Seed burial experiment at the Danish State Seed Testing Station, 1934–1983

Hans Arne Jensen, dr. agro.

Danish State Seed Testing Station 1962–1990; Danish Plant Directorate 1990–2003

DK-3660 Stenløse, Denmark

Lt.ha@jensen.mail.dk

The article below describes the termination of a burial experiment at the Danish Seed Testing Station, Copenhagen, from 1934 to 1983. The experiment was initiated in 1934 by the first President of ISTA from 1924 to 1937, Director Knud Dorph-Petersen. He had beside his tireless work for the development of international seed testing also a profound interest in research on seed biology. Colleagues from my first years at the Danish Seed Testing Station told me that he was a very unusual man, full of spirit and energy, and they knew a number of stories about him. For example, in 1934, at the beginning of the experiment, he held a burial speech for the seeds.

Introduction

During my work with archaeologically dated soil samples (Jensen 1986, 1987a, 1987b, 1988, 1991a, 1991b), I recorded both whole specimens and remains of seeds and fruits (macrofossils) of various species. Such records are useful both for evaluating the vegetation history of the excavated site and for identifying the plant associations utilized by the people in the settlements (e.g. grassland, arable). It is therefore of considerable interest to gain information on the time elapsing from sowing the seeds in an area until their complete disappearance from the soil.

The only way to obtain such information is to combine burial experiments with sieving out seed remains on a fine-meshed sieve after storage.

The objective of burial experiments is to mimic conditions in the field, and to evaluate under these semi-controlled conditions the longevity of the seed and the factors affecting it. In 1879, Dr. Beal initiated a study of the viability of 19 common weeds and one sample of Trifolium repens, in which the seeds were mixed with sand and buried in glass bottles. Kivilaan and Bandurski (1981) reported on the first 100 years, and Telewski and Zeewart (2002) on the germination results after 120 years of storage. These and similar burial experiments were summarized by Priestley (1986), Toole (1986), Baker (1989) and Thomson et al. (1997).

This article presents the burial experiment at the Danish State Seed Testing Station during the years 1934 to 1983. The Director, Knud Dorph-Petersen, initiated the experiment in 1934. The results for the periods 1934–1939 and 1934–1944 were published by Kjær (1940) and Kjær (1948), respectively, and Madsen (1962) prepared an overview for the period 1934–1960.

The present paper summarizes the findings, and reports specifically the germination results recorded in the years 1961–1983, after 27–49 years of burial. The results were held for years at the Seed Testing Station, but were only partially reported. The historical value and length of this

experiment are unique, and represent the type of endeavour that is rarely taken on; I have therefore decided to present it to you.

The experiment Dorph-Petersen buried 37 samples of 29 species (19 weeds and 10 crops) in clay pots, 20 cm below the soil surface. Each subsample consisted of 400 seeds. The weed seeds, collected in 1933, and the crop seeds, harvested in 1933, all originated from well-germinating seed lots. The seeds were buried in numbered clay pots, 8 cm high, and filled with soil up to 3 cm from the rim. The seeds were distributed on this soil surface, and the clay pots were then filled up to the rim with soil. They were placed with the seeds 20 cm below the surface of well-drained garden soil of pH 7.2 (Figure 1).

Figure 1. Excavated pre-numbered clay pots ready for examination of viable seeds

A plan for testing the seeds was prepared for each year until 1983 (Figure 2). For some species, the number of seeds available allowed for germination tests every year, for others only every five years or more (Dorph-Petersen 1934). The University weather station (No. 6193) is situated less than 500 m north of the experiment area, and recorded the climatic conditions continually from 1934 to 1983 (Cappelen 2014).

Figure 2. Left: plan of the location of the pots to be exhumed each year. Right: Section of the detailed plan for the burial experiment. From left: species number; laboratory number; species name (in Danish); number on pots to be exhumed from 1935 to 1983.

Throughout the whole experiment, the germinated seeds for each sample were counted for a period of two years. From 1935 to 1960, the germination tests of the exhumed seeds were in frames located in the garden of the Seed Testing Station. From 1961 to 1982, the germination tests were in boxes with sterile sand, placed in an unheated greenhouse.

