Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost Svetlana Yashina,Stanislav Gubin,Stanislav Maksimovich,Alexandra Yashina,Edith Gakhova, and David Gilichinskyb.2 Institutes of Cell Biophysics and PPhysicochemical and Biological Problems in Soil Science,Russian Academy of Sciences,Pushchino 142290,Russia Edited*by P.Buford Price,University of California,Berkeley,CA,and approved January 25,2012(received for review November 8,2011) Whole,fertile plants of Silene stenophylla Ledeb.(Caryophylla- However,to date,no viable flowering plant remains have been ceae)have been uniquely regenerated from maternal,immature discovered from these ancient permafrost sediments. fruit tissue of Late Pleistocene age using in vitro tissue culture and Outside the permafrost zone the longevity of seeds in soil has clonal micropropagation.The fruits were excavated in northeast- been studied during the last 45 y in many places,including ar- ern Siberia from fossil squirrel burrows buried at a depth of 38 m chaeological sites (10,11).At the moment,the oldest viable in undisturbed and never thawed Late Pleistocene permafrost seeds with the ability to germinate were radiocarbon dated to the sediments with a temperature of-7 C.Accelerator mass spec- first and eighth centuries of the Common Era.These are,re- trometry (AMS)radiocarbon dating showed fruits to be 31,800+ spectively,Phoenix dactylifera found near the Dead Sea (12)and 300 y old.The total y-radiation dose accumulated by the fruits Nelumbo nucifera found in northeastern China (13). during this time was calculated as 0.07 kGy;this is the maximal The burrows from which our study material derived were buried reported dose after which tissues remain viable and seeds still in permanently frozen loess-ice deposits on the right bank of lower germinate.Regenerated plants were brought to flowering and Kolyma River,northeastern Siberia(14).These deposits(Fig.14) fruiting and they set viable seeds.At present,plants of S.steno- are representative of a widely distributed,so-called Late Pleisto- phylla are the most ancient,viable,multicellular,living organisms. cene ice complex found throughout the eastern Arctic,which has Morphophysiological studies comparing regenerated and extant continuously accumulated during the last 60 ky.This complex plants obtained from modern seeds of the same species in the comprises icy silts with a network of large syngenetic polygonal ice same region revealed that they were distinct phenotypes of wedges and is richly fossil bearing (pollen,insects,plants,mac- S.stenophylla.The first generation cultivated from seeds obtained rofossil,and mammal fossils).The studied burrows (Fig.1B)were from regenerated plants progressed through all developmental found in three exposures:Zelyony Mys,Stanchikovsky Yar,and stages and had the same morphological features as parent plants. Duvanny Yar.The last represents a key cross-section of Late The investigation showed high cryoresistance of plant placental Quaternary environmental history in the eastern Arctic studied by tissue in permafrost.This natural cryopreservation of plant tissue many scientists.All burrows were found at depths of 20-40 m from over many thousands of years demonstrates a role for permafrost the present day surface and located in layers containing bones of as a depository for an ancient gene pool,i.e.,preexisting life, large mammals such as mammoth,wooly rhinoceros,bison,horse, which hypothetically has long since vanished from the earth's sur- deer,and other representatives of fauna from the age of Mam- face,a potential source of ancient germplasm,and a laboratory for moths as well as plant remnants.The deposits were formed under the study of rates of microevolution tundra-steppe conditions.The Late Pleistocene age marine iso- tope stage 3(MIS 3),a period between 60 and 27 ky ago during the plants of Late Quaternary Beringia ice complex paleoenvironment last glacial cycle of these layers,was previously confirmed by ra- ancient genetic resources natural cryobank diocarbon,oxygen-isotope,and palynological analyses of plant and bone remnants (15,16).The sediments have high ice content he long-term conservation of viable biological material is an (35-80%).The present day mean annual ground temperature on important scientific challenge.Low and extreme low tem- the studied watershed is-7C,and the depth of seasonal thawing perature preservation is often used (1-3)and the most wide- ranges,depending on the landscape features,from 40 to 70 cm. spread natural subzero depository,permafrost (~20%of the The ice complexes formed immediately on sedimentation,with earth's surface),is now under extensive investigation.This con- concurrent freezing from below and have remained undisturbed siderable frozen mass,up to several hundred meters deep,har- since their formation.No events of permafrost degradation were bors a vast variety of viable ecological and morphological observed in this continuous Late Pleistocene sequence.The microbial groups:anaerobic and aerobic,spore-forming and non- presence of vertical ice wedges demonstrates that it has been spore-forming bacteria,green algae and cyanobacteria,yeast. continuously frozen and never thawed.Accordingly,the fossil actino-and micromycetes,and protozoa(4,5).All have survived burrows and their content have never been defrosted since burial under permafrost conditions since the time of its formation. and simultaneous freezing (17) The age of retrieved and cultivated biota corresponds to the Seventy fossil burrows were found and more than 30 of them longevity of the permanently frozen state of the embedding have been investigated so far.The plant material from fossil strata and dates back tens to hundreds of thousands of years and burrows has been dated by the radiocarbon method as being 28 even more.Permafrost thawing due to anthropogenic or natural 32 ky old(Table 1).During burrow construction by animals,the impacts exposes relict life to modern ecosystems where the an- chambers with seed and fruit storage were built against the cient biota may resume physiological activity and may become frozen boundary of the permafrost sediments.This feature of part of present-day biological systems and processes.Micro- organisms and their metabolic end products,the pigments chlorophyll and phaeophytin,biologically active free intra-and Author contributions:S.Y,E.G.,and D.G.designed research;S.Y.,S.G.,S.M.,and AY. extracellular enzymes,biogenic gases and bacterial,archaeal, performed research;S.Y.and D.G.analyzed data;and S.Y.S.G.,and D.G.wrote the paper and fungal DNA occur in permafrost (6-8).Permanently frozen The authors declare no conflict of interest. deposits also contain ancient evidence of higher plants,e.g.. *This Direct Submission article had a prearranged editor. DNA preserved from 10 to 400 kya,for which plant DNA To whom correspondence should be addressed.E-mail:yashina@psn.ru. sequences are known (9)as well as viable spores of moss species. Deceased February 18,2012. 4008-4013|PWA5|March6,2012|vol.109|no.10 www.pnas.org/cgi/doi/10.1073/pnas.1118386109
Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost Svetlana Yashinaa,1, Stanislav Gubinb , Stanislav Maksimovichb , Alexandra Yashinaa , Edith Gakhovaa , and David Gilichinskyb,2 Institutes of a Cell Biophysics and b Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino 142290, Russia Edited* by P. Buford Price, University of California, Berkeley, CA, and approved January 25, 2012 (received for review November 8, 2011) Whole, fertile plants of Silene stenophylla Ledeb. (Caryophyllaceae) have been uniquely regenerated from maternal, immature fruit tissue of Late Pleistocene age using in vitro tissue culture and clonal micropropagation. The fruits were excavated in northeastern Siberia from fossil squirrel burrows buried at a depth of 38 m in undisturbed and never thawed Late Pleistocene permafrost sediments with a temperature of −7 °C. Accelerator mass spectrometry (AMS) radiocarbon dating showed fruits to be 31,800 ± 300 y old. The total γ-radiation dose accumulated by the fruits during this time was calculated as 0.07 kGy; this is the maximal reported dose after which tissues remain viable and seeds still germinate. Regenerated plants were brought to flowering and fruiting and they set viable seeds. At present, plants of S. stenophylla are the most ancient, viable, multicellular, living organisms. Morphophysiological studies comparing regenerated and extant plants obtained from modern seeds of the same species in the same region revealed that they were distinct phenotypes of S. stenophylla. The first generation cultivated from seeds obtained from regenerated plants progressed through all developmental stages and had the same morphological features as parent plants. The investigation showed high cryoresistance of plant placental tissue in permafrost. This natural cryopreservation of plant tissue over many thousands of years demonstrates a role for permafrost as a depository for an ancient gene pool, i.e., preexisting life, which hypothetically has long since vanished from the earth’s surface, a potential source of ancient germplasm, and a laboratory for the study of rates of microevolution. plants of Late Quaternary | Beringia ice