American Journal of Botany 89(2): 236–247. 2002.
LONG-LIVING LOTUS: GERMINATION AND SOIL
g-IRRADIATION OF CENTURIES-OLD FRUITS, AND
CULTIVATION, GROWTH, AND PHENOTYPIC
ABNORMALITIES OF OFFSPRING1
J. SHEN-MILLER,2,9 J. WILLIAM SCHOPF,3 GARMAN HARBOTTLE,4
RUI-JI CAO,5 SHU OUYANG,5 KUN-SHU ZHOU,6 JOHN R. SOUTHON,7
AND GUO-HAI LIU8
Department 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;
6
Beijing Institute of Geology, Beijing, China; 7Center of Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory,
Livermore, California 94551 USA; and 8Department of Geology, Liaoning Normal University, Dalian, Liaoning, China
2
Sacred lotus (Nelumbo nucifera) has been cultivated as a crop in Asia for thousands of years. An ;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 g-radiation of ;2.0 mGy/yr in the lotus-bearing beds; and (6) measured
stratigraphic sequences of the lakebed strata. The total g-irradiation of the old fruits of 0.1–3 Gy (gray, the unit of absorbed dosage
defined as 1 joule/kg; 1 Gy 5 100 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 g-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; g radiation; growth; imbibition; lotus; seed longevity.
Sacred lotus (Nelumbo nucifera) has been a prestigious crop
in China for nearly 5000 yr (Anonymous, 1987). 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).
In a previous paper, we reported a 67% germination rate for
six directly 14C-dated Xipaozi lotus fruits (obtained from Academia Sinica’s Beijing Institute of Botany) that ranged in
physical age from 400 to 1300 yr (Shen-Miller et al., 1995).
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
Manuscript received 10 April 2001; revision accepted 9 August 2001.
The authors thank R. S. Bandurski for suggesting study of soil dosimetry;
L. Knopoff, for earthquake data; farmer T. S. Li, for lotus fruits collected
from the topsoil in his cold frame; The Scotts Company, for support of field
work; K. O. Stetter, for generous hospitality at Regensburg Universität, and
his colleagues, V. Debus, A. Bresinsky, and P. Lindner, for help in lotus cultivation; G. Dodson, for modern lotus fruits; W. Y. Guo (farmer representative), Y. T. Li and S. W. Tien (engineers), and E. Chilenskas, for field assistance; W. G. Yang and W. M. Deng, for greenhouse assistance; and the reviewers for helpful comments. Work at BNL and LLNL were carried out
under US-DOE contracts DE-AC02-98CH10866 and W-7405-Eng-48, respectively.
9
Author for reprint requests (FAX: 310-825-0097; e-mail: shenmiller@
biology.ucla.edu).
1
236
February 2002]
SHEN-MILLER
ET AL.—OFFSPRING OF CENTURIES-OLD LOTUS FRUITS
237
Figs. 1–2. 1. Liaoning province, China, showing the location of Xipaozi Village (arrow, northeast of Pulandian), where old lotus fruits were collected. 2.
Farm fields at Xipaozi Village, showing the Anzihe River and, to the north, Curvy Dragon Hills and cold frames (white long structures on east side of the
Anzihe River).
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.
Our field group was officially hosted in Liaoning Province
by the Geology Department of Liaoning Normal University in
Dalian, China. This formal backing provided us entry into the
region, as did the presence in our group of three Academia
Sinica geologists. Old lotus fruits are highly treasured locally,
but rather than being valued as objects of scientific study they
are regarded by the villagers as precious oddities and a source
of pride (illustrated, for example, by the use of the name ‘‘Antique Lotus’’ both for a village restaurant and a locally produced banquet liquor). In view of the pervasive peat-mining
and the widespread removal of lotus-bearing clays along with
this fuel in the late 1950s, it was perhaps no small feat that
some four decades later we were still able to obtain 58 intact
fruits.
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 6 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).
