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Physiology and Development |
-irradiation of centuries-old fruits, and cultivation, growth, and phenotypic abnormalities of offspring1
2Department of Organismic Biology, Ecology, Evolution, University of California, Los Angeles, California 90095 USA; 3Department of Earth and Space Sciences, University of California, Los Angeles California 90095 USA; 4Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973 USA; 5Nanjing Institute of Geology and Palaeontology, Nanjing, Jiansu, China; 6Beijing Institute of Geology, Beijing, China; 7Center of Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California 94551 USA; 8Department of Geology, Liaoning Normal University, Dalian, Liaoning, China
Received for publication April 10, 2001. Accepted for publication August 9, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1300-yr-old lotus fruit, recovered from an originally cultivated but now dry lakebed in northeastern China, is the oldest germinated and directly 14C-dated fruit known. In 1996, we traveled to the dry lake at Xipaozi Village, China, the source of the old viable fruits. We identified all of the landmarks recorded by botanist Ichiro Ohga some 80 yr ago when he first studied the deposit, but found that the fruits are now rare. We (1) cataloged a total of 60 lotus fruits; (2) germinated four fruits having physical ages of 200–500 yr by 14C dating; (3) measured the rapid germination of the old fruits and the initially fast growth and short dormancy of their seedlings; (4) recorded abnormal phenotypes in their leaves, stalks, roots, and rhizomes; (5) determined 
-radiation of 2.0 mGy/yr in the lotus-bearing beds; and (6) measured stratigraphic sequences of the lakebed strata. The total -irradiation of the old fruits of 0.1–3 Gy (gray, the unit of absorbed dosage defined as 1 joule/kg; 1 Gy = 100 rad), evidently resulting in certain of the abnormal phenotypes noted in their seedlings, represents the longest natural radiobiology experiment yet recorded. Most of the lotus abnormalities resemble those of chronically irradiated plants exposed to much higher irradiances. Though the chronic exposure of the old fruits to low-dose -radiation may be responsible in part for the notably weak growth and mutant phenotypes of the seedlings, it has not affected seed viability. All seeds presumably repair cellular damage before germination. Understanding of repair mechanisms in the old lotus seeds may provide insight to the aging process applicable also to other organisms.
Key Words: abnormalities • dormancy • radiation • growth • imbibition • lotus • seed longevity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Virtually all parts of the lotus plant (seed, rhizome, leaf, stalk, petal, anther, pericarp, and fruit receptacle) are used as food or medicine (US-DHEW, 1974
; Shen-Miller et al., 1995
). The oldest cultivated fruit germinated and directly radiocarbon dated is that of a lotus >1000 yr old (Shen-Miller et al., 1995
). This unique find prompted us to journey to its source, a dry lake at Xipaozi Village, near Pulandian, Liaoning Province, in northeastern China (Fig. 1). Pollen entombed in the lotus-bearing lakebed dates from the late Holocene (Chen, Chien, and Zhou, 1965
). Lotus cultivation in the extensive basin evidently began well before 1000 yr ago (Shen-Miller et al., 1995
).
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). One other germinated lotus fruit of the same origin has a reported age of 466 yr (Priestley and Posthumus, 1982
). The present paper reports on the study of old fruits newly gathered from Xipaozi.
Xipaozi Village, China
Situated some 77 km north of the modern port city of Dalian (Liaoning Province, northeastern China) and 7 km northeast of the community of Pulandian (recently upgraded to city status; Fig. 1) is Xipaozi, the source of the old lotus fruits. A small farm village, Xipaozi is centered on the site of what was once a large lotus-filled lake, drained centuries ago into the Bo Hai Sea, and where, in the 1920s, Ichiro Ohga was the first to report the presence of old viable fruits (Ohga, 1923
).
We arrived at Xipaozi Village in the spring of 1996 with rucksacks, collecting gear, and detailed topographic maps. Just as Ohga (1927)
had depicted, along the northern boundary of the basin lie the Curvy Dragon Hills (Chuanlun San) and, coursing through the basin center, the southwesterly running Pulandian River (recently renamed the Anzihe) with its three deeply incised tributaries (Figs. 2 and 3). As shown on Ohga's 1927
map, the Shen-Da Line Railway still traverses the fields in the northwest quadrant of the basin (Fig. 3). As part of Chairman Mao's abortive "Great Leap Forward," a thorough mining in 1958 of the peat deposit and associated black clay layer underlying the lotus-bearing bed (Fig. 4) had lowered the Xipaozi farm fields by a meter or more over virtually the entire 4-km2 basin. Because of this disruption of the lakebed sediments, fruits of lotus can now be found exposed at the soil surface. More recently, as the fame of the site has spread, the local farm fields on the dry lake have become targeted for conversion to a tourist mecca; authentic old lotus fruits are becoming exceedingly rare.
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Ancient earthquakes
As shown in Table 1, the Pulandian region has been jolted repeatedly by great earthquakes. One such earthquake drained the Xipaozi lake into the Bo Hai Sea to the west (Fig. 1). Though it is uncertain which of the 11 strong temblors listed in Table 1 was responsible for draining the lake, the most likely, according to a Japanese geologist cited by Wester (1973)
, appears to have been an event of 1484 that registered 6.75 on the Richter scale. Three earthquakes of the same or greater magnitude have been recorded relatively near the lotus lake both before and after this event, with magnitudes ranging from 6.75 to 8.0 (Table 1). The lake-draining event may have occurred earlier (e.g., during the 1290 quake), or perhaps later (e.g., during the 1679 and 1888 quakes) than the date cited by Wester (1973)
. The youngest directly dated fruit found in our studies (oSL7, ungerminated, and used for analysis of a protein-repair enzyme; Shen-Miller et al., 1995
) is 104 ± 66 yr old (see MATERIALS AND METHODS), suggesting that the lake may then still have been extant. But the hypothesis that the 1484 quake caused the lake to drain fits an estimate made by Ohga (1923)
, based on the apparent rate of riverine down-cutting, that by 1923 the basin had been dry for some 400 yr (Wester, 1973
).