complex | paleoenvironment | ancient genetic resources | natural cryobank The long-term conservation of viable biological material is an important scientific challenge. Low and extreme low temperature preservation is often used (1–3) and the most widespread natural subzero depository, permafrost (∼20% of the earth’s surface), is now under extensive investigation. This considerable frozen mass, up to several hundred meters deep, harbors a vast variety of viable ecological and morphological microbial groups: anaerobic and aerobic, spore-forming and non– spore-forming bacteria, green algae and cyanobacteria, yeast, actino- and micromycetes, and protozoa (4, 5). All have survived under permafrost conditions since the time of its formation. The age of retrieved and cultivated biota corresponds to the longevity of the permanently frozen state of the embedding strata and dates back tens to hundreds of thousands of years and even more. Permafrost thawing due to anthropogenic or natural impacts exposes relict life to modern ecosystems where the ancient biota may resume physiological activity and may become part of present-day biological systems and processes. Microorganisms and their metabolic end products, the pigments chlorophyll and phaeophytin, biologically active free intra- and extracellular enzymes, biogenic gases and bacterial, archaeal, and fungal DNA occur in permafrost (6–8). Permanently frozen deposits also contain ancient evidence of higher plants, e.g., DNA preserved from 10 to 400 kya, for which plant DNA sequences are known (9) as well as viable spores of moss species. However, to date, no viable flowering plant remains have been discovered from these ancient permafrost sediments. Outside the permafrost zone the longevity of seeds in soil has been studied during the last 45 y in many places, including archaeological sites (10, 11). At the moment, the oldest viable seeds with the ability to germinate were radiocarbon dated to the first and eighth centuries of the Common Era. These are, respectively, Phoenix dactylifera found near the Dead Sea (12) and Nelumbo nucifera found in northeastern China (13). The burrows from which our study material derived were buried in permanently frozen loess-ice deposits on the right bank of lower Kolyma River, northeastern Siberia (14). These deposits (Fig. 1A) are representative of a widely distributed, so-called Late Pleistocene ice complex found throughout the eastern Arctic, which has continuously accumulated during the last 60 ky. This complex comprises icy silts with a network of large syngenetic polygonal ice wedges and is richly fossil bearing (pollen, insects, plants, macrofossil, and mammal fossils). The studied burrows (Fig. 1B) were found in three exposures: Zelyony Mys, Stanchikovsky Yar, and Duvanny Yar. The last represents a key cross-section of Late Quaternary environmental history in the eastern Arctic studied by many scientists. All burrows were found at depths of 20–40 m from the present day surface and located in layers containing bones of large mammals such as mammoth, wooly rhinoceros, bison, horse, deer, and other representatives of fauna from the age of Mammoths as well as plant remnants. The deposits were formed under tundra–steppe conditions. The Late Pleistocene age marine isotope stage 3 (MIS 3), a period between 60 and 27 ky ago during the last glacial cycle of these layers, was previously confirmed by radiocarbon, oxygen-isotope, and palynological analyses of plant and bone remnants (15, 16). The sediments have high ice content (35–80%). The present day mean annual ground temperature on the studied watershed is −7 °C, and the depth of seasonal thawing ranges, depending on the landscape features, from 40 to 70 cm. The ice complexes formed immediately on sedimentation, with concurrent freezing from below and have remained undisturbed since their formation. No events of permafrost degradation were observed in this continuous Late Pleistocene sequence. The presence of vertical ice wedges demonstrates that it has been continuously frozen and never thawed. Accordingly, the fossil burrows and their content have never been defrosted since burial and simultaneous freezing (17). Seventy fossil burrows were found and more than 30 of them have been investigated so far. The plant material from fossil burrows has been dated by the radiocarbon method as being 28– 32 ky old (Table 1). During burrow construction by animals, the chambers with seed and fruit storage were built against the frozen boundary of the permafrost sediments. This feature of Author contributions: S.Y., E.G., and D.G. designed research; S.Y., S.G., S.M., and A.Y. performed research; S.Y. and D.G. analyzed data; and S.Y., S.G., and D.G. wrote the paper. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. 1 To whom correspondence should be addressed. E-mail: yashina@psn.ru. 2 Deceased February 18, 2012. 4008–4013 | PNAS | March 6, 2012 | vol. 109 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1118386109
Fig.1.Late Pleistocene loess-ice deposits of Duvanny Yar,Kolyma Lowland.(A)General view.(B)Burrow of ground squirrel buried in permafrost deposits. storage chamber construction allowed high quality preservation water volume (21);this frozen environment is effectively a"bi- of their biological material. ologically dry"environment and favors conservation (22).There The storage chambers in the fossil burrows contain a great is only one previous report of plants (Lupinus arcticus)grown supply of plant seeds and fruits.The number of seeds and fruits from seeds deeply buried within lemming burrows in perma- reaches up to 600-800 thousand in some chambers. nently frozen silt in the central Yukon (23).However,after 40 y Direct in situ measurements of y-radiation were made in the these seeds were dated using the accelerator mass spectrometry borehole crossing the burrow buried horizon on Duvanny Yar. radiocarbon method (AMS)and it was concluded that they were The observation showed that in ice complex the ground radiation not of Pleistocene age (>10 ky old),but modern seeds that ap- level provided by natural radionuclides was 0.23 uGy/h or 2.01 parently contaminated the ancient sample (24). mGy/y on average.These data correlate well with an estimate of The aim of our studies was regeneration of plants from seeds ground radiation made using elemental analysis of the radioac- and fruits buried in the Late Pleistocene permafrost,28-32 ky tive elements in ice complex samples from other boreholes in this old and excavated from fossil burrows (Fig.1)of Urocitellus area:~2 mGy/y(18)and they are similar to the ground radiation paryii (an Arctic species of ground squirrel). levels reported for natural radionuclides surrounding the above- Previous studies(25)revealed the return of some physiological mentioned seeds of N.nucifera in China.In this case,the total activity to the cells and tissues of seeds and fruits of four plant y-radiation dose accumulated by 1,300-y-old fruits was calculated species from burrows in the permafrost deposits during in vitro at-3 Gy.On the basis of these data.the total y-radiation dose culture.Enlargement of the cotyledon was detected in a sedge accumulated by 28-to 32-ky-old fruits was calculated as 0.07 seed(Carex sp.,Cyperaceae)from burrow P-923 of Zelyony Mys kGy.This is now the maximal reported dose after which tissues locality (Table 1).Cell division activity was detected in several remain viable and seeds can still germinate. radicles of Arctous alpina (Ericaceae).The burrows P-1026 and Observations of seed viability in relation to environment show P-1068 (Stanchikovsky Yar exposure)with Arctous seeds are that their longevity increases as the seed storage temperature located at the same level and near burrow P-1010,for which and moisture decrease (19,20).This same situation is seen for radiocarbon analysis has been done.Radicle activation was also seed viability in the permafrost.First,at subzero temperatures detected in seeds of Rumex arcticus (Polygonaceae)from burrow the rates of biochemical reactions and biological processes be- P-1075.One Rumex seed germinated up to the cotyledon release come extremely slow and ensure preservation of the biological stage but the primary shoot did not develop because of shoot system.Second,in frozen ground,ice makes up 93-98%of total apical meristem degradation.The cotyledon explants isolated from this seedling were cultured on agar media and showed initiation of callusing but did not continue further.Eruptions of Table 1.Radiocarbon age of material taken from fossil burrows radicle and subsequent callusing occurred in vitro in Silene of Kolyma Lowland (Northern Yakutia) stenophylla Ledeb.seeds from burrow P-923 of the Zelyony Mys Cross-section Burrow Material Radiocarbon age,y exposure.However,further proliferation of callus tissue was aborted as was the case with R.arcticus.Primary root develop- Zelyony Mys P-923 Plant remnants 32,800±400 ment also stopped because of damaged embryo and storage tis- (IEMEZH 1178)* sues.Nevertheless,these preliminary results showed a real Zelyony Mys P-917 Plant remnants 30,500±700 possibility for obtaining material for whole plant regeneration. (IEMEZH 1179) In these earlier studies.we found that the seeds of S.sten- Stanchikovski Yar P.1010 Plant remnants 27,700±300 ophylla were the most promising for further investigation be- (GIN10874) cause they had some physiological vitality and viability.Seeds of Stanchikovski Yar P-820 Seeds and fruits 28,200±600 this species are dark in color and relatively small (0.6 mm wide. (IEMEZH 1190) 0.8 mm long).S.stenophylla is a perennial herbaceous plant from Duvanny Yar P.1075 Seeds and fruits 31,800±310 the family Caryophyllaceae.Seeds of wild species of this family (Beta157195) demonstrated high resistance to both nondeep and deep freezing *The laboratory number of the sample given by the institute that carried out (26).Abundant and numerous seeds and fruits of S.stenophylla the analysis is indicated in parentheses.IEMEZH,transliteration from the were found in most of the burrows.Many experiments were Russian abbreviation of the former name of the Institute of Ecology and carried out with seeds and with both open(ripe)and closed Evolution,Russian Academy of Sciences;GIN,Geological Institute,Russian (unripe)fruits of this species.Seeds taken from immature fruits Academy of Sciences;Beta,Beta Analytic. often had a funiculus,a seedstalk,by which the seed was Yashina et al PNAS|March6,2012|vol.1091no.10|4009