Japanese botanist—Ichiro Ohga’s estimated date for the
lake-draining event was based on observations reported to him
by a local farmer, ‘‘U. Liu’’ (Liu Guay San), who helped him
in his field work and collected most of the lotus fruits used in
Ohga’s studies (Ohga, 1927). 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: a-particles (helium nuclei), b-particles (nuclear
electrons), and X- and g-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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Figs. 3–5. 3. Sketch map showing the area covered by the old lotus lake basin at Xipaozi Village and the location of sites sampled: pit, well, farmer Liu’s
former field, and farmer Li’s cold frame. 4. Stratigraphy of the Xipaozi lake basin sediments measured at the pit and well, showing the stratigraphic position
of lotus fruits collected in situ, compared with sections previously measured by Chen, Chien, and Zhou (1965). 5. Stratigraphic section showing loess (fine
wind-blown silt) overlying lotus-bearing strata at the freshly excavated abandoned well at Xipaozi Village from which lotus fruits OL96-33 and OL96-34, wood
fragments, and soil samples were collected in situ.
MATERIALS AND METHODS
New-found fruits—Four lotus fruits (Nelumbo nucifera) were unearthed in
situ, two from each of two excavated sites (Fig. 4). A total of 56 other fruits
were gathered from the soil surface, about two-thirds of which were either
gifts or were purchased from local farmers (who collected them as they tilled
the land; fruits cost 10 yuan each, or about $1.25 US). Of the total, 20 fruits
(each having a carefully documented provenance) were collected by our team,
chiefly by combing a large area of farm fields on both sides of the Anzihe
River in the southern part of the basin. Our greatest success came after an
overnight rain, which washed away the dusty loess and made the eye-catching
shiny fruits easily visible on the soil surface near farmer Liu’s old homestead
(Fig. 3).
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 48C 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)
February 2002]
SHEN-MILLER
ET AL.—OFFSPRING OF CENTURIES-OLD LOTUS FRUITS
TABLE 1. China cataloga of selected strong earthquakes in the vicinity
of the Holocene lotus lake at Xipaozi Village, ;7 km northeast of
Pulandian (38–398N, 122–1238E), Liaoning Province, northeastern
China.
Year
Month and day
Latitude
(8N)
Longitude
(8E)
Magnitude
(Richter
scale)
Province
70 BC
1057 AD
1290b
1484c
1556d
1626
1668
1679b
1720
1830
1888b
June 1
Not recorded
September 27
January 29
January 23
June 28
July 25
September 2
July 12
June 12
June 13
36.3
39.5
41.5
40.4
34.5
39.4
35.3
40.0
40.4
36.4
38.5
119.0
116.3
119.3
116.1
109.7
114.2
118.6
117.0
115.5
114.2
119.0
7.0
6.75
6.75
6.75
8.0
7.0
8.5
8.0
6.75
7.5
7.5
Shantung
Hopeh
Liaoning
Hopeh
Shensi
Shansi
Shantung
Hopeh
Hopeh
Hopeh
Shantung
a
PDE (1964–1999); Lee, Wu, and Jacobson (1976).
Strong earthquakes particularly near Xipaozi Village.
c The earthquake suggested by the Japanese geologist cited by Wester
(1973) to have drained the Xipaozi lake into the Bo Hai Sea.
d An earthquake that resulted in the deaths of 830 000 people (Carruth, 1993).
b
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, MnSO4EDTA, ZnSO4-hydrate, and 0.1% chelated Fe. Optimum growth was observed
at 1003 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
239
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 1003 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 3 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 6 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’’ 5 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).