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). Under the Japanese occupancy of northeastern China in the 1920s, Ohga was appointed Government Botanist of the Kwang-Tung Leased Territory in South Manchuria and Professor of Botany at the Education Institute of the Southern Manchuria Railway in Dalian. During this period, Ohga carried out experiments on >7000 lotus fruits (Ohga, 1927
), specimens collected by farmer Liu from the soils of Liujia Zhuang (Liu's ancestral village; later, farmer Liu was regarded to be a Japanese collaborator and was executed).
Soil radiation
All soils are to some extent radioactive, due chiefly to spontaneous decay of mineral-bound potassium (40K), thorium (232Th), and uranium (235U and 238U). Some radioisotopes decay in a single step (e.g., 40K), whereas others break down via a "decay chain" made up of numerous radionuclides, both short-lived (having half-lives ranging from seconds to a few years) and long-lived (e.g., radium, 226Ra, from the breakdown of uranium, which has a half-life of 1600 yr). During decay, radionuclides emit energy into the surrounding soil in the form of heat and, of particular biologic importance, various kinds of radiation. For lotus fruits, entombed for a millennium in such soil, these types of radiation would represent continuous bombardment by a potentially mutagenic source: 
-particles (helium nuclei), ß-particles (nuclear electrons), and X- and -rays (Aitken, 1985
). It is thus not surprising that seedlings grown from the Xipaozi fruits display phenotypic, evidently mutational abnormalities. For damaged seeds to germinate, they need to repair cellular injury (to organelles, tRNA, DNA, membranes, and other cellular components; Bewley and Black, 1982, 1994
). Repair mechanisms in lotus, therefore, must be unusually effective, notably more so than those in other crops (Priestley, 1986
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The 60 newly collected fruits (58 intact plus two cracked), cataloged as OL96-1 through OL96-60 (OL, old lotus; 96, collection of 1996; and numbered sequentially in order of their acquisition), were stored in a 4°C chemical-free chamber in individual glass vials, each with a perforated cap to prevent build up of such volatiles as alcohols and aldehydes that may accelerate seed senescence (Zhang et al., 1995a, b
). Each of the fruits was characterized by its appearance, photographed, weighed, and tested for sedimentation in water (a test of viability). Lotus fruits are ellipsoidal (see Figs. 1 and 4 in Shen-Miller et al., 1995
) and relatively large (2 cm long by 1 cm wide). Fruits capable of germination usually have a dry mass of 0.7–0.9 g and sink in water, whereas fruits that float are most often nonviable (Shen-Miller et al., 1995
). Of the 58 intact fruits collected in 1996, the 40 having a mass >0.8 g sank; the 12 with a mass of 0.7 g also sank; and the four with a mass >0.8 g and two with a mass <0.7 g floated.
Nutrient requirements
Before beginning experiments on the newly collected Xipaozi fruits, a growth season (1997) was devoted to determining the soil and nutrient requirements of modern lotus. Serial dilutions were tested of a nutrient stock solution containing, per liter of tap water, 50.6 g of a commercial fertilizer (Stern's Miracle-Gro for tomatoes, with an N : P : K ratio of 18 : 18 : 21; Scotts Miracle-Gro Products, Port Washington, New York, USA) that contained the following major and minor nutrients: 18% N ([NH4 ] 2HPO4 4.4%, KNO3 6.0%, urea 7.6%), 18% available P ([NH4]2HPO4 and K3PO4), 21% soluble K2O, 0.5% soluble MgSO4, 0.05% each of CuSO4, MnSO4-EDTA, ZnSO4-hydrate, and 0.1% chelated Fe. Optimum growth was observed at 100x dilution of the stock solution (see RESULTS).
Germination (imbibition and mass gain)
Beginning early in the springs of 1998–2001, four Xipaozi fruits gathered by our team from known locations were tested, one each year, for germination and growth (OL96-44, OL96-53, OL96-50, and OL96-52, respectively, for 1998, 1999, 2000, and 2001). Each fruit sank quickly in water and each had a depressed spot at its style, a slight brown protuberance near its style, and a shiny pitted pericarp devoid of an outer opaque layer, which are characteristics quite similar to those of previously tested old viable fruits (see fig. 1 in Shen-Miller et al., 1995
). Fruit OL96-44, collected near the former farmhouse of farmer Liu (Fig. 3), had a dry mass of 0.85 g; OL96-53 and OL96-52, collected by farmer Li at his tomato cold frame (Fig. 3), dry masses of 0.76 g and 0.68 g, respectively; and OL96-50, collected west of the Anzihe River near farmer Liu's former farmland, a dry mass of 0.66 g (the lightest viable fruit thus far tested). In each of the four sets of experiments, modern lotus fruits were used as controls; these were fruits produced in 1996 by two lotus plants grown since 1951 at Kenilworth Aquatic Gardens in Washington, D.C., USA and germinated from two undated old fruits collected by Ohga at Xipaozi and given in 1950 by the Tuhuku Imperial University to paleobotanist R. W. Chaney of the University of California, Berkeley (Wester, 1973
). Germination procedures have previously been described (Shen-Miller et al., 1995
). Each fruit was weighed and filed at its "pore end" (see fig. 3 in Shen-Miller et al., 1995
) until the pink testa of the seed was reached, resulting in removal of 10–20 mg of pericarp (fruit coat). Each filed fruit was then soaked in tap water that had been standing overnight or resin filtered (to permit evaporation of chlorine or removal of chloramine, respectively). Each day during imbibition, the fruits were rinsed, blotted, weighed, and returned to freshly treated water. Upon germination, the dry pericarp of each fruit was peeled and retained for radiocarbon dating.