In 1983, with the aim of obtaining information on any seed remains left after 49 years of storage in soil, we washed in total 33 soil samples on a 0.4 mm sieve. All residue of the seeds originally placed in the pots was kept, and intact seeds germinated on a Jacobsen apparatus (pre-chilling at 10 °C for 7 days, 20 °C) until all seeds were either germinated or dead. We used the laboratory germination method, as previous tests of seeds from arable soil had indicated that this resulted in a higher number of seedlings compared to germination in the greenhouse (Jensen 1969). S.B. Madsen, Inspector at the Seed Testing Station, was in charge of the experiment from 1961 to 1972; the present author from 1973 to 1983.

Germination of samples 1961–1983 Table 1 contains the results of the germination tests for the period 1961–1983.

Table 1. Survey of experiments with buried seeds 1961-1983.

Percent plants in the field. - No seed available for examination. * Average of two samples (by mistake mixed before germination).

Species number

Species Number of years the seeds were buried 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

1 Rumex crispus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 Rumex crispus - - - - - - - - - - - - - - - - - - - - - - 0 3 Rumex acetosella 2 1 0 tr. 0 0 0 6 tr. 0 0 0 tr. tr. 0 tr. 0 0 0 0 0 0 0 4 Persicaria lapathifolia - - - 1 - - - - 0 - - - - 0 - - - - - - - - 0 5 Persicaria lapathifolia - - - - - - - - - - - - - - - - - - - - - - 0 6 Scleranthus annuus - - - 0 - - - - 0 - - - - 0 - - - - 0 - - - 0 8 Chenopodium album 68 58 46 40 0 44 tr. 4 29* tr. 4 16 42 15 0 31 - - 0 - - - 4 9 Chenopodium album 80 70 54 58 35 40 36 1 29* 26 36 30 43 27 20 48 tr. 31 tr. 34 17 0 22 10 Papaver rhoeas 17 6 2 2 1 1 0 3 1 1 0 1 1 3 0 1 tr. 1 1 0 0 0 0 11 Thlaspi arvense - - - 0 - - - - 3* - - - - tr. - - - - 0 - - - 0 12 Thlaspi arvense - - - 0 - - - - 3* - - - - 0 - - - - 0 - - - 0 13 Sinapis arvensis 9 7 1 0 3 3 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 14 Geranium dissectum - - - - - - - - - - - - - - - - - - - - - - 0 15 Geranium pusillum 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 Vicia hirsuta - - - 8 - - - - 0 - - - - 2 - - - - 0 - - - 0 17 Daucus carota - - - 0 - - - - 0 - - - - 0 - - - - 0 - - - 0 19 Plantago lanceolata - - - 0 - - - - - - - - - - - - - - - - - - 0 20 Plantago lanceolata - - - - - - - - - - - - - - - - - - - - - - 0 21 Plantago major 0 0 1 1 tr. tr. 0 0 0 1 0 0 0 3 0 0 tr. 0 0 tr. 0 0 0 24 Tripleurospermum

inodorum 0 0 0 1 tr. 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

25 Glebionis segetum - - - 0 - - - - 0 - - - - 0 - - - - 0 - - - 0 26 Circium arvense - - - - - - - - - - - - - - - - - - - - - 0

Species number

Species Number of years the seeds were buried 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

27 Cirsium arvense - - - 1 - - - - 0 - - - - 1 - - - - 1 - - - 0 28 Hordeum distichum - - - 0 - - - - 0 - - - - 0 - - - - - - - - 0 29 Triticum sativum - - - 0 - - - - 0 - - - - 0 - - - - - - - - 0 30 Lolium perenne - - - 0 - - - - 0 - - - - 0 - - - - 0 - - - 0 31 Phleum pratense - - - 0 - - - - 0 - - - - 0 - - - - 0 - - - 0 32 Dactylis glomerata - - - 0 - - - - 0 - - - - 0 - - - - 0 - - - 0 33 Brassica napus var.