Lake stratigraphy—To survey the landscape at Xipaozi Village, we were
accompanied by two geological engineers of the Liaoning Engineering Institute of Geology and Oceanography, Pulandian, Liaoning, China and a local
farmer. Although, as noted above, most of the basinal sediments were over-
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TABLE 2. Radiocarbon ages of viable fruits of lotus (Nelumbo nucifera) or their pericarps and of wood fragments, collected from the Holocene
lotus lake at Xipaozi Village, Liaoning Province, northeastern China, and of modern fruits from the Kenilworth Aquatic Garden, Washington,
DC, USA. Sample numbers listed in parentheses are laboratory analysis numbers for radiocarbon dating. Li and Liu are farmers from Xipaozi
Village (see Fig. 3, farm locations).
Sample
Source
Fruit oSL4 (UCLA#2387C)
Fruit oSL2c (UCLA#2387B)
Pericarp OL96-52d (CAMS#75444)
Pericarp OL96-44d (CAMS#51660)
Pericarp OL96-50d (CAMS#65056)
Pericarp oSL5c (CAMS#12775)
Pericarp OL96-53d (CAMS#56373)
Fruit M4e (UCLA#2387E)
Pericarp M8e (CAMS#12778)
Wood fragmentsd (CAMS#51480)
Not recorded
Not recorded
Li’s cold frame topsoil
Liu’s farm topsoil
Liu’s farm topsoil
Not recorded
Li’s cold frame topsoil
Not recorded
Not recorded
Excavated well
c
14
C agea (yr)
1350 6
705 6
410 6
380 6
280 6
270 6
200 6
Modern
Modern
100 6
220
145
40
40
40
60
50
40
Physical ageb (yr)
1288 6 271
676 6 98
466 6 94
464 6 91
408 6 67
400 6 72
192 6 152
(Shen-Miller et al., 1995)
(Shen-Miller et al., 1995)
175 6 135
Radiocarbon year (61 SD) based on a C half-life of 5568 6 30 yr (Libby, 1955), following the conventions of Stuiver and Polach (1977).
Mean age (61 SD) derived from the full range of calendar intercepts, at the time of germination or analysis, calibrated from the radiocarbon
age BP (before present, where ‘‘present’’ 5 AD 1950; Stuiver and Becker, 1993).
c Fruits collected at Xipaozi in 1952 by the Beijing Institute of Botany, Academia Sinica, Beijing, China (Shen-Miller et al., 1995).
d Specimens collected at Xipaozi in 1996, as reported here. Wood fragments were collected in a well ;0.35 m stratigraphically below the top of
the lotus-bearing gray clay (Fig. 4).
e Collected at Kenilworth Aquatic Garden in 1982 (M4) and 1983 (M8), produced by plants grown since 1951, from undated Xipaozi fruits of I.
Ohga’s collection given in 1950 to R. W. Chaney by the Tohuku Imperial University.
a
14
b
turned in an effort to extract peat for fuel, we located two sites that still have
complete sedimentary profiles. One was situated in the eastern part of the
basin (Fig. 3), an abandoned well (Figs. 4 and 5) where we measured the
section we had freshly exposed and collected in situ samples for soil radioactivity analysis as well as wood fragments and lotus fruits. The other was a
site near the center of the basin, a pit we excavated in which we measured
the stratigraphic section and collected fruits and soil samples in situ (Figs. 4
and 6).
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
g-ray intensities were interpreted using statistical program SPSS-X (SPSS,
1988), software specifically designed for use with the modified Marinelli beaker.
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 OL9634 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
RESULTS
Fig. 6. Excavation of the pit at Xipaozi Village from which lotus fruits
OL96-1 and OL96-3 and a soil sample were collected in situ.
Age determination by 14C analysis—All of the four Xipaozi
fruits collected in 1996 and tested for germination are viable.
As indicated in Table 2, 14C dating shows these fruits to range
from 192 to 466 yr in physical age. Table 2 also includes ages
of all viable fruits tested earlier (Shen-Miller et al., 1995; 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%
February 2002]
SHEN-MILLER
ET AL.—OFFSPRING OF CENTURIES-OLD LOTUS FRUITS
241
TABLE 3. Mass gain of old and modern fruits of lotus (Nelumbo nucifera) during imbibition and germination (61 SD), tested over four
seasons.
