in revised form January 4, 1984
DANIEL J. CANTLIFFE*, JEANNE M. FISCHER, AND TERRIL A. NELL Vegetable Crops Department (D. J. C., J. M. F.) and Ornamental Horticulture Department (T. A. N.), University ofFlorida, Gainesville, Florida 32611 ABSTRACr Lettuce (Lactau tiva L. cv Minetto) seeds were prmed in aerated solutios of 1% K3PO4 or water at 15C in the dark for various periods of time to determine the manner by which seed priming bypasses thermodormancy. Seeds which were not prmed did not germinate at 35°C, whereas the which were prime for 20 h in 1% K3PO4 or distilled H20 had up to 86% gIon. The rate of water uptake and respiration during primg were similar regadless of soak solution. Cell eion tio occurre in both water and 1% K3PO4 4 to 6 h prior to cell division. d sooner in water than K3PO4. Radicle protruBoth proc c sion (germiation) occured in the prming soluto at 21 h in water and 27 h in 1% K3P04. Respiration, radicle pt and cell division cotently occurred sooner in primd (redried) seeds compred to nonprmed seeds when they were imbibed at 25C. Cell division and elotio co after 10 h imbiion primed (redried) seeds imbibed at 35C. Neither proces d seeds. Respatory rtes were higher in both occum In primed and noprid seeds imbibed at 35°C compaed to those imbibed at 25C,t radicle prtusion did not occur in noprmed seeds a ad which were imbibed at 35C. It is apparent that cell e ntio division are hibited duig itemperatugre imbibition in mprimed lettuce seeds. Seed primi ppear to kad to the irreversible inititio of cell e ation, thus overcomin thermodmncy.
(19) postulated that the endosperm exerted a mechanical resistance to embryo expansion. However, Halmer et al. (12) reported that endosperm cell wall degradation is not a prerequisite for germination in lettuce. They based this opinion on the observation that an endosperm digestive enzyme, mannanase, was produced in quantity only after the radicle penetrated the endosperm layer. Guedes et al. (10) have reported that a progressive loosening of the endosperm layer occurred after 9 h of priming 'Minetto' lettuce seeds, and they postulated that this may be related to the 130 120 LD 100
t'1
.90 110
3000
Depending on cultivar, thermodormancy in lettuce seeds may be induced when germination temperatures exceed 28C (1). Seed priming is an effective method to overcome this dormancy (3, 9). Seed priming consists of soaking seeds under conditions that allow the seeds to imbibe water and initiate germination, but which do not permit radicle protrusion through the seed coat (13). This generally includes the use of an osmoticum (-5 to -20 bars), such as a salt solution or PEG and low temperature. The duration of the soak is important in order to ensure that radicle protrusion through the seed coat does not occur. The seeds can then be dried and stored or planted with conventional equipment. The mechanism by which seed priming bypasses thermodormancy is not understood. Several other methods can circumvent thermodormancy including treatment with cytokinins (22), ethylene (18), inased 02 concentration (5), and by damaging or removing the endosperm layer (7, 14). Ikuma and Thimann (14) proposed that the endosperm must be partially digested before radicle emergence would occur. Seveml researchers (15, 20, 21) showed that endosperm cells were extensively degraded as radicle growth began. Pavlista and Haber ' Florida Agricultural Experiment Station Journal Series No. 4935.
4
8
12
16 20 24 Time (hours)
28
32
36
40
FIG. 1. Water uptake as percentage increases in moisture of'Minetto' lettuce seeds placed in l1% K3P04 or distilled H20 at 15°C for periods of time to 40 h. Vertical bars represent SE. 3
2
^,250 -
o
3 4 5 6 89--o 7 o2
Time (hours) FIG. 2. Respiration of'Minetto' lettuce seeds during seed priming for 9 h at 1s5°C in either 1% K3P04 or distilled H20. Vertical bar represent SE.
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THERMODORMANCY IN LETTUCE
291
FIG. 3. Growth of 'Minetto' lettuce seed radicles imbibed at 35-C. A-C-E, nonprimed seeds. B-D-F, seeds primed in aerated 1% K3PO4 in the dark at 15C for 9 h. A-B, 12-h; C-D, 18-h; and E-F, 24-h germination period. Note the lack of radicle elongation on nonprimed seeds while elongation and growth patterns of primed seeds appear normal.
Plant Physiol. Vol. 75, 1984
CANTLIFFE ET AL.
292
I
at cn Co I I.-
0 z
z
w -J
z
c.)