storage chamber construction allowed high quality preservation of their biological material. The storage chambers in the fossil burrows contain a great supply of plant seeds and fruits. The number of seeds and fruits reaches up to 600–800 thousand in some chambers. Direct in situ measurements of γ-radiation were made in the borehole crossing the burrow buried horizon on Duvanny Yar. The observation showed that in ice complex the ground radiation level provided by natural radionuclides was 0.23 μGy/h or 2.01 mGy/y on average. These data correlate well with an estimate of ground radiation made using elemental analysis of the radioactive elements in ice complex samples from other boreholes in this area: ∼2 mGy/y (18) and they are similar to the ground radiation levels reported for natural radionuclides surrounding the abovementioned seeds of N. nucifera in China. In this case, the total γ-radiation dose accumulated by 1,300-y-old fruits was calculated at −3 Gy. On the basis of these data, the total γ-radiation dose accumulated by 28- to 32-ky-old fruits was calculated as 0.07 kGy. This is now the maximal reported dose after which tissues remain viable and seeds can still germinate. Observations of seed viability in relation to environment show that their longevity increases as the seed storage temperature and moisture decrease (19, 20). This same situation is seen for seed viability in the permafrost. First, at subzero temperatures the rates of biochemical reactions and biological processes become extremely slow and ensure preservation of the biological system. Second, in frozen ground, ice makes up 93–98% of total water volume (21); this frozen environment is effectively a “biologically dry” environment and favors conservation (22). There is only one previous report of plants (Lupinus arcticus) grown from seeds deeply buried within lemming burrows in permanently frozen silt in the central Yukon (23). However, after 40 y these seeds were dated using the accelerator mass spectrometry radiocarbon method (AMS) and it was concluded that they were not of Pleistocene age (>10 ky old), but modern seeds that apparently contaminated the ancient sample (24). The aim of our studies was regeneration of plants from seeds and fruits buried in the Late Pleistocene permafrost, 28–32 ky old and excavated from fossil burrows (Fig. 1) of Urocitellus parryii (an Arctic species of ground squirrel). Previous studies (25) revealed the return of some physiological activity to the cells and tissues of seeds and fruits of four plant species from burrows in the permafrost deposits during in vitro culture. Enlargement of the cotyledon was detected in a sedge seed (Carex sp., Cyperaceae) from burrow P-923 of Zelyony Mys locality (Table 1). Cell division activity was detected in several radicles of Arctous alpina (Ericaceae). The burrows P-1026 and P-1068 (Stanchikovsky Yar exposure) with Arctous seeds are located at the same level and near burrow P-1010, for which radiocarbon analysis has been done. Radicle activation was also detected in seeds of Rumex arcticus (Polygonaceae) from burrow P-1075. One Rumex seed germinated up to the cotyledon release stage but the primary shoot did not develop because of shoot apical meristem degradation. The cotyledon explants isolated from this seedling were cultured on agar media and showed initiation of callusing but did not continue further. Eruptions of radicle and subsequent callusing occurred in vitro in Silene stenophylla Ledeb. seeds from burrow P-923 of the Zelyony Mys exposure. However, further proliferation of callus tissue was aborted as was the case with R. arcticus. Primary root development also stopped because of damaged embryo and storage tissues. Nevertheless, these preliminary results showed a real possibility for obtaining material for whole plant regeneration. In these earlier studies, we found that the seeds of S. stenophylla were the most promising for further investigation because they had some physiological vitality and viability. Seeds of this species are dark in color and relatively small (0.6 mm wide, 0.8 mm long). S. stenophylla is a perennial herbaceous plant from the family Caryophyllaceae. Seeds of wild species of this family demonstrated high resistance to both nondeep and deep freezing (26). Abundant and numerous seeds and fruits of S. stenophylla were found in most of the burrows. Many experiments were carried out with seeds and with both open (ripe) and closed (unripe) fruits of this species. Seeds taken from immature fruits often had a funiculus, a seedstalk, by which the seed was Fig. 1. Late Pleistocene loess-ice deposits of Duvanny Yar, Kolyma Lowland. (A) General view. (B) Burrow of ground squirrel buried in permafrost deposits. Table 1. Radiocarbon age of material taken from fossil burrows of Kolyma Lowland (Northern Yakutia) Cross-section Burrow Material Radiocarbon age, y Zelyony Mys Р-923 Plant remnants 32,800 ± 400 (IEMEZH 1178)* Zelyony Mys Р-917 Plant remnants 30,500 ± 700 (IEMEZH 1179) Stanchikovski Yar Р-1010 Plant remnants 27,700 ± 300 (GIN 10874) Stanchikovski Yar Р-820 Seeds and fruits 28,200 ± 600 (IEMEZH 1190) Duvanny Yar Р-1075 Seeds and fruits 31,800 ± 310 (Beta 157195) *The laboratory number of the sample given by the institute that carried out the analysis is indicated in parentheses. IEMEZH, transliteration from the Russian abbreviation of the former name of the Institute of Ecology and Evolution, Russian Academy of Sciences; GIN, Geological Institute, Russian Academy of Sciences; Beta, Beta Analytic. Yashina et al. PNAS | March 6, 2012 | vol. 109 | no. 10 | 4009 PLANT BIOLOGY
attached to the placenta.These funiculi showed tissue enlarge- 2).As is typical for S.stenophylla as a perennial plant,flowering ment in vitro(27)and,importantly,these observations led us to and seed set occurred during the second year of the potted focus on investigations of immature fruit tissues of ancient Silene. plants'cultivation. Here,we describe the regeneration of whole plants from pla- Thirty-six ancient plants (12 from each fruit)and 29 extant cental tissue of such fruits. plants were morphologically tested.All ancient plants were morphologically identical.During vegetative development,the Results ancient and extant plants were morphologically indistinguishable Several immature fruits were found at a depth of 38 m in a seed from one another.However,at the flowering stage they showed conglomerate from the Duvanny Yar burrow P-1075 (Table 1). different corolla shape:petals of extant flowers were obviously Silene seeds and fruits were dominant in this burrow and were in wider and more dissected (Fig.3).Moreover,all flowers of the a state of good morphological preservation.AMS radiocarbon extant plants were bisexual (b)(Fig.34),whereas the primary dating showed them to be 31,800+300 y old (Beta-157195). flowers (two to three in number)of each ancient plant were strictly female (f)(Fig.3 B and C,f),and then bisexual flowers Organogenesis of Shoots and Microclonal Propagation.Our in vitro were formed on each ancient plant (Fig.3C,b). tissue culture method was adopted from one for the regeneration of ancient plants(28,29).Organogenesis of adventitious shoots Seed Production.The plants were tested for their sexual fertility. was induced in vitro directly from fragments of the placental It should be noted that S.stenophylla is allogamous and requires tissue of three immature uninjured fruits.A modification of cross-fertilization for sexual reproduction to occur.Flowers of Murashige and Skoog (30)and Anderson (31)nutrient media the ancient plants were pollinated artificially using pollen from was used for initiation of organogenesis and multiplication of other ancient plants;pollination of extant plants was performed shoots.Apart from ancient plants,those from extant seeds of the similarly.The time from artificial pollination of flowers to rip- same species,and from the same region,were grown in vitro as ening of first seeds took 8-9 wk.Laboratory germination of seeds a control.During micropropagation in vitro the ancient and taken from regenerated ancient plants was 100%and that of extant plants were different,with the ancient plants producing up seeds from control plants was 86-90%.The first generation to 1.5-2 times more buds,whereas the extant plants produced cultivated from seeds obtained from regenerated plants pro- roots more rapidly gressed through all developmental stages and showed the same morphological features as the parent plants.The taxonomic Vegetative Growth and Flowering.Rooted plants were transplanted identification of both ancient and extant regenerated plants was to plastic pots with appropriate soil and placed in a growth room verified at the Department of Higher Plants,Moscow State with controlled light and temperature.Under these conditions, University,Moscow,Russia.The ancient plants were considered plants grew and developed flowers and fruits and set seeds(Fig. to be a distinct phenotype from the extant plants. Discussion The present study uniquely demonstrates that viable placental tissue,from immature fruits of the flowering plant S.stenophylla B Fig.3.Flowering plants of Silene stenophylla.