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Dep. of Agron., Univ. of Missouri, Columbia, MO 65211
* Corresponding author (nelsoncj{at}missouri.edu)
Received for publication November 26, 2003.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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Abbreviations: Gt50, time to 50% of final germination • PEG, polyethylene glycol • RER, root elongation rate
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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Chemicals such as saponin (Guenzi et al., 1964; Marchaim et al., 1975), phenol-like compounds (Hall and Henderlong, 1989), medicarpin (Dornbos et al., 1990), and chlorogenic acid (Miller, 1996) have been implicated in alfalfa autotoxicity. There is no chemical that has been proven unequivocally to be the primary cause, but phenolics, including coumarin, o-coumaric acid, and trans-coumaric acid, give seedling growth responses that are consistent with those of water-soluble extracts of alfalfa tissue (Chon et al., 2002). Not knowing the active chemical involved makes it very difficult to fully understand alfalfa autotoxicity and to develop sound solutions.
Root growth is sensitive to the autotoxic chemical at low concentrations, more so than hypocotyl growth and seed germination (Chon et al., 2000, 2003). Moderate concentrations delay germination, usually determined by visual appearance of the radicle outside the seed coat, whereas higher concentrations reduce final germination percentage (Miller, 1996). A sequential analysis using path coefficients indicated that a water-soluble extract of dry alfalfa leaf tissue (4 g L–1) did not affect final germination percentage but increased time to germination by 16%, reduced hypocotyl length by 16%, and reduced root length by 85% at 120 h compared with a water control (Chon et al., 2003). Autotoxic inhibition of root length is associated with shorter cells and swelling of the tip due to continued lateral expansion of the vascular cylinder and cortex cell layers (Hedge and Miller, 1992; Chon et al., 2002). Root hair development is also reduced (Hedge and Miller, 1992).
Osmotic solutions induce stress in plants, primarily due to tissue dehydration and reduced cell expansion (Slatyer, 1967; Hsiao, 1973; Bewley and Black, 1994). Often the response of seed or seedlings to plant extracts in allelopathy assays is assumed to be due to chemical interference, but aqueous foliage extracts can also exert negative osmotic effects on test species (Bell, 1974). Wardle et al. (1992) concluded from studies using aqueous leaf extracts of four pasture grasses that allelopathy bioassays are more realistic when the control treatments have been adjusted to the same osmotic potential as the plant extract being tested. Part of the autotoxic effect on alfalfa may be osmotic as several osmotica are known to slow seed imbibition and time to emergence of alfalfa (Fick et al., 1988).
Delayed seed germination and slowed root growth by an autotoxic extract could be confounded with osmotic effects on rate of imbibition, delayed initiation of germination, and especially cell elongation (Black, 1989), the main factor that affects root growth before and after the tip penetrates the seed coat (Bewley and Black, 1994). Root growth within the seed of germinating dicots is generally more sensitive to osmotic stress than after the tip is exposed beyond the seed coat (Hegarty and Ross, 1978). Therefore, objectives were to distinguish effects of leaf extracts of alfalfa and osmotic solutions on seed imbibition, germination, and early seedling growth of alfalfa.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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General Bioassay Procedure
Difco Bacto agar solution (1.6%, w/v) was autoclaved for 25 min at 125°C and then equilibrated in a 50°C water bath along with a similar flask of stock extract and one of sterile deionized water (Chon et al., 2000). The warm stock extract (20 g L–1) was diluted appropriately with the warm deionized water and then mixed in a 1:1 ratio with the warm agar solution to give the desired final extract concentrations. About 10 mL of extract-agar or water-agar (control) were poured into 9-cm-diam. plastic Petri dishes and allowed to solidify for 4 h at room temperature.
Seed of Cody alfalfa were surface-sterilized for 15 min in sodium hypochlorite (5.25 g L–1), rinsed several times in deionized water, then imbibed in aerated, deionized water at 22°C for 10 or 12 h. Swelled seed were blotted in a folded paper towel for 30 min. Twenty or 25 swelled seed were evenly placed on the agar surface in each Petri dish. Dishes were covered, sealed by wrapping in parafilm, and placed in a growth chamber at 24°C during the 14-h light period and 22°C during the dark period. Dishes were illuminated (photosynthetic photon flux density of 400 µmol photons m–2 s–1) with a mixture of incandescent and fluorescent lamps.
Germinated seed (radicle 1 mm long) were counted at intervals over a defined period. Hypocotyl and root lengths were measured on all seedlings in each Petri dish at the end of each experiment, usually 132 h. Unless noted, all experiments had four replications. Data were subjected to analysis of variance. When the F test was significant (P < 0.05), means were separated based on the LSD at the 0.05 probability level.