F
z
2
FIG. 4. Cell division and cell elongation of 'Minetto' lettuce seeds during priming in 1% K3PO4 or distilled H20 at 15-C for a total duration of 45 h. Vertical bars represent sE. Cell division data represents the numbers of seeds out of 20 in which cell division was observed.
z
TIME (hours)
Table I. Per Cent ofPrimed or Nonprimed Lettuce Seeds Undergoing Cell Division during Germination at Two Temperatures Counts were made of at least 10 seeds at each time period for each treatment.
Hours of imbibition 8 10 12 14
25*C 35°C Nonprimed Primed Nonprimed Primed 0 0 10 80
% cell division 0 0 0 50 0 80 0 90
0 50 60 100
enhancement of germination at high temperatures. The relationship of this 'loosening' to those processes leading up to radicle growth, i.e. imbibition, ration, cell elongation and cell division, has not been studied. The objective of these experiments was to ascertain the mechanism by which seed priming prevents the establishment of thermodormancy in lettuce.
MATERIALS AND METHODS Seed Priming. Lettuce (Lactuca sativa L. cv Minetto) seeds were primed at 15C in the dark in aerated water or 1% K3PO4 for various durations. The seeds were placed in 200-mm test tubes with 20 ml of soak solution per g of seeds. After priming for the appropriate duration, the seeds were washed with 100 ml of glass disfilled H20 and surface moisture was removed by 2 to 3 min of suction. The seeds were dried at 5C and 45% RH, then stored under these conditions until used. Germination throughout the paper is defined as visible radicle
protrusion through the seed coat and pencarp.
Determination of Moisture Content. Seeds were primed as described above for various penrods of time to 40 h. At the appropriate time the seeds were removed, damp dried, and then weighed. Determination of Respiratory Activity. During priming, 100 seeds were placed in a Warburg flask containing 2 ml of 1% K3PO4 or water. The center well contained KOH. Respiratory activity (02-consumed) was measured at 15C on at least 6 replicate samples from 15 min to 9 h with a Gilson Differential Respirometer. During germination, nonprimed or primed seeds were placed in Warburg flasks and respiratory activity was determined on at least 6 replications as described above at 25 and 35C for 15 min to 27 h. Cell Division Counts. Seeds that were primed in water or 1% K3PO4 and seeds which had been previously primed or untreated before being germinated at 25 and 35C were p d for squash mounts after various time durations (O to 44 h) using the techniques of Dyer (6). A minimum of 10 samples at each time period and temperature were examined. Propionic orcein was used in place of lacto-propionic orcein. Ceil Eongation. Seeds were primed in water or 1% K3P04 at 150C for various periods of time to 45 h as described above. After rinsing they were placed in FAA solution (formaldehyde l0:glacial acetic acid 5:ethyl alcohol 50:water 35). Elongation was detrmined according to the procedures of Foard and Haber (8). The seeds were cut lengthwise with a razor blade and placed in 1% toluidine blue for 1 to 2 min. Measurements on at least 20 seeds per time period and soak treatment were made from the radicle tip to the apical meristem under a disseting micro-
i.~ sew ad
FIG. 5. Respiration measured as 02 uptake in primed wseds (1% K3PO4, 9 h at 15°C then redried to 5% moisture) and in nonprimed (raw) seeds during hydration in water over a 27-h
represent SE.
period
at
25"C.
Vertical bars
THERMODORMANCY IN LETTUCE
293
FIG. 6. Respiration measured as 02 uptake in primed (1% K3PO4, 9 h at 15C then redried) 'Minetto' lettuce seeds and in nonprimed (raw) seeds during hydration in water over a 27-h period at 35C. Vertical bars represent SE.
10
14 16 Tine (hours)
12
scope.