(A)Plant grown in vitro culture from seed of an extant plant.(B)Plant regenerated in vitro culture from tissue of fossil fruit with primary strictly female flower.(C)Plant Fig.2.Fruiting plants of Silene stenophylla regenerated from tissue of regenerated from tissue of fossil fruit with both female (f)and bisexual(b) fossil fruits.(Scale bar,50 mm.) flowers. 4010ww.pnas.org/cgi/doi/10.1073pna5.1118386109 Yashina et al
attached to the placenta. These funiculi showed tissue enlargement in vitro (27) and, importantly, these observations led us to focus on investigations of immature fruit tissues of ancient Silene. Here, we describe the regeneration of whole plants from placental tissue of such fruits. Results Several immature fruits were found at a depth of 38 m in a seed conglomerate from the Duvanny Yar burrow P-1075 (Table 1). Silene seeds and fruits were dominant in this burrow and were in a state of good morphological preservation. AMS radiocarbon dating showed them to be 31,800 ± 300 y old (Beta-157195). Organogenesis of Shoots and Microclonal Propagation. Our in vitro tissue culture method was adopted from one for the regeneration of ancient plants (28, 29). Organogenesis of adventitious shoots was induced in vitro directly from fragments of the placental tissue of three immature uninjured fruits. A modification of Murashige and Skoog (30) and Anderson (31) nutrient media was used for initiation of organogenesis and multiplication of shoots. Apart from ancient plants, those from extant seeds of the same species, and from the same region, were grown in vitro as a control. During micropropagation in vitro the ancient and extant plants were different, with the ancient plants producing up to 1.5–2 times more buds, whereas the extant plants produced roots more rapidly. Vegetative Growth and Flowering. Rooted plants were transplanted to plastic pots with appropriate soil and placed in a growth room with controlled light and temperature. Under these conditions, plants grew and developed flowers and fruits and set seeds (Fig. 2). As is typical for S. stenophylla as a perennial plant, flowering and seed set occurred during the second year of the potted plants’ cultivation. Thirty-six ancient plants (12 from each fruit) and 29 extant plants were morphologically tested. All ancient plants were morphologically identical. During vegetative development, the ancient and extant plants were morphologically indistinguishable from one another. However, at the flowering stage they showed different corolla shape: petals of extant flowers were obviously wider and more dissected (Fig. 3). Moreover, all flowers of the extant plants were bisexual (b) (Fig. 3A), whereas the primary flowers (two to three in number) of each ancient plant were strictly female (f) (Fig. 3 B and C, f), and then bisexual flowers were formed on each ancient plant (Fig. 3C, b). Seed Production. The plants were tested for their sexual fertility. It should be noted that S. stenophylla is allogamous and requires cross-fertilization for sexual reproduction to occur. Flowers of the ancient plants were pollinated artificially using pollen from other ancient plants; pollination of extant plants was performed similarly. The time from artificial pollination of flowers to ripening of first seeds took 8–9 wk. Laboratory germination of seeds taken from regenerated ancient plants was 100% and that of seeds from control plants was 86–90%. The first generation cultivated from seeds obtained from regenerated plants progressed through all developmental stages and showed the same morphological features as the parent plants. The taxonomic identification of both ancient and extant regenerated plants was verified at the Department of Higher Plants, Moscow State University, Moscow, Russia. The ancient plants were considered to be a distinct phenotype from the extant plants. Discussion The present study uniquely demonstrates that viable placental tissue, from immature fruits of the flowering plant S. stenophylla Fig. 2. Fruiting plants of Silene stenophylla regenerated from tissue of fossil fruits. (Scale bar, 50 mm.) Fig. 3. Flowering plants of Silene stenophylla. (A) Plant grown in vitro culture from seed of an extant plant. (B) Plant regenerated in vitro culture from tissue of fossil fruit with primary strictly female flower. (C) Plant regenerated from tissue of fossil fruit with both female (f) and bisexual (b) flowers. 4010 | www.pnas.org/cgi/doi/10.1073/pnas.1118386109 Yashina et al
is preserved in Late Pleistocene sediments.These sediments date placenta is involved in the regulation and transport of nutrient between 30,000 and 32,000 y in age and were deposited in an substances synthesized in its own tissues as well as in other tis- undisturbed permafrost environment.Under tissue culture and sues of a plant.The high concentration of organic substances, micropropagation,the placental tissue was able to differentiate including sucrose,responsible for the cryoresistance,is the likely and grow to become fertile adult plants reason that allowed high-quality preservation of fruits and their The principal points of discussion in support of this discovery contents in permafrost.The morphogenic factor,composed of are:The provenance and age of the fruits,the basis for preser- active phenolic compounds,is localized in the placenta (33). vation,the resilience of placental tissue,phenotypic plasticity, Phenolic compounds,accumulating in plant tissues under low and the opportunities provided by permafrost environments for temperatures,are well known for their protective function and preserving ancient and modern germplasm of higher plants. may be produced as a stress response.The high morphogenic Provenance.The fruits,from which placental tissue was obtained. potential of placental tissue of the fossil fruits observed here came from similar and immediately adjacent ice conglomerates may,therefore,result from special metabolic properties not only that were used to establish the age of the deposits.We dated the of the placental tissue itself but of the whole organism,which age of the seeds from the closed fruits and regenerated the plants developed under the cold,dry climatic conditions of Late from the placental tissue to which these very seeds were attached Pleistocene.Well-preserved placentae were observed only in in the process of their formation.Thus,we ensured "direct fruits with intact and undamaged pericarp.The good preserva- radiocarbon dating. tion of placenta is probably further due to the hermetic con- In view of the Porsild et al.(23)paper on seeds of Lupinus ditions in the immature fruit.Finally,immature tissues and from the Yukon and its refutation by Zazula et al.