napobrassica 0 0 0 0 0 0 0 0 0 tr. 0 0 0 0 0 0 0 0 0 0 0 0 0

34 Brassica rapa rapa 0 0 tr. 0 0 0 0 0 tr. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 Trifolium pratense 0 0 tr. 0 0 0 0 0 tr. 0 0 1 0 0 0 0 0 0 0 0 0 0 0 36 Trifolium repens 1 0 tr. tr. 0 0 1 0 0 0 0 tr. tr. 0 tr. 1 0 0 0 0 0 0 0 37 Medicago lupulina 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 tr. 0 0 0 0 0 0 0

Excluded from Table 1 are species of which no seeds were available for examination in the period. At the end of the test, the only species of which the samples contained living seeds was Chenopodium album. After 49 years of burial, 22 % of sample no. 9 germinated, and only 4 % of sample no. 8.

The samples of the following species, listed as they appear in Table 1, lost their capacity for germination between 1961 and 1983 (in parentheses: the last year of burial in which at least one seed germinated): Rumex acetosella (42 years); Persicaria lapathifolium (30); Papaver rhoeas (45); Thlaspi arvense (40); Sinapis arvensis (36); Vicia hirsuta (40); Plantago major (46); Tripleurospermum maritimum (32); Cirsium arvense (45); Brassica napus var. napobrassica (36); Brassica rapa rapa (35); Trifolium pratense (38); Trifolium repens (42); Medicago lupulina (42 years).

Table 2 presents the seed remains and the germination capacity found after 49 years of storage. The remaining 22 species mentioned in Table 1 did not germinate, and we found no remains larger than 0.4 mm. Thus, the germination capacity of these species was lost before 1961 (after 1 to 26 years of burial).

Table 2. Seed buried 1934–1983. Seed remains and germination percentage found after washing seed samples on a 0.4 mm sieve

Species number

Species Pieces of seeds (of 400 originally buried)

Germination (%)

8 Chenopodium album 242 (> half the original size) 4 9 Chenopodium album 326 (> half the original size) 22 10 Papaver rhoeas 2 (fragments) 0 16 Vicia hirsuta 1 (fragment) 0 33 Brassica napus var.

napobrassica 1 (fragment) 0

34 Brassica rapa rapa 1 (fragment) 0

Viability of species The following species retained their germination capacity:

For 40–49 years: Chenopodium album L.; Cirsium arvense (L.) Scop.; Medicago lupulina L.; Papaver rhoeas L.; Plantago major L.; Rumex acetosella L.; Thlaspi arvense L.; Trifolium repens L.; Vicia hirsuta (L.) Gray.

For 30–39 years: Brassica napus L. var. napobrassica (L.) Rchb.; Brassica rapa L. subsp. rapa (Brassica campestris L. var. rapifera Metzg.); Persicaria lapathifolia (L.) Delarbre (Polygonum tomentosum Schrank.); Sinapis arvensis L.; Trifolium pratense L.; Tripleurospermum inodorum (L.) Sch. Bip. (Matricaria inodora L.).

For 20–29 years (in part from Madsen 1962): Daucus carota L.; Geranium pusillum L. (Geranium pusillum Burm.); Phleum pratense L.; Rumex crispus L.

For 10–19 years (from Madsen 1962): Plantago lanceolata L.

For 0–9 years (from Kjær 1948): Agrostemma githago L.; Centaurea cyanus L.; Dactylis glomerata L.; Geranium dissectum L.; Glebionis segetum (L.) Fourr. (Chrysanthemum segetum L.); Hordeum vulgare

L. subsp. distichon (L.) Körn. (Hordeum distichum L.); Lolium perenne L.; Scleranthus annuus L.; Triticum aestivum L. (Triticum sativum Lam.).

Germination outdoors, in the greenhouse and in the laboratory The results reported above agree in general with those listed in the literature. The difference between species is in general a combined effect of the seed coat structure and seed dormancy.

As a rule, dry stored seeds lose their germination capacity before those buried in soil (Madsen, 1962, Roberts and Patricia, 1975). The limited storability at room temperature is confirmed by many experiments, including the germination of 50- and 60-year-old Danish seed collections (Jensen, 1971, Buchwald and Jensen, 1974).