Electron Microscopy. Primed and nonprimed seeds were gernated at 35C for 12, 18, and 24 h, fixed in FAA solution for 2 d, then dehydrated through an alcohol series 50%:75%:95%:100%:100% and critical point dried. Samples were randomly selected and then coated with 600 A pd/gold in a Technics Hummer V sputter coater. At least 10 seeds for each treatment at each time represented were viewed with a Hitachi S-450 Scanning Electron Microscope. Sanning
RESULTS AND DISCUSSION The rates of water uptake in lettuce seeds which were primed at 1 5C in either water or 1% K3P04 were similar over a 40 h period (Fig. 1). Respiration rates during the 9-h priming period also were similar in either water or 1% K3P04 (Fig. 2), which might be expected since water entry was not impeded by the salt solution. Although 1% K3P04 has an osmotic potential of approximately -5.1 bars, the rate of water uptake was not limited by this water potential gradient. 'Minetto' lettuce seeds which were not primed did not germinate at 35C (Fig. 3). Lettuce seeds soaked in 1% K3P04 had 56% germination at 35C when primed for 9 h and 86% germination when primed for 20 h (data not presented). Seeds primed in water had slightly different germination percentages (64 and 71%, respectively, for 9 and 20 h of priming) than those primed in salt solution. Previous reports (3, 9) stated that extending the duration (24 h) of the priming process gave rise to a high incidence ofabnormal seedlings when the seeds were germinated at 35°C. It was speculated that abnormal seedlings were caused by the occurrence ofcell division during priming and consequent damage during drying of the seeds. To determine when cell division and elongation commenced during priming, seeds were kept in the soak solution for periods up to 45 h. Radicle protrusion (germination) occurred at 21 h when lettuce seeds were primed in water and at 27 h when primed in 1% K3P04 (Fig. 4). Radicle protrusion was clearly via cell elongation since cell division did not begin for 5 to 9 h after protrusion was observed (i.e. 27 h in water and 36 h in K3P04). These results agree with those of Haber and Luippold (1 1) who reported that radicle protrusion during germination was the result of cell expansion and that cell division virtually played no role in this process. Guedes and Cantliffe (9) observed that during drying of lettuce seeds at room temperature after priming there was a slow loss of water over a 3- to 4 h-period. Under the conditions ofthe present experiment, drying was at 5C, thus the process was probably slower than previously reported and the chance for irreversible cell elongation was improved. Additional experiments showed
that germination at 35C was consistently higher, regardless of seed lot, if the soak duration in 1% K3P04 at 15C was extended to 20 h (3). Thus, from Figure 4, cell elongation would be well underway at this time. Under the conditions of this experiment priming at 15C initiated cell elongation before cell division in the priming solution. Subsequently, after the seeds were primed for 9 h and then redried, it took 10 h of soaking at 25 or 35C before cell division commenced (Table I). However, germination, as radicle protrusion, occurred before this (6 h at 25C, Fig. 5; and 9 h at 35°C, Fig. 6). Respiration, measured as 02 uptake was rapid in primed and nonprimed seeds germinated at 25C (Fig. 5); however, the rate of 02 uptake was continually higher throughout the germination period in primed seeds. Radicle protrusion (germination) in primed seeds occurred after 6 h imbibition, while it took slightly over 12 h in nonprimed seeds. Cell division at 25C commenced after 10 h imbibition in primed seeds and 12 h in nonprimed seeds (Table I). Thus, at 25C cell elongation continued to precede cell division in primed seeds compared to nonprimed seeds. At 35C nonprimed seeds did not germinate; however, radicle protrusion (germination) and cell division occurred after 10 h in primed seeds (Fig. 3, 6, and Table I). Respiration rates were high regardless of treatment; however, 02 uptake in primed seeds surpassed that which occurred in raw seeds (Fig. 6). Thus, respiration did not appear to be hindered during high temperature stress. Cantliffe (2) reported that ATP production was not inhibited in 'Grand Rapids' lettuce at 35°C although no radicle protrusion (germination) occurred. Hence, it does not appear that altered respiratory rates led to thermodormancy, nor that seed priming markedly alters the pattern of respiration. For radicle protrusion to occur, cell elongation must occur ( 11) and the protrusion process can go on without cell division. At threshold temperatures for thermodormancy, 28 to 30C, germination of 'New York' lettuce did not occur but cell division did (8, 11). We did not find cell division to occur for periods up to 96 h in nonprimed 'Minetto' lettuce seeds germinated at 35°C (Table I and data not shown). Foard and Haber (8) did not observe any cell division in 'New York' lettuce seeds germinated at 40°C. Thus, this distinction between cell division and elongation could only be observed at the 'threshold' temperature. Cell elongation in lettuce seeds is inhibited by high temperature (35C). For cell elongation to occur the osmotic potential of the radicle must be incrsed (17, 26). This could be caused by an increased accumulation of free amino acids in the radicle tip as previously reported (23-25). High temperature inhibits the accumulation of these free amino acids as does ABA (25). Kinetin reverses the effects of ABA and high temperature on both ger-
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CANTLIFFE ET AL.
mination and amino acid accumulation (25). ABA inhibits protease activity and thus might prevent amino acid accumulation. Chandra and Toole (4) reported that esterase activity coincided with radicle protrusion and that esterase was inhibited by high temperature. Several researchers have reported that ABA levels in lettuce seeds were higher under conditions of high temperatures (16). Possibly there is an interaction between esterase activity and ABA. Possibly the above processes are linked, and seed priming might initiate cell elongation (via amino acid accumulation at the radicle tip) so that high temperature could no longer inhibit germination thus circumventing thermodormancy. 1.