(24),showing organs are known to be more regenerative in vitro than mature that contamination of old seed by recent seed had occurred in tissues and organs (29). lemming burrows within Pleistocene deposits,we were careful to assess the possibility that this may have happened with our ma- Phenotypic Plasticity.Phenotypic plasticity,as a response to terial.We have high confidence that no contamination took temperature conditions and photoperiod,has been reported for place after the onset of freezing.The fossil burrows are well other members of Caryophyllaceae(34,35).The genotype of the below the seasonal thawing (active)layer and more than 35 m ancient plants may be different from that of the modern plants below the permafrost table.Further,we know of no animals that and may have developed under the severe climatic conditions of burrow deeper than the active layer.The permafrost table is the Late Pleistocene.The revealing of sexual dimorphism of ancient upper boundary of deep sediments within which physical and plants may also indicate that ontogeny of paleo-and extant biogeochemical exchanges occur and through which external plants were formed under different ecological conditions (36). factors and processes do not cross.The embedding over-and underlying sediments are firmly cemented together and are often Significance.Naturally occurring permanently frozen sediments totally filled in by ice.These sediments represent a closed system offer an important opportunity for the discovery of wild plant where there are no water-bearing horizons or water infiltration. species,preservation of biological material,studying the con- Thus,within the permafrost sediments the penetration of seeds ditions for cryopreservation,and developing germplasm collec- to the deeper frozen layers could not have occurred from out- tions.We consider it essential to continue permafrost studies in side;they exist in situ.Further,penetration along pores can also search of an ancient genetic pool,that of preexisting life,which be virtually ruled out because the diameter of the average pore is hypothetically has long since vanished from the earth's surface less than 0.1 mm,compared with seed diameters of 0.6-0.8 mm and diameters of 2-4 mm for fruits. Conclusion Late Pleistocene plant tissue of S.stenophylla,naturally pre- Basis for Preservation.The food storage chambers of squirrels are served in permafrost,can be regenerated using tissue culture and built adjacent to ice wedges and frozen sediment.The material micropropagation to form healthy sexually reproducing plants. within burrows and the embedding strata provide important ev- The source of the ancient tissue is the placentae of immature S. idence of rapid freezing and subsequent preservation without stenophylla fruits found in buried squirrel caches.At present, defrosting.Gubin et al.(17)have demonstrated that studied plants of S.stenophylla are the unique representative of ancient individual burrows have only a single entrance at ground level higher plants to be cultivated successfully.Their high cryore and the fossil burrows were sealed by loess and became rapidly sistance over many thousands of years demonstrates the value of and permanently frozen.As cited in the Introduction,it is well permafrost sediments as a cryodepository,a potential source of known that representatives of nearly all single-cell and lower ancient germplasm,and a natural laboratory for studies of cry- plant biota can survive over long geological periods at subzero popreservation of ancient genetic resources and rates of micro- temperatures and biologically dry milieu of the permafrost.The evolution.In this sense,development of the Svalbard Global long-term subzero temperature regime of the permafrost pre- serves living tissue through stabilization via dehydration.which Seed Vault(Spits Bergen,Norway)is of great interest and im- leads to a considerable decrease of biochemical and metabolic portance.Duplicates of seeds of cultivated plants from different activities.Thus,it is reasonable to expect that higher plant tis- germplasm collections are to be stored in this institution (www. sues,especially those associated with reproductive processes,can nordgen.org/sgsv/). be expected to survive better within the permafrost sediments The squirrel burrows with seeds have been identified within than in any other known habitat.It is also possible that low doses Late Pleistocene ice complex not only in eastern Siberia,but also of radiation also might have an effect on the viability of bi- in Alaska and Yukon (37,38).This indicates that the whole ological objects (13,32) Beringia has a great potential as storage of ancient life preserved in permafrost.Further investigations of morphological,func- Tissue Resilience.Among the various approaches used to seek the tional,biochemical,and cytogenetic features of regenerated viability of seed and fruit material in this study,only the placental plants,as well as their seminal progeny,and comparison with tissues from immature fruits had sufficient vitality to be cultured extant species will allow us to obtain new data on the mecha- and grown to whole,healthy,fertile adult plants.An explanation nisms of cryoresistance of plant cells,their adaptation potential, for the success with placental tissue may lie with the high met- and additional information about environmental conditions of abolic activity of these tissues as reported by Pontovich (33).The Late Pleistocene. Yashina et al PNAS|March6.2012|vol.1091no.10|4011
is preserved in Late Pleistocene sediments. These sediments date between 30,000 and 32,000 y in age and were deposited in an undisturbed permafrost environment. Under tissue culture and micropropagation, the placental tissue was able to differentiate and grow to become fertile adult plants. The principal points of discussion in support of this discovery are: The provenance and age of the fruits, the basis for preservation, the resilience of placental tissue, phenotypic plasticity, and the opportunities provided by permafrost environments for preserving ancient and modern germplasm of higher plants. Provenance. The fruits, from which placental tissue was obtained, came from similar and immediately adjacent ice conglomerates that were used to establish the age of the deposits. We dated the age of the seeds from the closed fruits and regenerated the plants from the placental tissue to which these very seeds were attached in the process of their formation. Thus, we ensured “direct radiocarbon dating.” In view of the Porsild et al. (23) paper on seeds of Lupinus from the Yukon and its refutation by Zazula et al. (24), showing that contamination of old seed by recent seed had occurred in lemming burrows within Pleistocene deposits, we were careful to assess the possibility that this may have happened with our material. We have high confidence that no contamination took place after the onset of freezing. The fossil burrows are well below the seasonal thawing (active) layer and more than 35 m below the permafrost table. Further, we know of no animals that burrow deeper than the active layer. The permafrost table is the upper boundary of deep sediments within which physical and biogeochemical exchanges occur and through which external factors and processes do not cross. The embedding over- and underlying sediments are firmly cemented together and are often totally filled in by ice. These sediments represent a closed system where there are no water-bearing horizons or water infiltration. Thus, within the permafrost sediments the penetration of seeds to the deeper frozen layers could not have occurred from outside; they exist in situ. Further, penetration along pores can also be virtually ruled out because the diameter of the average pore is less than 0.1 mm, compared with seed diameters of 0.6–0.8 mm and diameters of 2–4 mm for fruits. Basis for Preservation. The food storage chambers of squirrels are built adjacent to ice wedges and frozen sediment. The material within burrows and the embedding strata provide important evidence of rapid freezing and subsequent preservation without defrosting. Gubin et al. (17) have demonstrated that studied individual burrows have only a single entrance at ground level and the fossil burrows were sealed by loess and became rapidly and permanently frozen. As cited in the Introduction, it is well known that representatives of nearly all single-cell and lower plant biota can survive over long geological periods at subzero temperatures and biologically dry milieu of the permafrost. The long-term subzero temperature regime of the permafrost preserves living tissue through stabilization via dehydration, which leads to a considerable decrease of biochemical and metabolic activities. Thus, it is reasonable to expect that higher plant tissues, especially those associated with reproductive processes, can be expected to survive better within the permafrost sediments than in any other known habitat. It is also possible that low doses of radiation also might have an effect on the viability of biological objects (13, 32). Tissue Resilience. Among the various approaches used to seek the viability of seed and fruit material in this study, only the placental tissues from immature fruits had sufficient vitality to be cultured and grown to whole, healthy, fertile adult plants. An explanation for the success with placental tissue may lie with the high metabolic activity of these tissues as reported by Pontovich (33). The placenta is involved in the regulation and transport of nutrient substances synthesized in its own tissues as well as in other tissues of a plant. The high concentration of organic substances, including sucrose, responsible for the cryoresistance, is the likely reason that allowed high-quality preservation of fruits and their contents in permafrost. The morphogenic factor, composed of active phenolic compounds, is localized in the placenta (33). Phenolic compounds, accumulating in plant tissues under low temperatures, are well known for their protective function and may be produced as a stress response. The high morphogenic potential of placental tissue of the fossil fruits observed here may, therefore, result from special metabolic properties not only of the placental tissue itself but of the whole organism, which developed under the cold, dry climatic conditions of Late Pleistocene. Well-preserved placentae were observed only in fruits with intact and undamaged pericarp. The good preservation of placenta is probably further due to the hermetic conditions in the immature fruit. Finally, immature tissues and organs are known to be more