Fruits
Olda
N
Dry mass (g)
Net gain (%), day 1
Total net gain (%)
Germination (d)
0.74
40
136
3.5
4
6
6
6
6
Controlsb
0.07
13
8
0.5
1.27
74
163
17
12
6 0.12
6 28
6 36
6 15
a Fruits OL96-52 (466 yr old), OL96-44 (464 yr old), OL96-50 (408
yr old), and OL96-53 (192 yr old), collected at Xipaozi, China, as reported here.
b Modern fruits, tested during the same seasons as each of the old
fruits, collected in 1996 from the Kenilworth Aquatic Garden (see Table
2, footnotee).
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 6 8% and 163 6 36%, respectively).
OL96-44 and OL96-53 germinated 4 d after imbibition and
OL96-50 and OL96-52 on day three. The modern controls,
tested under the same conditions as the corresponding old
fruits, were ;1.73 heavier (having an average dry mass of
1.27 g); exhibited a mass gain ;1.83 greater on day one; but
required 53 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 ;48C, 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 1003 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 (253 dilution of the stock solution), browning and drying of younger
leaves begin around their entire peripheries and advance in-
Figs. 7–8. Phenotypic effects of nutrient toxicity on leaves of modern
lotus seedlings. 7. Drying young leaf of a seedling grown at three times the
optimum nutrient concentration, showing a prominent, wavy, grayish-brown
peripheral border. 8. Older leaf with dark-green veins and pale-green tissue,
signs of an uptake of excess nitrogen.
ward, forming a wavy pattern, layer upon layer (Fig. 7). At
lower concentrations (503 and 753 dilution of the stock),
yellow or brown necrotic spots appear on the peripheries of
younger leaves and similarly advance inward. At suboptimal
levels (.1003 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).
Growth—For the offspring of both old and modern fruits,
seedlings germinated in late spring (e.g., mid-March to April)
exhibited faster growth than those sprouted in January–February. Two unique features of the lotus that should be emphasized are the green embryo axis of a mature fruit and the
absence of roots from such embryos at germination (see fig.
10 in Shen-Miller et al., 1995). 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
242
AMERICAN JOURNAL
OF
BOTANY
[Vol. 89
Figs. 9–12. Abnormal leaf phenotypes of seedlings grown from old lotus fruits (Figs. 9–11) and normal modern control (Fig. 12). 9. The seedling grown
from 400-yr-old fruit oSL5 on day 55 after germination, showing prominent, pale, wedge-shape variegation on leaf no. 6 (left center). 10. A leaf of the seedling
grown from 464-yr-old fruit OL96-44 on day 105 after germination, photographed at an oblique angle to show in its central oval the presence of varicose veins,
thick cabbage-like wrinkles, and a central pale ‘‘nose’’ (subtended by a spindly stalk, not shown), and in the foreground, its down-turned rim (under water).
11. The plant grown from 192-yr-old fruit OL96-53, ;4.5 mo after germination, with leaves that are prostrate on the water surface, rather than standing, and
are small (the largest of which, centermost, ;15 cm in diameter, has been perforated by insects). 12. The modern control plant for the seedling from OL9653, ;4.5 mo after germination, having large standing leaves up to ;25 cm in diameter.
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).
In southern California, lotus seedlings enter dormancy in
December, when night temperatures drop to ;58C and daylength shortens to ,10 h. In late February, new growth normally begins when night temperatures persist at ;118C, 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
;128C, 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 Califor-
nia, lotus requires a temperature of ;108C to break winter
dormancy. Lotus plants can safely weather freezing air temperatures of2308C 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 con-
February 2002]
SHEN-MILLER
ET AL.—OFFSPRING OF CENTURIES-OLD LOTUS FRUITS
TABLE 4. Representative data illustrating trends during early growth
of lotus seedlings of old (from Xipaozi, Liaoning, China) and modern control fruits (from Washington, DC, USA). Abbreviations:
Dag 5 days after germination; Dai 5 days after imbibition.