Effects of Extracts on Seed Imbibition
Seven replicates of 10 air-dry seed each were weighed as zero-time samples. Air-dry seed were imbibed in aerated deionized water (control) or alfalfa extracts of 0.5, 2, and 8 g L–1 at 21°C. Ten seed were sampled randomly from the seven replicates at 1 and 2 h after immersion and then every 2 h for 24 h. Seed were blotted between folded paper towels for 30 s to remove surface moisture and weighed. Water uptake was calculated as the increase in seed weight.
Release of Autotoxins during Imbibition
In the solutions above and other earlier bioassays on filter paper, a brown exudate was noted during imbibition of alfalfa seed. To determine if the exudate had autotoxic activity, 2 g of dry seed was soaked in 100 mL of aerated deionized water at 22°C for 0 (control), 6, 12, 24, and 48 h. The extracts were filtered through Whatman no. 1 paper, centrifuged, and mixed with agar for bioassay. Final concentration was that extracted from 10 g L–1. The data indicated the toxic activity was released mainly during the first 6 h so the experiment was repeated with sampling at 0, 1, 2, 3, 4, 5, 6, 12, and 24 h. Data for the common treatments were very similar to the first experiment; only the data from the second experiment are reported.
Since the extract from imbibing seed was toxic, samples of 0.1, 0.2, 0.4, 0.8, and 1.6 g of dry seed were allowed to imbibe for 24 h in 100 mL of aerated deionized water at 22°C. The water control and exudate solutions were filtered through Whatman no. 1 paper, centrifuged, and mixed with agar for assay as above. Final concentrations in agar were 0 (water control), 0.5, 1, 2, 4, and 8 g dry seed L–1. Seed for the bioassay were imbibed in water for 12 h and transferred to the extract-agar treatments. Lengths of roots and hypocotyls were measured at 132 h.
Autotoxin Effects on Germination of Imbibed Seed
Several seed were placed in aerated water at 21°C to imbibe for 12 h. Then 20 imbibed seed were placed on agar-extract at 0, 4, and 8 g L–1 in Petri dishes as above and placed in the growth chamber at 21°C. Seed were examined for germination at 16 (4 h after transfer), 20, 24, 36, 48, 60, 72, 84, and 96 h. The cumulative germination data were plotted and time to 50% of final germination (Gt50) for each replicate determined by interpolation. Root and hypocotyl lengths were recorded at 132 h. The experiment with four replications was repeated. Data were similar, so they were combined for presentation.
Extract Effects during Phases of Germination
Previous data showed that germination of alfalfa in control treatments occurred about 20 h after the initiation of imbibition, with extract treatments causing a delay (Chon et al., 2000). The objective was to determine the relative autotoxic effect during imbibition and early stages of germination compared with later stages of the germination process. Seed were imbibed for 10 h at 21°C with aerated water (control) or leaf extracts of 4 and 8 g L–1. The effect of extract on early stages of germination was tested by transferring seed imbibed in each treatment to agar containing water for the rest of the bioassay. Conversely, effects on late stages of germination were tested by imbibing seed for 10 h in water before transferring to agar containing 0 (water control), 4, or 8 g L–1 extract for the remaining time of the bioassay. Germinated seed in all treatments were counted at 16 (6 h after transfer), 20, 24, 30, 36, 48, 60, 72, 84, and 96 h. Cumulative data were plotted, and Gt50 for each replication was determined as above. Hypocotyl and root lengths were measured at 132 h. Final root length (minus 1 mm at Gt50) and time duration from Gt50 to 132 h were used to calculate RER.
Osmotic Effects of Extracts
Agar was prepared as above with leaf extract to give final concentrations of 0, 1, 2, 4, and 8 g L–1 or with solutions of PEG 8000 to give final concentrations of 0, 20, 40, and 80 g L–1. Osmotic potentials of extracts were measured in agar pieces using the isopiestic technique with a thermocouple psychrometer (Boyer and Knipling, 1965). Alfalfa seed were imbibed in water for 10 h, and then 20 imbibed seed were placed on the agar as above. Cumulative germination was recorded and Gt50 calculated. Root lengths were measured after 132 h and RER calculated.