Cultivation
The germinated seeds were each potted in a 3 : 1 soil mix of UCLA garden clay to greenhouse soil (the latter containing equal amounts of spagnum moss, washed sand, and sandy loam). Although animal manure has been suggested as a useful addition to such soil mixes (Anonymous, 1987
; Wester, 1973
), it was not so in our experience; the addition of commercial steer manure, regardless of concentrations tested, proved fatal to the young control seedlings. Clay is a crucial component for nutrient retention in aquaculture (Speichert, 2000
); a hard clay contains all the minor nutrients necessary for water culture (Speichert, 2001
). Thus, clay is a suitable soil medium for lotus culture. Lotus grows best in an acidic soil of pH 4.6 (Anonymous, 1987
; Wester, 1973
).
Each of the potted plants was placed on a greenhouse bench; the soil around the seedlings was covered with small lava chips; and each of the pots was placed within a larger pot filled with a 100x dilution of the nutrient stock solution. The pH of the water and nutrient solution was 5.0. The seedlings were immersed in the solution to a depth of 5 cm. After several months, at the 10–12 leaf stage of growth, each seedling was transplanted into a larger and deeper pot (45 x 52.5 cm); placed in a sunny area outdoors; and filled with nutrient solution to a depth of 15 cm. Nutrient solutions were continuously maintained in the pots, including during winter dormancy.
Exceptional care is crucial to the maintenance of young lotus seedlings at germination; even a light touch to any of their first three plumules (juvenile leaves) can cause blackening and drying within hours. Blooms of algae can also inhibit growth, especially of young seedlings (a difficulty overcome by absorbing the algal scum onto paper towels; or in transplants, by skimming off the algal layer by filling a pot until it overflowed; and under conditions of severe algal infestation, by scooping away or siphoning off all the water, wiping clean the inner pot surface, and refilling the pot with fresh nutrient solution). Occasional aeration of the water (splashing by hand) seems to deter algal growth. Heavy algal growth can promote the rotting of floating leaves and depletion of nutrients required for seedling growth. In healthy plants, when rhizomes (underground stems) and nodal roots are effective in their absorption of nutrients, water in the pot clears, and a layer of biofilm forms at the soil surface. Often, it takes 2–3 seasons of plant growth before the pot water becomes continuously clear.
Seedling growth measurement
Each day, beginning on the first day of planting, detailed data were recorded on the emergence of plumules, nodal leaves, roots, and rhizomes; the development of rhizome nodes; and the height of stalks and diameter of leaf blades. Abnormal phenotypes were systematically noted and documented.
Radiocarbon dating
The 14C dating of lotus fruits was carried out by use of accelerator mass spectrometry (AMS), which for analysis requires only a small fraction (10–50 mg) of a 300-mg peeled lotus pericarp. Wood fragments collected in situ from a measured stratigraphic section were also dated by AMS. In preparation for dating, all specimens were first acid extracted (1 mol/L HCl), to remove soil carbonate; then, sequentially, base extracted until colorless (1 mol/L NaOH), to remove humic organics; acid washed (1 mol/L HCl), to remove trapped CO2; and rinsed with water and dried in a vacuum at room temperature. At the AMS Center of Lawrence Livermore National Laboratory, Livermore, California, USA (Davis et al., 1990
), the cleaned specimens were combusted and the 14C-containing gases were collected and analyzed. Radiocarbon ages were derived by use of a 14C half-life of 5568 ± 30 yr (Libby, 1955
) and by following the conventions of Stuiver and Polach (1977)
and Stuiver and Becker (1993)
as summarized by Shen-Miller et al. (1995)
.
Because of temporal variability in the production of 14C in the atmosphere (arising from variation of the solar-wind shielding of galactic cosmic rays), the 14C calibration curve used to determine 14C ages also fluctuates. Derived from decadal samples, the curve plots years BP (before present, for which "present" = AD 1950) vs. calendar years AD (Stuiver and Becker, 1993
); and because of the fluctuations, the year BP of a given sample can fall in more than one calendar year. To provide a conservative estimate of reported ages, all physical ages recorded in this paper (including recalculation of ages earlier reported by Shen-Miller et al., 1995
; see Table 2) are mean ages based on the full range of calendar intercepts (from the earliest to the latest AD).
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Soil radioactivity
Soil samples from three horizons were collected and sealed in plastic bags for analysis of soil radioactivity: (1) gray clay, from the horizon containing in situ fruit OL96-1, stratigraphically 0.05 m below the top of the lotus-bearing gray clay in the pit (Fig. 4); (2) black clay, encasing in situ fruit OL96-34, 0.2 m below the top of the gray clay bed in the excavated well (Fig. 4); and (3) black clay, 0.05 m below fruit OL96-34 in the well (Fig. 4). Radioactivities of the samples were analyzed at Brookhaven National Laboratory, Upton, New York, USA. Equipment, techniques, and numerical methods of data reduction for these analyses are those described by Harbottle (1993)
and Harbottle and Evans (1997)
; methods used to calculate radiation dosages are those of Aitken (1985)
. Xipaozi soil samples having a dry mass of 500–800 g (except sample 2, above, which was increased to this mass by the addition of silica sand) were measured for radioactivity by means of a Germanium counter, counting over a 3-d period in a modified Marinelli beaker precalibrated by the use of standard radionuclides discussed by Hill, Hine, and Marinelli (1950)
and Harbottle (1993)
. Measured -ray intensities were interpreted using statistical program SPSS-X (SPSS, 1988
), software specifically designed for use with the modified Marinelli beaker.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; including ages recalculated for conformity, as discussed in MATERIALS AND METHODS). OL96-44, the first of the newly collected Xipaozi fruits tested (an experiment carried out at Regensburg Universität, Regensburg, Germany, 1998), has an age of 464 yr. The other three fruits, OL96-53, OL96-50, and OL96-52, tested later at University of California, Los Angeles (Los Angeles, California, USA) have ages of 192, 408, and 466 yr, respectively. As expected, the control fruits, obtained from the Kenilworth Aquatic Garden, are dated to be modern (Shen-Miller et al., 1995
). Shredded wood fragments collected from the wall of the excavated abandoned well (Fig. 3) have an age of 175 yr.