regenerative in vitro than mature tissues and organs (29). Phenotypic Plasticity. Phenotypic plasticity, as a response to temperature conditions and photoperiod, has been reported for other members of Caryophyllaceae (34, 35). The genotype of the ancient plants may be different from that of the modern plants and may have developed under the severe climatic conditions of Late Pleistocene. The revealing of sexual dimorphism of ancient plants may also indicate that ontogeny of paleo- and extant plants were formed under different ecological conditions (36). Significance. Naturally occurring permanently frozen sediments offer an important opportunity for the discovery of wild plant species, preservation of biological material, studying the conditions for cryopreservation, and developing germplasm collections. We consider it essential to continue permafrost studies in search of an ancient genetic pool, that of preexisting life, which hypothetically has long since vanished from the earth’s surface. Conclusion Late Pleistocene plant tissue of S. stenophylla, naturally preserved in permafrost, can be regenerated using tissue culture and micropropagation to form healthy sexually reproducing plants. The source of the ancient tissue is the placentae of immature S. stenophylla fruits found in buried squirrel caches. At present, plants of S. stenophylla are the unique representative of ancient higher plants to be cultivated successfully. Their high cryoresistance over many thousands of years demonstrates the value of permafrost sediments as a cryodepository, a potential source of ancient germplasm, and a natural laboratory for studies of crypopreservation of ancient genetic resources and rates of microevolution. In this sense, development of the Svalbard Global Seed Vault (Spits Bergen, Norway) is of great interest and importance. Duplicates of seeds of cultivated plants from different germplasm collections are to be stored in this institution (www. nordgen.org/sgsv/). The squirrel burrows with seeds have been identified within Late Pleistocene ice complex not only in eastern Siberia, but also in Alaska and Yukon (37, 38). This indicates that the whole Beringia has a great potential as storage of ancient life preserved in permafrost. Further investigations of morphological, functional, biochemical, and cytogenetic features of regenerated plants, as well as their seminal progeny, and comparison with extant species will allow us to obtain new data on the mechanisms of cryoresistance of plant cells, their adaptation potential, and additional information about environmental conditions of Late Pleistocene. Yashina et al. PNAS | March 6, 2012 | vol. 109 | no. 10 | 4011 PLANT BIOLOGY
A Fig.4.Immature fruit of Silene stenophylla from burrow buried in permafrost more than 30,000 y ago.(A)Dissected fruit showing seeds and placenta(P).(B) Fragment of placenta with seeds at different developmental stages.(Scale bars,1 mm.) Materials and Methods phosphate and supplemented with ascorbic acid 2 mg glycine 2 mg1 Biological Samples.Many seeds and fruits from collection sites in permafrost myo-inositol 100 mg l-,adenine 30 mg-,kinetin 0.5 mg,BAP 1mg-1 have well preserved coats (testa and pericarp),which allow their taxonomic GA 2 mg I-,IAA 0.1 mg I-,1%sucrose,and vitamins (41). identification (39).Our collection of extant seeds and fruits of Kolyma lowland plants,along with seed collections of the Botanical Institute,Rus- Methods.Preparation of tissue samples for culture.Immature fruits were care- sian Academy of Sciences,St.Petersburg,Russia,were used for identification fully cleaned,washed with tap water,and successively surface sterilized with of ancient seeds.To date,38 species are identified.The identity of S.sten- 0.1%solution of corrosive sublimate(mercury bichloride HgCl2)with addition ophylla seeds was also confirmed by scanning electron microscopy using of Tween-80(0.4 mL in 1,000 mL of solution)and with 70%ethanol.The TESLA BS-300 (40). tissue was washed with sterile distilled water after each disinfectant.Right The accumulation of seeds and fruits in fossil burrows appeared as icy after disinfection,fruits were dissected under sterile conditions.A dissected, conglomerates.Seeds and fruits excavated from fossil burrows were imme- immature fruit of 5.stenophylla,having placental tissue and a fragment of diately transferred to sterile bags,kept frozen in the field,and transported the placenta retained with attached seeds at different developmental frozen to the laboratory at a temperature of-10C.Samples selected for the stages,is shown in Fig.4.Fragments of the placenta were cultivated in vitro study were defrosted and examined by light microscopy to select un. on media 1-3.Reagents were purchased from Sigma-Aldrich damaged firm seeds and fruits for investigation. Explant culture.Explants,isolated from placentae of three different immature fruits,were cultured on the three different media (1-3)described above. Media for Culture in Vitro.The following media were used:Medium 1: Organogenesis was observed on both rich-and poor-nutrient media.Suc- Murashige and Skoog basal nutrient medium(MS)rich in nutrients was cessful initiation of shoots on different media occurred,presumably due to supplemented with vitamins (41),ascorbic acid 2 mg I-,glycine 2 mg -1, high morphogenic and physiological potential of the placental tissue.The myo-inositol 100 mg-,casein hydrolysate 500 mg-,6-benzylaminopurine first shoot initiated from placental tissue is shown in Fig.5A.The first (BAP)2 mg I-,kinetin 1 mg I-,dichlorophenoxyacetic acid (2,4-D)2 mg I- formed shoots were etiolated as a result of being cultivated under dark gibberellic acid (GA)1 mg I,and 3%sucrose.Medium 2:same as medium 1, conditions.When the cultures were transferred to sufficient light,shoots to which 10%coconut milk was added.Medium 3:a nutrient-poor medium, synthesized chlorophyll and made active growth.After 5-20 d,numerous consisting of the basal MS medium with sucrose concentration reduced to adventitious shoots were being formed.On the poor medium,primary shoot 1%and supplemented only with kinetin (0.2 mg and indole-3-acetic acid initiation was retarded up to 7-9 d,compared with explants cultivated on (IAA)(0.1 mg I-).Modified Anderson basal nutrient medium (ABM)(31), both rich media(medium 1 and medium 2).Morphogenesis took place only with monobasic sodium phosphate replaced by monobasic potassium in placental tissues from unripe fruits. 6 B Fig.5.Clonal micropropagation of Silene stenophylla regenerated from placenta tissue of immature 30,000-y-old fruits buried in permafrost deposits.(A) Initial shoot initiated from placental tissue in vitro.(B)Stages of clonal micropropagation from primary shoots to rooted plants.(Scale bars,20 mm.) 4012ww.pnas.org/cgi/doi/10.1073pna5.1118386109 Yashina et al
Materials and Methods Biological Samples. Many seeds and fruits from collection sites in permafrost have well preserved coats (testa and pericarp), which allow their taxonomic identification (39). Our collection of extant seeds and fruits of Kolyma lowland plants, along with seed collections of the Botanical Institute, Russian Academy of Sciences, St. Petersburg, Russia, were used for identification of ancient seeds. To date, 38 species are identified. The identity of S. stenophylla seeds was also confirmed by scanning electron microscopy using TESLA BS-300 (40). The accumulation of seeds and fruits in fossil burrows appeared as icy conglomerates. Seeds and fruits excavated from fossil burrows were immediately transferred to sterile bags, kept frozen in the field, and transported frozen to the laboratory at a temperature of −10 °C. Samples selected for the study were defrosted and examined by light microscopy to select undamaged firm seeds and fruits for investigation. Media for Culture in Vitro. The following media were used: Medium 1: Murashige and Skoog basal nutrient medium (MS) rich in nutrients was supplemented with vitamins (41), ascorbic acid 2 mg l−1 , glycine 2 mg l−1 , myo-inositol 100 mg l−1 , casein hydrolysate 500 mg l−1 , 6-benzylaminopurine (BAP) 2 mg l−1 , kinetin 1 mg l−1 , dichlorophenoxyacetic acid (2,4-D) 2 mg l−1 , gibberellic acid (GA) 1 mg l−1 , and 3% sucrose. Medium 2: same as medium 1, to which 10% coconut milk was added. Medium 3: a nutrient-poor medium, consisting of the basal MS medium with sucrose concentration reduced to 1% and supplemented only with kinetin (0.2 mg l−1 ) and indole-3-acetic acid (IAA) (0.1 mg l−1 ). Modified Anderson basal nutrient medium (ABM) (31), with monobasic sodium phosphate replaced by monobasic potassium phosphate and supplemented with ascorbic acid 2 mg l−1 , glycine 2 mg l−1 , myo-inositol 100 mg l−1 , adenine 30 mg l−1 , kinetin 0.5 mg l−1 , BAP 1 mg l−1 , GA 2 mg l−1 , IAA 0.1 mg l−1 , 1% sucrose, and vitamins (41). Methods. Preparation of tissue samples for culture. Immature fruits were carefully cleaned, washed with tap water, and successively surface sterilized with 0.1% solution of corrosive sublimate (mercury bichloride HgCl2) with addition of Tween-80 (0.4 mL in 1,000 mL of solution) and with 70% ethanol. The tissue was washed with sterile distilled water after each disinfectant. Right after disinfection, fruits were dissected under