I)
Fruits
First rhizome
internode
expansion
(Dag)
Fruits
First roots
initiation
(Dag)
OL96-44 (464 yr)a
Modernb
43
78
OL96-53 (192 yr)a
Modernb
8
13
First plumule
emergence
(Dai)
Fruits
Third plumule
diameter
(cm)c (12 Dag)
II)
Fruits
OL96-50 (408 yr)a
Modernb
3
9
OL95-52 (466 yr)a
Modernb
4.0
0.3
a
Collected from Xipaozi in 1996, as reported here.
Collected from Kenilworth Aquatic Garden in 1996, (see Table 2,
footnotee).
c Measured from the tip of one marginal spicule (e.g., Fig. 10, left)
to the other through the center of the nose.
b
trols, 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.)
The seedling from the 464-yr-old OL96-44 also grew faster
than its control (Table 4), but it produced leaves that never
enlarged beyond juvenile size and it had exceedingly long thin
stalks (;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 ;498N, as compared to ;348N
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
243
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 .148C 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 midJune 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 g-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 OL9644 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 smallleafed. 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.
244
AMERICAN JOURNAL
TABLE 5. Gamma radiation (in micrograys per year)a from soil radionuclides in the lotus-bearing horizons of the Holocene lotus lake
bed sediments at Xipaozi Village, Liaoning Province, northeastern
China.
Soil
b
1
2c
3d
Ave
K
444
456
421
440
Th
Th
U
U
(pre Tn)e (post Tn)f (pre Rn)g (post Rn)h
317
346
308
324
498
563
503
521
48.0
38.6
37.8
41.5
331
409
333
358
Si
Sj
GSk
1638
1813
1603
1685
1788
1963
1753
1835
1847
2022
1812
1894
1 Gy (gray) 5 100 rad.
Gray clay containing fruit OL96-1, stratigraphically ;0.05 m below
the top of the bed in the pit (Fig. 4).
c Black clay encasing fruit OL96-34, ;0.2 m below the top of the
gray clay bed in the well (Fig. 4).
d Black clay ;0.05 m below fruit OL96-34 in the well (Fig. 4).
e Converted from 228Ac.
f Converted from 232Th.
g Converted from 238U.
h Subtracted 25% due to 226Ra emanation.
i Total micrograys per year.
j Added cosmic ray radiation (150 mGy), following Aitken (1985).
k Grand total, including a correction factor for inherent fruit radioactivity of 59 mGy/yr, measured in lotus fruit OL96-7.
OF
Soil g-irradiation
(mGy/yr 6 1 SD)
Viable fruits
6
6
6
6
6
6
6
oSL4c
oSL2c
OL96-52d
OL96-44d
OL96-50d
oSL5c
OL96-53d
1.9
1.9
1.9
1.9
1.9
1.9
1.9
b
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 g-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 g-ray emission of 1.9 6 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 5 100 rad; Aitken, 1985; Hall, 2000).
[Vol. 89
TABLE 6. Soil g-irradiation of viable old fruits of lotus (Nelumbo nucifera) from Xipaozi Village, Liaoning Province, northeastern China.
a
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.
BOTANY
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Age 6 1 SD
(yr)a
1288
676
466
464
408
400
192
6
6
6
6
6
6
6
271
98
94
91
67
72
152
Total g-irradiation
(Gy)b
1.83–3.12
1.04–1.55
0.67–1.12
0.67–1.11
0.60–0.95
0.59–0.94
0.07–0.69
a
Physical age (Table 2).
1 Gy (gray) 5 100 rad.
c Collected at Xipaozi in 1952 by the Beijing Institute of Botany,
Academia Sinica (Shen-Miller et al., 1995).
d Collected at Xipaozi in 1996, as reported here.
b
Stratigraphy—In Fig. 4, the stratigraphic sequences measured at the newly measured pit and well are compared with
those previously reported by Chen, Chien, and Zhou (1965).
