Evaluation of Seed Germination of Six Rare Stipa Species following Low Temperature Stress (Cryopreservation)

Cryopreservation is one way to preserve rare, endangered species. However, during the cryopreservation process, plant cells undergo considerable stress, which may lead to cell death. In our work, orthodox Stipa seeds of six rare species were cryopreserved: S. sareptana, S. ucrainica, S. tirsa, S. dasyphylla, S. adoxa, and S. pulcherríma. Short-term cryopreservation (14 days) stimulated germination of all Stipa species studied. Prolonged cryopreservation (70 days and more) decreased the germination of all Stipa seeds except S. sareptana. The decrease in germination progressed over time as a result of the cumulative stress of cryopreservation rather than the initial stress. To stimulate germination, seeds were stratified and treated with GA3, KNO3, NaOH, and H2O2. After four years of seed cryopreservation, it was possible to obtain seedlings of all the Stipa species studied with 30 days of stratification and 180 days of germination. After five years of cryopreservation and seed treatment with 30% NaOH for one hour, the best germination was obtained in S. adoxa and S. pulcherrima. After treatment with 5% H2O2 for 20 min, the best germination was obtained in S. sareptana, S. ucrainica, and S. dasyphylla. S. sareptana seeds germinated in all the aforementioned experiments. S. sareptana has a non-deep physiological dormancy and is the most widespread and drought-tolerant Stipa species studied. The best habitat adaptation and stress tolerance correlated with this species’cryotolerance. S. sareptana was recommended for further cryopreservation, while storage protocols for the other Stipa species studied need further improvements.


Introduction
One of the ways to preserve rare, endangered plant species and unique genotypes is cryopreservation.Cryopreservation is a process of low-temperature storage (−135 • C) of biological objects with the potential of restoring their biological functions after thawing.As a rule, cryopreservation is carried out at −196 • C in a cryobank with liquid nitrogen [1].The establishment of cryobanks of germplasm: seeds, pollen, embryos, tissue, and cell cultures and other genetic material is included in national programs for the conservation of genetic phytoresources in many countries [2][3][4].At present, about 400 botanical gardens in the world have seed banks containing more than 300 thousand stored samples [5].
Cryopreservation of hydrated plant cells, tissues, and organs is a very extreme process due to the formation and growth of ice crystals during freezing and dehydration and recrystallisation during subsequent thawing, resulting in cold, mechanical, water, osmotic, and oxidative stress [6].The formation of ice crystals inside cells can be prevented in several ways: reducing the cooling rate, desiccation, and vitrification.To date, the most widespread dehydration technique is vitrification, which is the conversion of intracellular water into a glassy matrix during ultra-fast freezing.Before vitrification, the explants should be incubated in special solutions such as the Plant Vitrification Solution (PVS) series developed by A. Sakai [7,8].PVS2 and PVS3 are the most commonly used options [9,10].Vitrification protocols have been developed and improved over time.Currently, they include encapsulation dehydration [11], encapsulation vitrification [12,13], drip vitrification [14,15], cryoplast protocols [16,17], vacuum infiltration vitrification (VIV) [18,19], and a new cryo-mesh protocol [20].All cryopreservation protocols are based on dehydration to 10-20% of original weight [21].However, dehydration may lead to dysfunction in membranes and changes in their barrier properties [22][23][24], as well as destruction of cell organelles [25,26].As a result of dehydration, the concentration of dissolved compounds, mainly inorganic ions and metabolites with toxic effects, is significantly increased in the cell.Dehydration results in conformational changes in protein macromolecules, causing cross-links within them and, thus, leads to non-reversible biochemical changes [27][28][29].Changes in protein and lipid properties lead to metabolic disorders, in particular, to suppression of the biochemical and photochemical activity of chloroplasts and mitochondria [30,31], formation of reactive oxygen species (ROS) [32][33][34] and, as aresult, to programmed cell death [35,36].Thus, the aim of hydrated plant cells'cryopreservationis to minimize the stress effects of dehydration.In some cases, preliminary plant cold acclimation may be helpful [37].
In the case of plant material containing a minimal amount of water (pollen, orthodox seeds), cryopreservation is not that difficult.Such material, previously dried, can be placed directly in liquid nitrogen and then thawed in air under normal conditions [38,39].Since ultra-low temperatures presumably stop cell metabolism and chemical and physical reactions, it has been suggested that seeds can be stored for decades under such conditions [40].Despite these assumptions, NCGRP monitoring data show that a decrease in seed viability during cryogenic storage can be detected within 10-25 years for 15 out of 42 seed species stored in liquid nitrogen (−196 • C) [41].The degradation process progressed over time, demonstrating that the decrease in viability was due to aging stress rather than initial stresses.The authors concluded that despite the glassy matrix forming, slow movement of molecules within the seeds continued.It appears that molecular mobility is an important factor controlling the kinetics of the aging process.The nature and extent of molecular motion vary considerably depending on moisture levels, temperature, and on the hydration levels of the seeds as well [41].The combination of extreme drying and extreme cooling may result in abnormal temperature responses in aging kinetics [42].Some authors have reported physiologically dormant seeds having difficulties germinating after cryopreservation [43].Therefore, the primary objective during cryopreservation of orthodox seeds is to monitor germination consistently.Seed germination after cryopreservation may be influenced by the moisture content levels of the seed, the degree of seed dormancy, the weather conditions during seed formation, the location of collection, and the storage conditions until cryopreservation [41,43].
In our work, orthodox Stipa seeds of the following six rare species were cryopreserved: S. sareptana, S. ucrainica, S. tirsa, S. dasyphylla, S. adoxa, and S. pulcherríma.The Stipa genera includes more than 300 species of grasses.These perennial grasses have adapted to arid conditions with a low humidity and extreme temperature fluctuations.The caryopsis of Stipa is a single structure.It is traditionally referred to as a 'seed' in scientific publications [44,45].The Stipa is becoming a rare and protected species due to the development of steppes for agriculture and grazing [46].At present, Stipa, as individual dominants, can be used as indicators of steppe communities and for their classification [47].In addition, populations of some Stipa species may be used as indicators of global climate change [48].Unfortunately, the system of protected areas cannot ensure the conservation of the biological diversity of Stipa species in Russia [49].In this regard, the aims of our research were to create a seed bank of six Stipa species; to test the germination of frozen seeds at time intervals; and to investigate the effect of stratification and different seed stimulants on their germination.