The experiment in agar was repeated with the same PEG 8000 and extract concentrations. In addition, Whatman no. 1 filter paper was used as a test medium; it is commonly used as such yet is known to reduce the osmotic potential due to paper exclusion of PEG (Emmerich and Hardegree, 1991). Five milliliters of the above solutions were added to the filter paper in each dish. The treatments, i.e., the liquid after wetting the filter paper and the agar blocks containing extract, were sampled for measuring osmotic potentials as above. Twenty seed, imbibed for 10 h in aerated water, were placed on the wetted filter paper or the agar treatments, dishes were sealed, and seed incubated as above. Seed were examined regularly for germination and Gt50 calculated. Root length at 132 h was converted to RER. Responses to PEG 8000 treatments in the two experiments were very similar, so only the data from the second experiment, which included the filter paper treatments, are reported.
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THEORY |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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Autotoxins should have little toxic effect on early imbibition as both living and nonliving seed imbibe water at a similar rate (Bewley and Black, 1994), but imbibition could be affected by the osmotic effect (Wardle et al., 1992), mainly during the latter stages of Phase I, which may delay or prevent reaching the critical water content (Fig. 1). Autotoxicity may affect the activation or increase in metabolic components, namely activation of critical enzyme systems and initiation of mitochondrial development to allow the seed to pass from Phase I to Phase II. Physical and physiological seed dormancy, generally not a factor with modern alfalfa cultivars, must also be overcome.
Root growth within the seed during Phase II is mainly by cell elongation using stored energy sources in the cortex cells of the root for early cell wall synthesis until the main storage organs are activated. The O2 consumption increases after root cells begin to elongate within the seed. Root growth within the seed is relatively slow as mitosis, a high-energy-requiring process, does not become very active until the seed coat is ruptured, after which O2 consumption increases dramatically and the seedling transitions to Phase III (Bewley and Black, 1994).
Cell elongation is usually more sensitive than cell division to metabolic and, especially, osmotic properties of the autotoxin (Hsiao, 1973). Therefore, Phase II can be affected by toxicity factors on membranes and metabolic systems and by osmotic factors needed for turgor to expand the root cells and to overcome physical resistances of other seed parts for elongation growth (Bradford, 1995). Root elongation in Phase III can be altered by both the metabolic factors and osmotic factors needed for mitosis and active cell elongation, especially the toxic factors as continued growth becomes more dependent on mitosis.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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Control seed reached Gt50 and entered Phase III at about 20 h, at which time the fresh weight of the seed was about 5.6 mg (Fig. 2). In another study at 22°C, the Gt50 for seed imbibed and germinated in water was about 20 h (Table 1). In a third study at 22°C, the Gt50 was delayed to 23 h at an extract concentration of 0.5, to 30 h at 1.0, and to 32 h at 4.0 g L–1 (data not shown).
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Release of Autotoxin from Seed
Seed exuded autotoxic substance(s) during imbibition, most of which was released during the first 6 h (Fig. 3). This coincides with the period of most rapid imbibition (Fig. 2) and mainly before the germination process commences. Imbibing seed of crownvetch (Coronilla varia L.), sweetclover (Melilotus alba Medik.), and red clover (Trifolium pratense L.) also release chemicals during the first 4 to 6 h, probably phenolics, that are allelopathic to seedling growth of several species, especially alfalfa (McKee et al., 1971). Similar to other tissue extracts, the growth response to the seed extract depended on concentration (Fig. 4), but at equal sample weights, the extract from alfalfa seed was much less toxic to root growth of alfalfa than was the extract from alfalfa leaves (compare with Table 1; Chon and Nelson, 2001). These seed exudates may influence bioassays if large numbers of nonimbibed seed are used in each Petri dish, increasing the potential for roots of adjacent seedlings to intercept toxic areas around other germinated seed (McKee et al., 1971). They may also influence germination and root growth of closely spaced seed in the field.
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Final germination percentages were similar to the control when seed were imbibed in extract before transfer to water-agar medium (Table 1), but Gt50 was delayed by about 1 h, perhaps due to the delay in onset of germination. Subsequent responses of RER and hypocotyl length were both enhanced slightly by the extract treatments used in imbibition. Some residual autotoxin likely remained in the seed after the transfer and at low concentrations can stimulate root elongation (Chon et al., 2000). In contrast, seed imbibed in water for 10 h before transfer to extract treatments had a 6- to 9-h delay in reaching Gt50, probably due to effects of the toxicity component on metabolism and growth, but with no effect on final germination percentage, which is less sensitive to the extract. Similar to earlier studies (e.g., Chon et al., 2000), the autotoxin treatment after water imbibition had little effect on hypocotyl length whereas RER was markedly decreased (Table 1).