Imbibition, mass gain, germination
Within 10 min after immersion in water, the filed ends of all old fruits became crenulated and crumbled into fine shreds. Modern fruits, in contrast, bore no wrinkles and remained intact throughout germination. Table 3 summarizes lotus fruit mass gain during imbibition. Over four seasons of testing, mass gain during imbibition and days to germination are closely reproducible among old fruits, but varied greatly among the modern controls (see DISCUSSION). Mass gain by old fruits was slower than that of controls and was essentially uniform throughout imbibition; the average mass gain by control fruits was 74% after 1 d of imbibition, compared to a gain of 40% in the old (Table 3). Interestingly, regardless of initial mass or rate of gain during imbibition, the total amounts of gain at germination for the old and modern fruits were statistically indistinguishable (136 ± 8% and 163 ± 36%, respectively).
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1.7x heavier (having an average dry mass of 1.27 g); exhibited a mass gain 1.8x greater on day one; but required 5x as long to germinate (17 d compared with 3.5 d). Among modern fruits tested, the three most extreme cases of prolonged germination time were 22, 33, and 58 d, all of which had been stored prior to imbibition at 4°C, as had been all other lotus fruits. The fruits that germinated in 22 and 58 d had, respectively, dry masses of 1.31 and 1.08 g and remained afloat for the first 3 d and 10 d of imbibition, respectively. The third of these slow-germinating fruits initially weighed 1.44 g and sedimented immediately upon immersion in water. None of these fruits developed bacterial growth during their prolonged germination (and for this reason, they were kept under observation). After germination, all three of these modern fruits developed into normal healthy plants having many standing leaves.
Nonviable control fruits remained buoyant, had a high rate of mass gain (131% within the first 1–2 d of imbibition), lost mass shortly thereafter, and became moldy as their cell contents leaked into solution. Most of these modern nonviable fruits had large cracks in their dry pericarps. (But not all of the cracked fruits were nonviable; even some that lacked pericarps remained viable after as long as 30 mo of storage.)
Nutrient requirements
Growth of modern control lotus plants was tested with serial dilutions of the nutrient stock solution (see MATERIALS AND METHODS). Lotus seedlings at germination are rootless and their plumules, at this stage of development, are relatively insensitive to nutrient concentration. A 100x dilution of the stock solution was selected as an overall optimum concentration for all stages of growth. But once roots are formed and begin to absorb nutrients, young lotus leaves become extremely sensitive to nutrient concentration; toxicity symptoms become visible within hours of nutrient application. At a high nutrient concentration (25x dilution of the stock solution), browning and drying of younger leaves begin around their entire peripheries and advance inward, forming a wavy pattern, layer upon layer (Fig. 7). At lower concentrations (50x and 75x dilution of the stock), yellow or brown necrotic spots appear on the peripheries of younger leaves and similarly advance inward. At suboptimal levels (>100x dilution of the stock), all leaves are small and uniformly pale green to yellow. At high nitrogen levels, older leaves of seedlings become greenish-yellow with dark green veins (Fig. 8; Alley, 1996
).
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). The visible spectrum of the green pigment in the embryo axis shows it to be chlorophyll (J. Shen-Miller, unpublished). One to three weeks are required by a lotus seedling to develop fibrous roots at the base of its plumules. After all plumules have emerged, a rhizome apex appears near the base of the first plumule. Once this apex extends, a node is formed in the rhizome and fibrous roots extend from the node. New apices then continue to produce new rhizomes, nodes, and roots; a new leaf emerges from each new node. Each newly emergent leaf is initially tightly rolled with its two halves rolled toward the center and bearing a spicule-like tip at each end. Initially, a young rolled blade and its stalk form a straight line, the blade parallel to, and partially extended from, the stalk (see fig. 12 in Shen-Miller et al., 1995
). As a leaf unfolds, the young blade turns to become perpendicular to its stalk. All nodal leaves have a longer life span and grow to a larger size than any of the plumules.
Lotus leaf blades are more or less circular when fully unfolded and have radial veins that converge centrally to a pale green region, a tissue known as the "nose" (e.g., Figs. 9 and 10) where air exchange takes place between the blade and the underground rhizome (Anonymous, 1987
). Directly beneath the nose is the leaf stalk that contains numerous air ducts. The stalk serves as a conduit for air and nutrient transport. To prevent plugging of these airways, stalks should always be pruned above the water level. (Once water-logged, rhizomes directly beneath an incorrectly pruned stalk die; Anonymous, 1987
.) In a first-year seedling, all early emergent stalks are prostrate and have leaves that float on the water surface; emergence of standing leaves is a sign of healthy root and rhizome growth that contribute eventually to the development of a healthy plant (Anonymous, 1987
).