Germination in garden soil was the method assumed to be close to germination in the field. The germination results from 1935 to 1959 show, however, considerable variation between years (Madsen 1962), and due to this observation, the germinations from 1960 to 1982 were performed in a greenhouse. In some years, germination in an unheated greenhouse led to poorer results, which were most likely also due to less optimal germination conditions.

For future research, I recommend to wash out the buried seeds and germinate them in the laboratory. In an examination of the content of viable seeds in Danish arable soil, this method provided considerably higher results compared to both outdoor germination and germination in an unheated greenhouse (Jensen 1969, Jensen and Kjellsson 1995).

Activity of earthworms Germination tests of ancient archaeologically dated soil samples (of which some are several hundred years old) have proved that they can contain viable seeds of a number of species (Ødum 1965). Some authors raised the question whether in such studies earthworms may have moved seed from the surface to the examined soil layers and thus affected the results (Godwin 1968, Baker 1989), and McRill and Sagar (1973), McRill (1974) found that earthworms eat and transport seeds. The role of earthworms in the present study is therefore considered.

In some of the buried pots, I observed activity of earthworms. It is likely that they removed some seeds from the clay pots, but the size of this activity is unknown. Transport of living seeds by earthworms to the pots is unlikely, as the surface of the garden soil was free of weeds. Furthermore, the numbers on the clay pots allowed verification of the species planted in 1934, and thus any germinated seeds not originally planted in the pot could be discarded.

Remains of seeds after 49 years burial in soil According to Table 2, washing soil samples on a 0.4 mm sieve disclosed the remains of seeds and fruits of only five species (six samples). After 49 years of burial in arable soil, we found no remains of the other 22 species (27 samples) mentioned in Table 1. This is important when discussing archaeological findings of macrofossils in arable soil. Previously, a close relation was demonstrated between the media examined (grain storage, burnt house, wet areas) and the finds of macrofossils (Jensen 1987b).

If the washing method is used in future burial experiments, valuable information can be available for the evaluation of archaeologically dated macrofossils.

The results are also relevant for examination of the seed bank in arable soil. The numbers of living and dead seeds in the seed bank at the time of sampling are, as suggested by Cavers and Benoit (1989), Jensen (1987b), Roberts (1970), under continuous influence by various factors.

Factors adding to the seed bank:

– seeds left from previous years;

– seeds produced at the site in a given year;

– seeds transported to the site by wind, water, animals, by sowing crop plants, manure, tools etc.

Factors subtracting from the seed bank:

– germinated seeds;

– seeds removed by wind, water, animals, harvest of crops, tools;

– seeds decomposed in the soil.

Dead seeds of Chenopodium album, found in large numbers in this experiment, were in most cases fragile, empty and without visible outside damage. Similar empty seeds of e.g. Chenopodium album and Stellaria media are frequent in archaeologically dated soil samples (Jensen 1986). This suggests that both the microfauna and microflora are active in the decomposing seeds in soil (Chee-Sanford and Fu, 2010).

McRill and Sagar (1973) and McRill (1974) report on earthworms and Martinková et al. 2006) on ground beetles as active in transporting and eating buried seeds.

Accordingly, I suggest that these soil organisms add to the factors which reduce the densities of seeds in soil.

Summary This article presents the burial experiment at the Danish State Seed Testing Station from 1934 to 1983, and specifically the germination results from 1961 to 1983. K. Dorph-Petersen buried 37 samples of 29 species in clay pots, 20 cm below the soil surface. After 49 years, two samples of Chenopodium album germinated at 22 % and 4 %. Seeds of eight species died after 40-49 years, of six after 30-39 years, of four after 20-29 years, and of one after 10–19 years, and nine species lived less than 10 years. In 29 seed samples stored in soil for 49 years, washing on a 0.4 mm sieve revealed 326 and 242 seeds of Chenopodium album (in 2 samples), two seeds of Papaver rhoeas, and one seed each of Brassica napus, Brassica rapa, and Vicia hirsuta of the original 400 seeds buried. No seeds remained of the other samples, an important information for archaeological botany. The roles of earthworms, beetles and the microflora in depleting the seed bank in arable soil are briefly discussed.

Acknowledgements Sincere thanks for valuable comments to the Head of Department Grethe Tarp, Dr. Johannes Jørgensen and Dr. Sabine Karg.

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