The transition from Phase I to Phase II (Fig. 1) appears to be slightly less than 10 h for these treatments. The autotoxin likely enters the seed during imbibition as the delay in Gt50 response after transfer from water to the extract indicates the autotoxin readily passes through the seed coat during late imbibition and Phase II (Fig. 1) even though the uptake of water is rather slow (Fig. 2). During the most rapid stages of imbibition, most of the water is used to expand the cotyledons due to their high protein concentration (Sheaffer et al., 1988), perhaps in a way that allows metabolism of the associated autotoxin or its sequestration away from the root apex. Effects on cotyledon growth or final size were not measured. Later, as the cotyledons slow in water uptake and expansion, Phase II begins, and the embryo axis, mainly the root, is likely the major sink for water and the toxin. Similar to citrus (Zekri, 1993), the effects of the autotoxin are mainly osmotic during the imbibition period but mainly toxic after Phase II begins. The toxin likely reduces RER while within the seed to further delay Gt50 (Chon et al., 2003).
Osmotic Effects on Time to 50% of Final Germination and Root Elongation Rate
Respective osmotic potentials of the treatment media for each PEG 8000 solution were proportionately lower on paper than when mixed with agar (Fig. 5), likely because of exclusion of PEG by the filter paper (Emmerich and Hardegree, 1991). The PEG was thoroughly mixed with the agar solution during preparation and therefore should be more evenly distributed throughout the media. In contrast with PEG, the osmotic potentials of the extract concentrations were proportionately higher on filter paper than in agar. The reason is not known, but the autotoxin does adsorb to organic substances such as activated charcoal (Jensen et al., 1984).
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Both media showed RER during Phase III (Fig. 1) was stimulated slightly by the decreased osmotic potential of PEG (Fig. 5B). In these cases, the roots were observed to be thinner than the controls, a common response to low levels of water stress (Sharp et al., 1988). This contrasts with the response to the autotoxin of enlarged root tips and added cortex cell layers (Hedge and Miller, 1992; Chon et al., 2002). In contrast with PEG, RER in extracts on both media was markedly decreased as concentration increased. This response easily offsets the slight osmotic stimulation, indicating the reduced RER was nearly all due to the toxicity component. Further, as RER was slowed by the extract, the root tips were observed to be enlarged in diameter, especially at high extract concentrations, showing the characteristic signs of autotoxicity.
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INTERPRETATION AND CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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There was a release of autotoxin to the external solution from imbibing seed, probably from being pressed from the seed coat due to the internal pressure of the swelling cotyledons, but it is not clear whether the autotoxin is being taken up. There was little effect of imbibition for 10 h in extract or water on either Gt50 or RER, suggesting the autotoxin was either not taken up during early imbibition, was sequestered, or had no effect. Thus, the exudates may be a defense mechanism against adjacent seedlings. If exposed to the autotoxin following 10 h of imbibition in water, however, the Gt50 was delayed, indicating the toxin was taken up after the seed had swelled and quickly reached the embryo axis where germination processes and early root growth were commencing. After root tip emergence, the toxin had a much greater effect on RER than did the osmotic effect.
With high concentrations of extract, it was noted regularly that roots of seed that germinated grew to a length of about 4 to 6 mm, stopped elongating, and developed the characteristic enlarged tip in response to the toxin. This indicates that longitudinal growth of the embryonic root cells had been exhausted, cell division was not fully activated, and the cell supply was limiting growth, likely due to disruption of mitosis by the autotoxic chemical. The young cortex cells then grew laterally to develop the characteristic root form of affected seedlings with no further elongation of the root (Hedge and Miller, 1992; Chon et al., 2002). This response in the field would allow the seedling to survive for a few days or weeks in a weakened condition and then either die or form root branches behind the tip to exist as a less thrifty autoconditioned plant (Jennings and Nelson, 2002a).
In conclusion, the osmotic components of the leaf extract affected seed imbibition (Phase I), but in Phase II, and especially in Phase III, the toxic factor was the major influence on RER. The seed release of autotoxic activity during the first few hours of imbibition is largely negated by preimbibing the seed in water for 10 to 12 h (Chon et al., 2000), leaving a slight delay in Gt50 and a major reduction in RER and root length in seedling bioassays used to separate germplasm responses (Chon et al., 2003). Therefore, correcting for osmotic effects of leaf extracts would not be necessary for an alfalfa bioassay that uses preimbibed seed and is based on root length.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORYRESULTS AND DISCUSSIONINTERPRETATION AND CONCLUSIONSREFERENCES |
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