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5°C and daylength shortens to <10 h. In late February, new growth normally begins when night temperatures persist at 11°C, and day length increases to >11 h. In mid-June to early August, flowers have bloomed in four modern controls after 2–3 seasons of growth (though such blooms have yet to be produced by offspring of old fruits). Floral initiation began in April, when day length reached 13 h and night temperatures were 12°C, and the first flower buds appeared soon thereafter, in early May. Lotus thus appears to be a long-day plant having a photoperiod of 13 h. (In China, lotus also blooms during the summer months; Anonymous, 1987
.) In southern California, lotus requires a temperature of 10°C to break winter dormancy. Lotus plants can safely weather freezing air temperatures of–30°C and lower, as long as their soil bed remains unfrozen, as shown by their widespread presence in China across vast latitudes from north to south (Anonymous, 1987
).
Growth of offspring from old fruits
All offspring of old lotus fruits showed faster initial growth than their modern controls. Table 4 summarizes representative data illustrating the early rapid growth of the lotus offspring of old fruits; e.g., in the 464-yr-old OL96-44, the first rhizome internode expansion occurred 35 d before that of its modern control; and the widths of the third plumule on day 12 of growth were, for the offspring of the 466-yr-old fruit (OL96-52) and its modern control, respectively, 4 and 0.3 cm. Similar trends of early rapid growth were observed in offspring of the 400-yr-old oSL5 (table 3 in Shen-Miller et al., 1995
) as well as in studies of other Xipaozi fruits (Chang, 1978
; Wester, 1973
). Seedling leaves, once emerged, expanded and quickly reached maximum diameters, but they were always smaller than those of their controls, an aspect of growth noted in all plants from the OL96 group here tested. The oSL5 seedling (whose germination was reported in Shen-Miller et al., 1995
) developed abnormal phenotypes (see below) and after 7 mo of growth became splindly (due probably to inappropriate cultivation practices and poor rhizome and root development). It was transplanted in the fall, but failed to emerge the following spring. (On the basis of this experience, fall transplanting of first-year plants seems inadvisable.)
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30 cm) that were brittle and prostrate. It succumbed after 5 mo of very weak growth, whereas its control (though also initially spindly) produced standing leaves after 6 mo. Low light intensity, low humidity, water alkalinity, low ambient temperature, use of an inappropriate soil mix, and short day length all evidently contributed to its early demise. This seedling was germinated and grown in a greenhouse in Regensburg, Germany (latitude 49°N, as compared to 34°N for Los Angeles where seedling growth was more successful). The offspring of the 192-yr-old OL96-53 had one season of growth (1999). Like the seedling from OL96-44, it, too, grew faster than its control (Table 4); and before it was transplanted, it had 2–3 short standing leaves. The OL96-53 seedling was the first of those tested here from an old fruit that recovered from winter dormancy, a dormancy that was shorter than the controls (and one that ended in late January 2000). But, after sprouting early in the spring and producing five small pale yellowish spindly leaves, it, too, quickly succumbed. Its demise may have been due to an inadequate nutrient regulation during fall and spring cultivation that affected the health of its root and rhizome. Water in the pot where the plant underwent dormancy was very murky, containing algal and much bacterial growth.
The 408-yr-old OL96-50 fruit produced a seedling that grew slightly faster than its control (Table 4). This seedling had 2–3 short standing leaves before it was transplanted, but none thereafter. The plant endured one season of growth, became fully dormant by mid-December 2000, broke dormancy 2 wk later, and produced two small shoots by the first week of January 2001 (having prostrate stalks 6–8 cm long). This exceptionally early termination of dormancy, seen also in OL96-53, may have been hastened by the warm winter of the Los Angeles area, having day temperatures of >14°C from late December to early January 2001 (although during this period, none of the controls broke dormancy until 2 mo later). Algal growth (Chroococcus, Euglena, etc.), accumulated on the pot wall and in the water surface overlying the plant of OL96-50 during dormancy and spring growth, has been repeatedly removed and the pot replenished with fresh nutrient solution. In July 2001, it still had no standing leaves. To prevent rot, all leaves have been propped up by bamboo sticks.
The most recently sprouted 466-yr-old OL96-52 fruit produced a seedling, evidently healthy in all respects, that by mid-June 2001 (80 d after germination) had 12 light green leaves. The latest emerging of these leaves were small and pale green, having red veins and bronze lower surfaces. At transplanting in late June, the youngest rhizomes, pinkish cream in color, were 3–4 cm wide and 5–6 cm long. By early July, all prostrate leaves had been propped up (by use of bamboo supports) and one stout shoot had emerged.
Abnormalities
Although continuous exposure over hundreds of years to -radiation of 2 mGy/yr (see below) appears not to have affected the viability of the old lotus fruits tested, the following numerous phenotypic abnormalities, presumably expressed mutations, have been observed in seedling leaves, stalks, roots, and rhizomes.
oSL5
The seedling of the 400-yr-old oSL5, shown in Fig. 9, exhibited many abnormal phenotypes, both in its plumules and its nodal leaves, abnormalities unknown or not at all common in the control seedlings. Each of the leaf phenotypes observed in this offspring (wrinkled, speckled, red-patched, pale-wedged, or spindly with brittle stalks) has been documented individually in mutant maize plants (Neuffer, Coe, and Wessler, 1997
). All these phenotypes, however, were present in the single seedling of oSL5, as follows: The first plumule was injured and died (due to accidental touch); the second plumule had prominent veins, with interveinal tissue thick and rubbery having roughness like that of a cabbage leaf (cf. Fig. 10); other leaves had red and pale specks and red veins. The sixth leaf had a wrinkled periphery and prominent variegation with tiny red specks in a pie-shaped pale wedge (Fig. 9, left center) that spanned five red radial veins that in color differed from the light green veins of the remainder of the leaf. This plant produced no standing leaves after 7 mo of growth before winter dormancy.