Although the thickness of the fine loess topsoil above the lotus-bearing layer varies from place to place, that of the lotusbearing 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
The demonstration of exceptional seed longevity in lotus
(Nelumbo nucifera) combined with the recent collection of old
fruits from the Holocene dry lakebed at Xipaozi present a
unique opportunity to study the development of seedlings derived from fruits hundreds of years in age, revealing characteristics of their germination, growth, phenotypic abnormalities, and dormancy. Use of the highly sensitive AMS technique
for age determination has made such developmental studies
possible, because the removal from germinated fruits of the
dry pericarps for radiocarbon dating does not affect subsequent
seedling growth.
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
February 2002]
SHEN-MILLER
ET AL.—OFFSPRING OF CENTURIES-OLD LOTUS FRUITS
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 seedstorage 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 ;48C 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
245
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 g-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-g-emitting nuclides (Harbottle,
1993). This approach, especially useful because of its capability to measure all relevant parameters, shows that the lotusbearing beds at Xipaozi emit g-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) 3 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
246
TABLE 7.
AMERICAN JOURNAL
OF
BOTANY
[Vol. 89
Low-dose ionizing radiation: exposure and response. Abbreviations: ; 5 converted to gray (Gy) from röntgens (R).
Total irradiation
Gya
Organisms
Exposure
Response
References
Old Lotus (dry fruits)
0.1–1.0
Apple (seedling)
;0.8/d (initial season)
Antirrhinum majus (Seedling)
;1.8/d (60 d)
Snapdragon (seedling)
;4.9/d (60 d)
Sedum (seedling)
;5.0/d (60 d)
Various seedlingsb
;.3.0/d (weeks)
Mung bean (seedling)
;1.0
Chronic (200–500 yr) Thick/pink/chimeric leaves, curved This paper
rims
Chronic
Chimeric leaves (following season) Gunckel and Sparrow,
1954
Chronic
Leaf thickness (significant inGunckel and Sparrow,
crease)
1954
Chronic
Thick, leathery leaves, curved rims Gunckel and Sparrow,
1954
Chronic
Pink leaves
Gunckel and Sparrow,
1954
Chronic
50% tumor per plant fresh mass
Grosch and Hopwood,
1979
Acute
IAA biosynthesis (45% inhibition) Gordon, 1957
Lilium longifolium (seedling)
;8.0
Acute
Lethal
Trout (embryo)
;0.5
Acute
50% lethality
Fruit fly (embryo)
;1.9
Acute
50% lethality
Mice (embryo)
;2.0
Acute
79% lethality
Microorganisms
;1000
Acute
Lethal (food pasteurization)
Human (adult) (localized)
2.0/d (70 Gy in 7 wk)
Fractionated
Tumor cell death (cancer therapy)
Grosch and
1979
Grosch and
1979
Grosch and
1979
Grosch and
1979
Grosch and
1979
Hall, 2000
Hopwood,
Hopwood,
Hopwood,
Hopwood,
Hopwood,
Gy (gray) 5 100 rad 5 100 R (röntgens); Grosch and Hopwood (1979); Hall (2000).
b Arabidopsis, beans, Crepis, ferns, Nicotiana, peas, snapdragon.
a
and Sparrow, 1954). Thickening of the leaf blade is particularly common after chronic g- 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 g-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
g-radiation of 3 Gy accumulated over 1300 yr.
The low dose chronic exposure of buried lotus fruits to 2
mGy/yr of background radiation for hundreds of years is an
experiment that cannot be duplicated in a laboratory. Table 7,
however, shows a comparison of the ‘‘low-dose’’ responses of
lotus and other plants and organisms relevant to leaf abnormalities, IAA biosynthesis, tumor formation, lethality, food
processing, and cancer therapy. In general, the larger a cell’s
nuclear volume, the greater the radiosensitivity of the organism. Using the interphase chromosome volume of sensitive
cells as an index, radiosensitivity can be ordered from greater
to lesser sensitivity as follows: mammals and plants . birds
. fish . reptiles . insects . mosses/lichens/algae/bacteria/
viruses (Linsley, 1997; 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 g-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.