sterile conditions. A dissected, immature fruit of S. stenophylla, having placental tissue and a fragment of the placenta retained with attached seeds at different developmental stages, is shown in Fig. 4. Fragments of the placenta were cultivated in vitro on media 1–3. Reagents were purchased from Sigma-Aldrich. Explant culture. Explants, isolated from placentae of three different immature fruits, were cultured on the three different media (1–3) described above. Organogenesis was observed on both rich- and poor-nutrient media. Successful initiation of shoots on different media occurred, presumably due to high morphogenic and physiological potential of the placental tissue. The first shoot initiated from placental tissue is shown in Fig. 5A. The first formed shoots were etiolated as a result of being cultivated under dark conditions. When the cultures were transferred to sufficient light, shoots synthesized chlorophyll and made active growth. After 5–20 d, numerous adventitious shoots were being formed. On the poor medium, primary shoot initiation was retarded up to 7–9 d, compared with explants cultivated on both rich media (medium 1 and medium 2). Morphogenesis took place only in placental tissues from unripe fruits. Fig. 4. Immature fruit of Silene stenophylla from burrow buried in permafrost more than 30,000 y ago. (A) Dissected fruit showing seeds and placenta (P). (B) Fragment of placenta with seeds at different developmental stages. (Scale bars, 1 mm.) Fig. 5. Clonal micropropagation of Silene stenophylla regenerated from placenta tissue of immature 30,000-y-old fruits buried in permafrost deposits. (А) Initial shoot initiated from placental tissue in vitro. (В) Stages of clonal micropropagation from primary shoots to rooted plants. (Scale bars, 20 mm.) 4012 | www.pnas.org/cgi/doi/10.1073/pnas.1118386109 Yashina et al
Microclonal propagation.Shoots were multiplied through microclonal propa- and decreased night temperature (8-12 C)for 70-90 d.Adapted vegeta- gation in vitro on modified ABM(Fig.5B).For micropropagation,the me- tive plants were then transplanted to a potting medium with mineral dium was switched from MS to ABM because of high shoot extension on MS nutrients,peat,sand,and modern cryozem soil taken from an area with media containing high levels of nitrogen.Plants cultivated from different contemporary S.stenophylla.To initiate the generative stage of plant de- fruits were maintained as separate lineages.Apart from ancient plants, velopment,lighting conditions close to those Arctic values were main- those from extant seeds of the same species,and from the same region, tained (5,000 Ix,18/6 h and then 20/4 light/dark period)at a constant were grown in vitro as a control lineage.All cultivation stages of ancient material were repeated in the control lineage. temperature of 18C. Green shoots were rooted on half-strength modified ABM without the organic compounds mentioned above and further grown under low light ACKNOWLEDGMENTS.This paper is dedicated to Dr.David Gilichinsky,the (2,000 Ix,16/8 h light/dark period)at 22-26 C.Rooted plants were trans- longtime Head of Geocryology Lab.A pioneer in studying microorganisms in ferred to plastic pots with high-moor peat(50%)and sand (or vermiculite, Siberian and Antarctic permafrost,his achievement attracted scientists from all over the world to research on permafrost life systems.We thank Dr.N.V. 50%)and placed in a growth room.Plants were watered as needed with Obrucheva for discussions and encouragement,Dr.A.G.Devyatov for distilled water and fertilized using ABM diluted 1:4.To provide adaptation verification and identification of the plants,and Professors P.J.Webber, for aseptic conditions,regenerated plants were cultivated in high humidity M.M.Webber,and M.A.Holland for help with scientific editing. 1.Veprintsev BN,Rott NN(1979)Conserving genetic resources of animal species.Nature 23.Porsild AE,Harington CR,Mulligan GA (1967)Lupinus arcticus Wats.grown from 280:633-634. seeds of Pleistocene a 2.Withers LA(1985)Cryopreservation and storage of germplasm.Plant Cell Culture.A Practical Approach,ed Dixon RA (IRL,Oxford),pp 169-191. Lupinus arcticus plants were grown from modern not Pleistocene seeds.New Phytol 3.Stanwood PC(1985)Cryopreservation of seed germplasm for genetic conservation 182:788-792. Cryopreservation of Plant Cells and Organs,ed Kartha KK(CRC,Boca Raton,FL),pp 25.Yashina 5G,Gubin SV.Shabaeva EV,Egorova EF,Maksimovich SV (2002)Viability of 199-226. higher plant seeds of late pleistocene age from permafrost deposits as determined by 4.Gilichinsky DA,Rivkina EM (2011)Permafrost microbiology.Encyclopedia of Geo in vitro culturing.Dok/Biol Sci 383:151-154. biology,eds Reitner J.Thiel V(Springer.New York),. 26.Tikhonova VL,Yashina SG(1997)Long-term storage of endangered wild plant seeds 5.Steven B,Leveille R,Pollard WH,Whyte LG(2006)Microbial ecology and biodiversity Physiology and General Biology Reviews,ed Turpaev TM(Harwood Academic Pub in permafrost.Extremophiles 10:259-267. lishers,Amsterdam),pp 1-33. 6.Vorobyova E,et al.(1997)The deep cold biosphere:Facts and hypothesis.FEMS Mi 27. Yashina SG,Gakhova EN,Gubin SV(2003)Permafrost as a natural cryobank of late crobiol Rev 20:277-290. Pleistocene and modern plant germplasm.Permafrost.Extended Abstracts Reporting 7.Lydolph MC,et al.(2005)Beringian paleo logy inferred from permafrost-preserved Current Res rch and Ne Information,eds Haeberli W,Brandova D(Glaciology anc fungal DNA.Appl Environ Microbiol 71:1012-1017. Geomorphodynamics Group,Geography Department,University of Zurich,Switzer- Vishnivetskaya TA,et al.(2006)Bacterial community in ancient Siberian permafrost as and,Pp187-188. characterized by culture and culture-independent methods.Astrobiology 6:400-414. 28.Butenko RG(1964)Culture of Isolated Tissues and the Physiology of Plant Morpho- 9.Willerslev E,et al.(2003)Diverse plant and animal genetic records from Holocene and genesis(Nauka,Moscow)in Russian. Pleistocene sediments.Science 300:791-795. 29. Tisserat B(1985)Embryogenesis,organogenesis and plant regeneration.Plant Cell 10.Odum S(1965)Germination of ancient seeds:Floristical observations and experiments Culture.A Practical Approach,ed Dixon RA (IRL,Oxford),pp 79-126. with archaeologicaly dated soil samples.Dansk Botanisk Arkiv 24:1-70. 30.Murashige T,Skoog F(1962)A revised medium for rrapid growth and bioassays with 11.Priestly DA(1986)Seed Ageing:Implications for Seed Storage and Persistence in the tobacco tissue cultures.Physio/Plant 15:473-497. Soil(Comstock Associates,Ithaca,NY). 31.Anderson WC(1978)Rooting of tissue cultured rhododendrons.Proc Inter Plant Prop 12.Sallon S,et al.(2008)Germination,genetics,and growth of an ancient date seed. SoC28:135-139. Science 320:1464. 32.Kuzin AM (2002)Role of Natural Radioactivity and Secondary Biogenic Radiation in 13.Shen-Miller J,et al.(2002)Long-living lotus:Germination and soil y-irradiation of a Phenomenon of Life (Nauka,Moscow)in Russian. centuries-old fruits,and cultivation,growth,and phenotypic abnormalities of off- 33.Pontovich VE(1970)Culture of tissues of reproductive organs.Culture of Plant Or- spring.Am J Bot 89:236-247. gans,Tissues and Cells,ed Butenko R (Nauka,Moscow).pp 7-20 in Russian. 14.Gubin SV,Khasanov BF(1996)Fossil burrows of mammals in the loess-ice deposits of 34.Macdonald SE,Chinnappa CC Reid DM (1984)Studies on Stellaria longipes Goldie the Kolyma-Indigirka lowland.Dok/Biol Sci 346:26-27. complex:Phenotypic plasticity.I.Response of stem elongation to temperature and 15.Sher AV(1974)Pleistocene mammals and stratigraphy of the Far Northeast USSR and photoperiod.Can J Bot 62:414-419. North America.Int Geo/Rev 16:1-284. 35. Chinnappa CC,Donald GM,Sasidharan R,Emery RJN(2005)The biology of Stellaria 16.Kaplina TN,Lozhkin AV(1984)Age and history of accumulation of the"Ice Complex" longipes (Caryophyllaceae).Can J Bot 83:1367-1383. of the Maritime Lowlands of Yakutia.Late Quaternary Environme nts of the Soviet 36. Khryanin VN (2002)Role of phytohor nes in sex differentiation in plants.Russ J Union,eds Velichko AA,Wright HE,Barnosky CW(University of Minnesota Press, Plant Physio/49:545-551. Minneapolis),pp 147-151. 37.Gaglioti BV,Barnes BM,Zazula GD,Beaudoin AB,Wooller MJ (2011)Late Pleistocene 17.Gubin SV.et al.(2003)[The possible contribution of late pleistocene biota to bio- paleoecology of arctic ground squirrel (Urocitellus pamryin)caches and nests from diversity in present permafrost zonel.Zh Obshch Bio/64:160-165. Interior Alaska's mammoth steppe ecosystem USA.Quat Res7673-382. 18.McKay CP(2001)The Deep Biosphere:Lessons for Planetary Exploration.Subsurface 38.Zazula GD,Froese DG,Elias SA.Kuzmina Mathewes RW (2007)Arctic ground Microbiology and Biogeochemistry,eds Fredrekson JK,Fletcher M (Wiley-Liss,New squirrels of the mammoth-steppe:Paleoecology of Late Pleistocene middens (24 000- York,pp315-327. 29 450Cyr BP),Yukon Territory,Canada.Quat Sci Rev 26:979-1003. 19.Ellis RH,Roberts EH(1980)Improved equations for the prediction the seed longevity. 39.Gubin SV,Maximovich SV,Zanina OG,Stakhov VL(2011)Morphogenetics of plant Ann Bot (Lond)45:13-30. remains from paleosols and rodent burrows buried in permafrost of the Late Pleis- 20.Murdoch AJ,Ellis RH (2000)Dormancy,Viability and Longevity/CAB Intemational tocene 32-28,000 BP).Plant Archaeogenetics,ed Gyulai G(Nova Science,New York). Seeds:The Ecology of Regeneration in the Plant Communities,ed Fenner N(CAB p11-21. International,Wallingford,UK),2nd Ed,pp 183.214 Chapter 8. 40. Basli GA,et al.(2009)Light and scanning electron microscopic analysis of Silene 21.Andersen DM(1967)Ice nucleation and substrate-ice interface.Nature 216:563-566. stenophylla seeds excavated from Pleistocene-Age (Kolyma).Anadolu Univ J Sci 22.Gilichinsky DA Wagener S,Vishnivetskaya TA (1995)Permafrost microbiology.Per Technol10:161-167. mafrost and Periglacial Processes 6:281-291. 41.White PR(1963)The Cultivation of Animal and Plant Cells (Ronald Press,New York). Yashina et al. PNAs|March6.2012|vol.109|no.10|4013