OL96-44
The seedling grown from the 464-yr-old OL96-44 had very poor growth, but in comparison with that grown from fruit oSL5 showed fewer abnormalities (an apparent relative normalcy of the OL96-44 plant difficult to interpret because of its premature leaf death). As noted earlier, this Regensburg-grown seedling was extremely spindly and small-leafed. Leaf characteristics noted in oSL5 were also present here, e.g., a cabbage-textured thick blade (Fig. 10) having a prominent demarcation of the central oval surrounding the nose and a red rim, curved downward into water (Fig. 10, foreground). All of the stalks were spindly, brittle, and dried shortly after being lifted out of water. This seedling also produced no standing leaves; it died after 5 mo of growth.
OL96-53 and OL96-50
Seedlings of both of these fruits (192- and 408-yr-old, respectively) were smaller than their controls. During summer growth they exhibited intense red coloration in the veins and on both the upper and lower surfaces of some of the leaves and pale pink noses. They, too, shared the leaf abnormalities described above. Unlike their controls, no standing leaves were present toward end of the growing season (Figs. 11 and 12). In the first year of development, both seedlings exhibited poor rhizome and root growth. After a very short dormancy, OL96-50 sprouted in January 2001 and continued to produce leaves having pale pink noses, veins intensely red to purple, and interveinal tissues purplish green in color. As redness receded during growth, the leaves became dark green, darker than those of their neighboring controls (such darkness being a trait of shaded leaves). Leaves of the seedling of OL96-50 were smaller than those of the control and, as late as early August 2001, it had produced no standing leaves.
OL96-52 (466 yr)
The recently germinated seedling of this fruit developed early leaves that had holes along the periphery of the oval tissue surrounding the nose. The tissue beneath the oval, normally pale reddish-green, was dark grayish brown, rubbery and puckered, and the leaf stalks were brown having a rough scab-like texture. Ovals similarly surrounded by holes have been observed in leaves of control seedlings, but were never accompanied by the other abnormalities present in the OL96-52 offspring.
Soil -radiation
Tabulated in Table 5 are the amounts of radioactive elements (40K, 232Th, 238U) and their decay products (228Ac and 226Ra) measured in the Xipaozi lakebed samples. The levels of radioactivity measured in each of the three Xipaozi samples are virtually identical and, as shown in Table 6, yield an average rate of -ray emission of 1.9 ± 0.1 mGy/yr. The old viable fruits have a total absorption range of 0.1–3.0 Gy (gray, the unit of specific energy imparted and adsorbed, where 1 Gy = 100 rad; Aitken, 1985
; Hall, 2000
).
|
|
. Although the thickness of the fine loess topsoil above the lotus-bearing layer varies from place to place, that of the lotus-bearing gray clay zone is fairly uniform. The peat bed underlying the lotus-bearing clay, mined for fuel in the 1950s, is 
0.5 m thick; the pervasive peat-mining of this unit and the resultant overturning of strata throughout much of the Xipaozi basin explain why local farm children have been able to find lotus fruits exposed on the topsoil of the lake, as previously noted (and regarded as something of a mystery) in a Chinese report (Anonymous, 1962
). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Measurement of soil radiation in the lotus-bearing layer of the lake sediments contributes data about the potentially mutagenic environment in which the old fruits were buried. It remains to be explained, however, what the underlying bases may be that protect lotus fruits from damage and/or contribute to the repair of cellular damage accrued over hundreds of years, enabling them to remain viable much longer and to germinate at a much higher rate than any other aged seeds known.
The various lotus fruits from Xipaozi thus far tested range in age from 200 to 1300 yr and have an overall germination rate of 80% (in 10 fruits tested). In comparison with these fruits and a reputedly viable 620-yr-old seed of Canna compacta reported by Lerman and Cigliano (1971)
, experimentally buried seeds tested by Kivilaan and Bandurski (1981
; see also Brown, 2001
) and germination testing and comprehensive listings of such tests compiled by Milberg (1990, 1994)
show that the four next longest known survivors, all weeds, include a malvacean and two scrophulariaceans (with ages of 100 yr and germination rates of 1–42%) and a 129-yr-old hard-coated geraniacean (gathered after a forest burn, having a germination rate of 30%). One of the Xipaozi fruits given to R. W. Chaney was dated by W. F. Libby to be 1040 yr in age (Libby, 1955
; Wester, 1973
); the viability of this fruit, however, was not tested, and the accuracy of its dating has been disputed (Goodwin and Willis, 1964
). Other claims of exceptional long-term viability, based on circumstantial evidence rather than direct radiocarbon dating, are equivocal and subject to question (Bewley and Black, 1982, 1994
; Priestley, 1986
; Shen-Miller et al., 1995
). According to data gathered from 13 worldwide seed-storage stations and collated by Priestley (1986)
, seeds of other cash crops, e.g., barley, corn, oats, potato, rice, soybean, and wheat, have half-lives of only 3–13 yr.
The rate of mass gain during germination differs distinctly between the old lotus fruits tested and their modern controls. During imbibition, the old fruits germinated as soon as they reached a maximum mass. Although this gain was not statistically different from the controls, the modern fruits took a much longer time to sprout. The reasons for this difference, not yet understood, could presumably be clarified by a detailed comparison of cellular events occurring during such imbibition.