February 2002]
SHEN-MILLER
ET AL.—OFFSPRING OF CENTURIES-OLD LOTUS FRUITS
Toward these ends, we invite those interested to join with us
in studies of the recently collected fruits from Xipaozi.
LITERATURE CITED
AITKEN, M. J. 1985. Thermoluminescence dating: studies in archeological
science. Academic Press, London, UK.
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., AND M. BLACK. 1982. Plant physiology and biochemistry of
seeds: (2) viability, dormancy and environmental control. Springer Verlag, Berlin, Germany.
BEWLEY, J. D., AND 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, AND 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, AND J. S. VOGEL. 1990. LLNL-UC AMS facility and
research program. Nuclear Instruments and Methods in Physics Research
B 52: 269–272.
FREELING, M., AND V. WALBOT [EDS.]. 1994. The maize handbook. Springer
Verlag, New York, New York, USA.
GOODWIN, H., AND E. H. WILLIS. 1964. The viability of lotus seeds (Nelumbo
nucifera Gaertn.). New Phytologist 63: 410–412.
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.
GROSCH, D. S., AND L. E. HOPWOOD. 1979. Biological effects of radiations,
2nd ed. Academic Press, New York, New York, USA.
GUNCKEL, J. E., AND 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., AND C. V. EVANS. 1997. Gamma-ray methods for determining natural and anthropogenic radionuclides in environmental and soil
science. Radioactivity and Radiochemistry 8: 38–46.
HILL, R. H., G. F. HINE, AND L. D. MARINELLI. 1950. The quantitative determination of gamma-ray radiation in biological research. American
Journal of Roentgenology 63: 160–169.
KIVILAAN, A., AND R. S. BANDURSKI. 1981. The one hundred year-period
for Dr. Beal’s seed viability experiment. American Journal of Botany 68:
1290–1292.
LEE, W. H. C., F. T. WU, AND C. JACOBSON. 1976. A catalog of historical
earthquakes in China compiled from recent Chinese publications. Bulletin
of Seismology Society of America 66: 2003–2016.
LERMAN, J. B., AND E. M. CIGLIANO. 1971. New carbon-14 evidence for six
hundred years old Canna compacta seed. Nature (London) 232: 568–
570.
LIBBY, W. F. 1955. Radiocarbon dating, 2nd ed. University of Chicago Press,
Chicago, Illinois, USA.
247
LINSLEY, G. 1997. Radiation and the environment: assessing effects on plants
and animals. IAEA Bulletin 39 (No. 1).
LITTLE, J. B. 1998. Radiation-induced genomic instability. International Journal of Radiation Biology 74: 663–671.
MILBERG, P. 1990. Hur lange kan ett fro leva? (What is the maximum longevity of seeds?). Svensk Botany Tidskr 84: 323–325 (English abstract).
MILBERG, P. 1994. Germination of up to 129 year old, stored seed of Geranium bohemicum (Geraniaceae). Nordic Journal of Botany 14: 27–29.
MOMIYAMA, M., K. TOMOKAZU, K. FURUKAWA, Y. KAMUYA, AND M. SATO.
1999. Effects of g-irradiation on elongation and indole-3-acetic acid level of maize (Zea mays) coleoptiles. Environmental and Experimental Botany 41: 131–143.
NEUFFER, G., E. H. COE, AND S. R. WESSLER. 1997. Mutants of maize. Cold
Spring Harbor Laboratory Press, Plainview, New York, USA.
OHGA, I. 1923. On the longevity of seeds of Nelumbo nucifera. Botanical
Magazine 37: 87–95.
OHGA, I. 1927. On the age of ancient fruit of the Indian lotus which is kept
in the peat bed in south Manchuria. Botanical Magazine 41: 1–6.