Microclonal propagation. Shoots were multiplied through microclonal propagation in vitro on modified ABM (Fig. 5B). For micropropagation, the medium was switched from MS to ABM because of high shoot extension on MS media containing high levels of nitrogen. Plants cultivated from different fruits were maintained as separate lineages. Apart from ancient plants, those from extant seeds of the same species, and from the same region, were grown in vitro as a control lineage. All cultivation stages of ancient material were repeated in the control lineage. Green shoots were rooted on half-strength modified ABM without the organic compounds mentioned above and further grown under low light (2,000 lx, 16/8 h light/dark period) at 22–26 °C. Rooted plants were transferred to plastic pots with high-moor peat (50%) and sand (or vermiculite, 50%) and placed in a growth room. Plants were watered as needed with distilled water and fertilized using ABM diluted 1:4. To provide adaptation for aseptic conditions, regenerated plants were cultivated in high humidity and decreased night temperature (8–12 °C) for 70–90 d. Adapted vegetative plants were then transplanted to a potting medium with mineral nutrients, peat, sand, and modern cryozem soil taken from an area with contemporary S. stenophylla. To initiate the generative stage of plant development, lighting conditions close to those Arctic values were maintained (5,000 lx, 18/6 h and then 20/4 light/dark period) at a constant temperature of 18 °C. ACKNOWLEDGMENTS. This paper is dedicated to Dr. David Gilichinsky, the longtime Head of Geocryology Lab. A pioneer in studying microorganisms in Siberian and Antarctic permafrost, his achievement attracted scientists from all over the world to research on permafrost life systems. We thank Dr. N. V. Obrucheva for discussions and encouragement, Dr. A. G. Devyatov for verification and identification of the plants, and Professors P. J. Webber, M. M. Webber, and M. A. Holland for help with scientific editing. 1. Veprintsev BN, Rott NN (1979) Conserving genetic resources of animal species. Nature 280:633–634. 2. Withers LA (1985) Cryopreservation and storage of germplasm. Plant Cell Culture. A Practical Approach, ed Dixon RA (IRL, Oxford), pp 169–191. 3. Stanwood PC (1985) Cryopreservation of seed germplasm for genetic conservation. Cryopreservation of Plant Cells and Organs, ed Kartha KK (CRC, Boca Raton, FL), pp 199–226. 4. Gilichinsky DA, Rivkina EM (2011) Permafrost microbiology. Encyclopedia of Geobiology, eds Reitner J, Thiel V (Springer, New York), pp 726–732. 5. Steven B, Léveillé R, Pollard WH, Whyte LG (2006) Microbial ecology and biodiversity in permafrost. Extremophiles 10:259–267. 6. Vorobyova E, et al. (1997) The deep cold biosphere: Facts and hypothesis. FEMS Microbiol Rev 20:277–290. 7. Lydolph MC, et al. (2005) Beringian paleoecology inferred from permafrost-preserved fungal DNA. Appl Environ Microbiol 71:1012–1017. 8. Vishnivetskaya TA, et al. (2006) Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods. Astrobiology 6:400–414. 9. Willerslev E, et al. (2003) Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science 300:791–795. 10. Odum S (1965) Germination of ancient seeds: Floristical observations and experiments with archaeologicaly dated soil samples. Dansk Botanisk Arkiv 24:1–70. 11. Priestly DA (1986) Seed Ageing: Implications for Seed Storage and Persistence in the Soil (Comstock Associates, Ithaca, NY). 12. Sallon S, et al. (2008) Germination, genetics, and growth of an ancient date seed. Science 320:1464. 13. Shen-Miller J, et al. (2002) Long-living lotus: Germination and soil γ-irradiation of centuries-old fruits, and cultivation, growth, and phenotypic abnormalities of offspring. Am J Bot 89:236–247. 14. Gubin SV, Khasanov BF (1996) Fossil burrows of mammals in the loess-ice deposits of the Kolyma-Indigirka lowland. Dokl Biol Sci 346:26–27. 15. Sher AV (1974) Pleistocene mammals and stratigraphy of the Far Northeast USSR and North America. Int Geol Rev 16:1–284. 16. Kaplina TN, Lozhkin AV (1984) Age and history of accumulation of the ‘‘Ice Complex’’ of the Maritime Lowlands of Yakutia. Late Quaternary Environments of the Soviet Union, eds Velichko AA, Wright HE, Barnosky CW (University of Minnesota Press, Minneapolis), pp 147–151. 17. Gubin SV, et al. (2003) [The possible contribution of late pleistocene biota to biodiversity in present permafrost zone]. Zh Obshch Biol 64:160–165. 18. McKay CP (2001) The Deep Biosphere: Lessons for Planetary Exploration. Subsurface Microbiology and Biogeochemistry, eds Fredrekson JK, Fletcher M (Wiley-Liss, New York), pp 315–327. 19. Ellis RH, Roberts EH (1980) Improved equations for the prediction the seed longevity. Ann Bot (Lond) 45:13–30. 20. Murdoch AJ, Ellis RH (2000) Dormancy, Viability and Longevity/CAB International Seeds: The Ecology of Regeneration in the Plant Communities, ed Fenner N (CAB International, Wallingford, UK), 2nd Ed, pp 183.214 Chapter 8. 21. Andersen DM (1967) Ice nucleation and substrate-ice interface. Nature 216:563–566. 22. Gilichinsky DA, Wagener S, Vishnivetskaya TA (1995) Permafrost microbiology. Permafrost and Periglacial Processes 6:281–291. 23. Porsild AE, Harington CR, Mulligan GA (1967) Lupinus arcticus Wats. grown from seeds of Pleistocene age. Science 158:113–114. 24. Zazula GD, Harington CR, Telka AM, Brock F (2009) Radiocarbon dates reveal that Lupinus arcticus plants were grown from modern not Pleistocene seeds. New Phytol 182:788–792. 25. Yashina SG, Gubin SV, Shabaeva EV, Egorova EF, Maksimovich SV (2002) Viability of higher plant seeds of late pleistocene age from permafrost deposits as determined by in vitro culturing. Dokl Biol Sci 383:151–154. 26. Tikhonova VL, Yashina SG (1997) Long-term storage of endangered wild plant seeds. Physiology and General Biology Reviews, ed Turpaev TM (Harwood Academic Publishers, Amsterdam), pp 1–33. 27. Yashina SG, Gakhova EN, Gubin SV (2003) Permafrost as a natural cryobank of late Pleistocene and modern plant germplasm. Permafrost. Extended Abstracts Reporting Current Research and New Information, eds Haeberli W, Brandova D (Glaciology and Geomorphodynamics Group, Geography Department, University of Zurich, Switzerland), pp 187–188. 28. Butenko RG (1964) Culture of Isolated Tissues and the Physiology of Plant Morphogenesis (Nauka, Moscow) in Russian. 29. Tisserat B (1985) Embryogenesis, organogenesis and plant regeneration. Plant Cell Culture. A Practical Approach, ed Dixon RA (IRL, Oxford), pp 79–126. 30. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. 31. Anderson WC (1978) Rooting of tissue cultured rhododendrons. Proc Inter Plant Prop Soc 28:135–139. 32. Kuzin AM (2002) Role of Natural Radioactivity and Secondary Biogenic Radiation in a Phenomenon of Life (Nauka, Moscow) in Russian. 33. Pontovich VE (1970) Culture of tissues of reproductive organs. Culture of Plant Organs, Tissues and Cells, ed Butenko R (Nauka, Moscow), pp 7-20 in Russian. 34. Macdonald SE, Chinnappa CC, Reid DM (1984) Studies on Stellaria longipes Goldie complex: Phenotypic plasticity. I. Response of stem elongation to temperature and photoperiod. Can J Bot 62:414–419. 35. Chinnappa CC, Donald GM, Sasidharan R, Emery RJN (2005) The biology of Stellaria longipes (Caryophyllaceae). Can J Bot 83:1367–1383. 36. Khryanin VN (2002) Role of phytohormones in sex differentiation in plants. Russ J Plant Physiol 49:545–551. 37. Gaglioti BV, Barnes BM, Zazula GD, Beaudoin AB, Wooller MJ (2011) Late Pleistocene paleoecology of arctic ground squirrel (Urocitellus parryii) caches and nests from Interior Alaska’s mammoth steppe ecosystem, USA. Quat Res 76:373–382. 38. Zazula GD, Froese DG, Elias SA, Kuzmina S, Mathewes RW (2007) Arctic ground squirrels of the mammoth-steppe: Paleoecology of Late Pleistocene middens (24 000- 29 450 14C yr BP), Yukon Territory, Canada. Quat Sci Rev 26:979–1003. 39. Gubin SV, Maximovich SV, Zanina OG, Stakhov VL (2011) Morphogenetics of plant remains from paleosols and rodent burrows buried in permafrost of the Late Pleistocene (32 - 28,000 BP). Plant Archaeogenetics, ed Gyulai G (Nova Science, New York), pp 11–21. 40. Başli GA, et al. (2009) Light and scanning electron microscopic analysis of Silene stenophylla seeds excavated from Pleistocene-Age (Kolyma). Anadolu Univ J Sci Technol 10:161–167. 41. White PR (1963) The Cultivation of Animal and Plant Cells (Ronald Press, New York). Yashina et al. PNAS | March 6, 2012 | vol. 109 | no. 10 | 4013 PLANT BIOLOGY