That the old fruits consistently germinated faster than the controls may be related to differences in fruit maturation. To harvest the modern fruits, large fruit receptacles, each bearing 20 green fruits, were collected from lotus plants at the Kenilworth Aquatic Garden. Collected in early October, the fruits were stored at 4°C for several weeks, were removed from the receptacles and then washed and dried on the laboratory bench at ambient temperature. Within 2–3 d, the fruits became dark brown and some developed wide cracks. In contrast, the old fruits from Xipaozi, collected from the now dry lakebed sediments, had matured and separated from their receptacles under normal conditions before they were deposited onto the lakebed. In essence, the old fruits were "vine ripened," a process likely to have played a role in their maturation, particularly of their pericarps, and, thus, in their eventual germination. Whether the longer sprouting time required by the modern fruits (4 d to as long as 58 d, in comparison with 3–4 d for the old fruits) reflects a period necessary for maturation and/or cellular repair needs to be investigated. Harvest of thoroughly ripened modern lotus fruits is planned for the fall of 2001 at the Kenilworth Aquatic Garden.
The high sensitivity and rapid response of lotus seedlings to their surroundings, whether grown from old or modern fruits, are striking. For example, plumules rapidly blackened after just a slight touch; symptoms of toxicity soon appeared upon exposure to high concentrations of nutrients; leaves and long spindly stalks quickly dried after being lifted out of water; and in tall leaves, interveinal tissues dried quickly if the level of standing water appreciably decreased. The rapidity of such responses permitted rapid correction of cultivation practices and facilitated growth experiments, particularly those related to optimization of nutrients.
The rapid germination of old fruits, as well as the initial rapid development of the leaves and rhizomes of their seedlings here reported, have been noted previously for undated Xipaozi fruits studied by others (Wester, 1973
; Chang, 1978
). However, with few exceptions (e.g., the old fruits germinated at the Kenilworth Garden and the Beijing Institute of Botany), such growth has not been sustained enough to give rise to long-lived healthy plants. Presumably, the seedlings incorporate aberrant changes inherent in the old fruits, expressed particularly in weak root and rhizome growth and by the presence of dark green leaves and pink veins and noses, results, perhaps, of inadequate photosynthesis and food mobilization. Early termination of winter dormancy is another response noted in two offspring of the old fruits tested that differs from the longer dormancy time typical of the modern lotus.
At the time of sprouting, a mature lotus embryo axis has three visible plumule initials (see fig. 10 in Shen-Miller et al., 1995
). Abnormalities reported here in seedlings—the lack of standing leaves, variegation in nodal leaves, red coloration in summer leaves, and leaf abnormalities in the second season of growth—all occurred during the later stages of growth and were abnormalities in tissues produced by later cell division. This observation is consistent with the hypothesis that in mammalian cells, radiation-induced genomic instability can accumulate over generations of cell replication (Little, 1998
).
Throughout growth, seedlings of all old fruits tested exhibited distinct phenotypic characters that mimicked those of mutant maize, in which they are known as rough sheath, lesions, red/brown midribs, speckles, bronze, and brittle stalks and have been shown to reflect, respectively, the expression of mutant genes rs1, les8, bm1, spc2, bz2, and bk2 (Freeling and Walbot, 1994
; Neuffer, Coe, and Wessler, 1997
). Maize mutations are known to have counterparts in such crops as barley, soybean, tomato, and wheat, as well as in Arabidopsis, petunia, and snapdragon (Neuffer, Coe, Wessler, 1997
). Abnormalities observed in the lotus offspring here studied were plentiful, symptoms that were almost entirely absent from controls grown at the same time under the same conditions. As in mutant maize, certain of the lotus abnormalities may be hormonally promoted; for example, pink leaf pigmentation in maize reflects production of the stress hormone ABA (Walbot et al., 1994
). Red coloration can be present in modern lotus during early spring and late fall, when air temperatures are relatively low. But offspring of old fruits often show a high degree of redness during peak summer growth, possibly reflecting lingering stress that is perhaps expressed also by their lack of standing leaves resulting in inadequate photosynthesis and concomitant poor rhizome development.
Special detection equipment for quantitative determination of soil -radiation, together with use of the Rutherford-Bateman differential equations of radioactive decay, have permitted accurate estimation of the amounts of the major radionuclides present in the Xipaozi soil and close approximation of the concentrations of other non--emitting nuclides (Harbottle, 1993
). This approach, especially useful because of its capability to measure all relevant parameters, shows that the lotus-bearing beds at Xipaozi emit -radiation at a rate comparable to the mean background radiation value of 1997 for the continental United States, 2–3 mGy/yr (US-DOE-BNL, 1999
). The permissible irradiation level for humans is a person's "Age (yr) x 0.01 Gy" (Hall, 2000
).
The low-level chronic irradiation to which the Xipaozi fruits have been subjected, regarded safe for humans, seems likely to be responsible for the aberrations presumed to underlie certain of the abnormal phenotypes of the seedlings grown from the old fruits. Indeed, some of the abnormalities noted in the leaves of lotus offspring have been observed also in those plants chronically subjected to a much higher irradiance level; for example, leaf thickening, puckering, marginal curving, chimera formation, and pink to red coloration (Table 7; Gunckel and Sparrow, 1954
). Thickening of the leaf blade is particularly common after chronic - or X-radiation (e.g., in 20 documented species of mono- and dicotyledonous plants; Gunckel and Sparrow, 1954
). After being subjected to an entire season of -irradiation of 0.8 Gy/d, apple seedlings produced white segmented leaves the following season (Table 7; analogous to the lotus shown in Fig. 9). The similarity of leaf abnormalities between these modern plants and the lotus offspring subjected to very different levels of irradiance (Table 7) is striking, although the prevalence of abnormalities may be less in the lotus due to a lower overall exposure. The low growth vigor of the offspring of the old lotus fruits, reflected in their aberrant rhizome development and, perhaps, inadequate photosynthetic capacity, remains a major concern for their effective cultivation. Nevertheless, the viability of lotus embryos has evidently been little affected by exposure to a total maximum dose of -radiation of 3 Gy accumulated over 1300 yr.