PDE (PRELIMINARY DETERMINATION OF EPICENTERS). 1964–1999. Bi-weekly updates by National Earthquake Information Service (NEIS). US Geological Survey, Washington, D.C., USA.
PRIESTLEY, D. A. 1986. Seed aging. Comstock Cornell University Press, Ithaca, New York, USA.
PRIESTLEY, D., AND M. A. POSTHUMUS. 1982. Extreme longevity of lotus
seeds from Pulantien. Nature (London) 299: 148–149.
SHEN-MILLER, J., P. LINDNER, G. FREY, G. HARBOTTLE, AND K. O. STETTER.
2000. Long living lotus: new plants from old embryos, chimeric embryo
protein. Plant Biology 2000, 22 and 52. American Society Plant Physiologists Meeting (Mini symposium and poster abstracts), San Diego, California, USA.
SHEN-MILLER, J., M. B. MUDGETT, J. W. SCHOPF, S. CLARKE, AND R. BERGER. 1995. Exceptional seed longevity and robust growth: ancient Sacred
Lotus from China. American Journal of Botany 82: 1367–1380.
SHEN-MILLER, J., J. W. SCHOPF, G. HARBOTTLE, R. J. CAO, S. OUYANG, S.
K. ZHOU, P. LINDNER, J. R. SOUTHON, AND K. O. STETTER. 1999. Sacred
Lotus of exceptional seed longevity: phenotypically abnormal plant of
350 y-old fruit, soil radioactivity, and heat stable proteins. Plant Biology
1999, 63. American Society of Plant Physiologists Annual Meeting (poster abstract), Baltimore, Maryland, USA.
SHEN-MILLER, J., J. W. SCHOPF, M. THOLANDI, AND G. HARBOTTLE. 1997.
Sacred Lotus seeds of exceptional longevity: a call for research participation. Seed Biology and Technology: Applications and Advances, 76.
Symposium Abstract (poster). Fort Collins, Colorado, USA.
SPEICHERT, G. 2000. Getting down and dirty about potting up water plants.
Water Gardening 5: 40–43.
SPEICHERT, G. 2001. Fertilizer for pond plants. Water Gardening 6: 38–40.
SPSS. 1988. SPSSX user’s guide, 3rd ed. SPSS, Chicago, Illinois, USA.
STUIVER, M., AND B. BECKER. 1993. High precision calibration of the radiocarbon time-scale AD 1950–6000 BC. Radiocarbon: Calibration 1993
35: 35–63.
STUIVER, M., AND H. A. POLACH. 1977. Discussion: reporting of 14C data.
Radiocarbon 19: 355–363.
US-DHEW. 1974. A barefoot doctors’ manual. U.S. Department Health, Education, and Welfare Publishing, National Institute of Health (NIH) 75–
695, Washington, D.C., USA.
US-DOE-BNL. 1999. Site environmental report 1997. U.S. Department of
Energy-Brookhaven National Laboratory, Upton, New York, USA.
WALBOT, V., M. I. BENITO, J. B. BODEAU, AND J. N. NASH. 1994. Abscisic
acid induces pink pigmentation in maize aleurone in the absence of
Bronze2. Maydica 39: 19–28.
WESTER, H. V. 1973. Further evidence on age of ancient viable lotus seeds
from Pulantien deposit, Manchuria. HortScience 8: 371–377.
ZHANG, M., Y. NAKAMURA, S. TSUDA, T. NAGACHIMA, AND Y. ESASHI.
1995a. Enzyme conversion of volatile metabolites in dry seeds during
storage. Plant Cell Physiology 36: 157–164.
ZHANG, M., H. YAJIMA, Y. UMEZAWA, Y. NAKAGAWA, AND Y. ESASHI.
1995b. GC-MS identification of volatile compounds evolved by dry
seeds in relation to storage conditions. Seed Science and Technology 23:
59–68.