2. 3. 4.
5. 6. 7.
8. 9. 10.
LITERATURE CITED BoRTHwiCK HA, WW RoBBINS 1928 Lettuce seed and its germination. Hilgrdia 11: 275-304 CANTFFE DJ 1976 Changes in ATP in lettuce germinated at different tempenaue in the presence of growth regulators. Plant Physiol 57: 38 CANTLIFFE DJ 1981 Seed priming of lettuce for early and uniform emergence under conditions of environmental stress. Acta Hortic 122: 29-38 CHANDRA GR, VK TOoLE 1976 Release of estse during the germination of lettuce seed (Lactuca sativa L). Plant Physiol 57: 37 COME D, T TmAouI 1972 Interrelated effects of imbibition, temperature, and oxygen on seed germination. In W Heydecker, ed, Seed Ecology. Pennsylvania State University Press, University Park, pp 157-169 Dma AF 1963 The use of lactoprpionic orcein in rapid squash methods for chromosome prepaations. Stain Techol 38: 85-89 EvENAI M, G NEUMANN 1952 The grmination of lettuce seed IL The influence of fruit coat, seed coat and endosperm upon germination. Bull Res Counc Isr 2: 75-77 FoARD DE, AH HABER 1966 Mitosis in thermodormant lettuce seeds with reference to histologial location, localized expansion, and seed storage. Planta 71: 160-170 GUEDEs AC, DJ CANTIFPE 1980 Germination of lettuce seeds at high temperature after seed priming. J Am Soc Hortic Sci 105: 777-781 GUEDEs AC, DJ CANTUFFE, TA NELL 1981 Morphological changes during
Plant Physiol. Vol. 75, 1984
lettuce seed priming and subsequent radicle development. J Am Soc Hortic Sci 106: 121-126 11. HABER AH, HJ LUIPPOLD 1960 Separation of mechanisms initiating cell division and cell expansion in lettuce seed germination. Plant Physiol 35: 168-173 12. HALMER P, JD BEWLEY, TA THORPE 1976 An enzyme to degrade lettuce endosperm cell walls. Appearance of a mannanase following phytochrome
and gibberellin-induced germination. Planta 130:189-196 13. HEYDECKER W, P COOLBEAR 1977 Seed treatments for improved performancesurvey and attempted prognosis. Seed Sci Technol 5: 353-425 14. IKUMA H, KV THIMANN 1963 The role of the seed-coats in germination of photosensitive lettuce seeds. Plant Cell Physiol 4: 169-185 15. JoNEs RL 1974 The structure of the lettuce endosperm. Planta 121: 133-146 16. MCWHA JA 1976 Changes in abscisic acid levels during imbibition and germination of non-dormant and thermodormant lettuce seeds. Aust J Plant Physiol 3: 849-851 17. NABOltS MW, A LANG 1971 The growth physics and water relations of redlight-induced germination in lettuce seeds. Planta 101: 1-25 18. NEGM FB, OE SmITH 1978 Effects of ethylene and carbon dioxide on the germination of osmotically inhibited lettuce seed. Plant Physiol 62: 473-476 19. PAVLISTA AD, AH HABER 1970 Embryo expansion without protrusion in lettuce seeds. Plant Physiol 45: 636-637 20. PAVLISTA AD, JG VALDOVINOS 1978 Changes in the surface appearance of the endosperm during lettuce achene germination. Bot Gaz 139: 171-179 21. PSARAS G, K GEORGHIou, K MITRAKOs 1981 Red-lght-induced endosperm preparation for radicle protrusion of lettuce embryos. Bot Gaz 142: 13-18 22. SMITH OE, W YEN, JM LYoNs 1968 The effects of ldnetin in overcoming high temperature dormancy of lettuce seed. Proc Am Soc Hortic Sci 93:444 453 23. TAKEBA G 1980 Changes revealed by a tracer technique in the amino acid metabolism of thermodormant and nondormant New York lettuce seeds. Plant Cell Physiol 21: 1627-1638 24. TAKEBA G 1980 Accumulation of free amino acids in the tips of nonthermodormant embryonic axes accounts for the increase in the growth potential on New York lettuce seeds. Plant Cell Physiol 21: 1639-1644 25. TAKEBA G 1980 Effects of temperature, red light and hormones on the accumulation offree amino acids in osmotically growth-inhibited embryonic axes of New York lettuce seeds. Plant Cell Physiol 21: 1645-1649 26. TAKEBA G, S MATSUBARA 1979 Measurement of growth potential of the embryo in New York lettuce seed under various conditions of temperature, red light, and hormones. Plant Cell Physiol 20: 51-61