|
; Table 7). One of the most radiosensitive reactions yet observed is the inhibition of the activity of indoleacetaldehyde oxidase in IAA biosynthesis of X-irradiated mung bean seedlings, an inhibition commencing at exposure levels as low as 0.1 Gy (Gordon, 1956, 1957
). This response may be a result of oxidation of the sulfhydryl groups of the oxidase by radiation-generated peroxides and free radicals (Bandurski, personal communication). A marked and immediate decrease in IAAld oxidase level and activity was found in corn coleoptiles after -irradiation at 3 kGy (Momiyama et al., 1999
); the conversion of conjugated IAA to IAA, however, was unaffected. Aqueous IAA (at physiological level, but not in vivo) was readily degraded at 30 Gy (Momiyama et al., 1999
).
Our earlier paper on the sprouting and dating of ancient lotus fruits from Xipaozi (Shen-Miller et al., 1995
) generated a flurry of interest. The public was evidently enthralled by what the press dubbed the discovery of a "fountain of youth." But the potential significance of our investigations does not center on the demonstration of longevity per se. Rather, our interests focus on questions posed by the continued viability of fruits over hundreds of years of aging, questions that wait to be answered by the long-living lotus.
For the present, a principal priority is development of appropriate methods to assure and to maintain vigorous growth of the seedlings of old fruits. Numerous other studies are underway or in the planning stages (Shen-Miller et al., 1999
). The relatively large size of lotus embryos provides ample material for investigation: a single lotus embryo axis, that has, for example, a dry mass of 30–50 mg, yields soluble proteins sufficient for scores of SDS-PAGE mini-gels (Shen-Miller et al., 2000
). Additionally, the genetic mechanism underlying the long-term viability of lotus are ripe for study; lotus has eight pairs of chromosomes (Anonymous, 1987
; Shen-Miller et al., 1997
), but its genome has yet to be sequenced. If understanding of the workings of lotus seed aging proves transferable to other organisms, it would provide promising means to prolong the shelf life of seeds of other economic crops and even to mitigate the effects of aging in animals, including humans. Toward these ends, we invite those interested to join with us in studies of the recently collected fruits from Xipaozi.
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FOOTNOTES |
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9 Author for reprint requests (FAX: 310-825-0097; shenmiller{at}biology.ucla.edu
).
|
LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Alley M. 1996 Dwarf lotus: small wonders of the Nelumbo world. Water Gardening 1: 16-23
Anonymous. 1962 Mysterious seeds. People's Pictorial 8: 37 (in Chinese)
Anonymous. 1987 China Lotus. Wuhan Botanical Institute, Academia Sinica, Science Publishing Association, Wuhan, Hepeh, China (in Chinese)
Bewley J. D. M. Black 1982 Plant physiology and biochemistry of seeds: (2) viability, dormancy and environmental control. Springer Verlag, Berlin, Germany
Bewley J. D. M. Black 1994 Seeds: physiology, development, and germination, 2nd ed. Plenum Press, New York, New York, USA and London, UK
Brown K. 2001 Patience yields secrets of seed longevity. Science 5510: 1884-1885
Carruth G. 1993 The encyclopedia of the world facts and data. Harper Collins, New York, New York, USA
Chang Y. J. 1978 A thousand year-old lotus has awaken. Fossil 1: 22-23 (in Chinese)
Chen C. H. S. M. Chien K. S. Zhou 1965 Palynological analysis of the Holocene Nymphaceae seed-bearing deposits at the vicinity in Liaotung Peninsula. Quaternaria Sinica 4: 167-173 (in Chinese)
Davis J. C. I. D. Procter J. R. Southon M. W. Caffee D. W. Heikkinen M. L. Roberts T. L. Moore K. W. Turteltaub D. E. Nelson D. H. Loyd J. S. Vogel 1990 LLNL-UC AMS facility and research program. Nuclear Instruments and Methods in Physics Research B 52: 269-272[CrossRef]
Freeling M. V. Walbot [Eds.] 1994 The maize handbook. Springer Verlag, New York, New York, USA
Goodwin H. E. H. Willis 1964 The viability of lotus seeds (Nelumbo nucifera Gaertn.). New Phytologist 63: 410-412[CrossRef][ISI]
Gordon S. A. 1956 The biogenesis of natural auxin. In R. L. Wain and F. Wightman [eds.], The chemistry and mode of action of plant growth substances, 65–75. Butterworth Scientific Publications, London, UK
Gordon S. A. 1957 The effects of ionizing radiation on plants: biochemical and biophysical aspects. Quarterly Review of Biology 32: 3-14[CrossRef][Medline]
Grosch D. S. L. E. Hopwood 1979 Biological effects of radiations, 2nd ed. Academic Press, New York, New York, USA
Gunckel J. E. A. H. Sparrow 1954 Aberrant growth in plants induced by ionizing radiation. Brookhaven Symposium on Biology 6: 252-279
Hall E. J. 2000 Radiobiogy for the radiobiologist, 8th ed. J. P. Lippincott, Philadelphia, Pennsylvania, USA
Harbottle G. 1993 A Marinelli beaker modified for easier mathematical modeling for self absorption in environmental radioactivity measurements. Radioactivity and Radiochemistry 4: 20-31
Harbottle G. C. V. Evans 1997 Gamma-ray methods for determining natural and anthropogenic radionuclides in environmental and soil science. Radioactivity and Radiochemistry 8: 38-46