Description of the Stipa Species Studied
Seeds of six species of Stipa were collected in 2015 from the virgin steppes on the territory of the "Botanical Garden of the Southern Federal University" (Rostov-na-Donu) [50].The Botanical Garden preserves the collection of rare and endangered plant species of the Rostov region and the Don floodplain (Figure 1; Table 1) [51,52].For our experiments we used three species of Stipa: S. ucrainica P. Smirn., S. dasyphylla (Lindem.)Trautv., and S. pulcherrima K. Koch, which are listed in the Red Data Book of the Russian Federation.The other three species: S. sareptana A. Becker, S. tirsa Steven, and S. adoxa Klokov and Ossyczn., are protected by the Red Data Books of various regions of the Russian Federation and other European countries.

Description of the Stipa Species Studied
Seeds of six species of Stipa were collected in 2015 from the virgin steppes on the territory of the "Botanical Garden of the Southern Federal University" (Rostov-na-Donu) [50].The Botanical Garden preserves the collection of rare and endangered plant species of the Rostov region and the Don floodplain (Figure 1; Table 1) [51,52].For our experiments we used three species of Stipa: S. ucrainica P. Smirn., S. dasyphylla (Lindem.)Trautv., and S. pulcherrima K. Koch, which are listed in the Red Data Book of the Russian Federation.The other three species: S. sareptana A. Becker, S. tirsa Steven, and S. adoxa Klokov and Ossyczn., are protected by the Red Data Books of various regions of the Russian Federation and other European countries.The number in the table corresponds to the number on the map.This number is the location of the Stipa seeds collection site.

Seed Cryopreservation
The seeds were gathered by hand.The long pinnate awns were removed.The palea and lemma were not separated from the caryopsis to avoid damaging the seeds.The  The number in the table corresponds to the number on the map.This number is the location of the Stipa seeds collection site.

Seed Cryopreservation
The seeds were gathered by hand.The long pinnate awns were removed.The palea and lemma were not separated from the caryopsis to avoid damaging the seeds.The collected seeds were placed in labeled paper bags and stored in a dark at room temperature (20-25 • C) and 40-60% humidity for several months until the start of the experiment.About 50 seeds of each Stipa species were used in the studies.
Biological storage (XB-0.5;manufactured by Ural Compressor Plant, Ekaterinburg, Russian Federation) was used for seed cryopreservation.The seeds were placed in cryoampoules (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) before freezingand then the cryoampoules were placed in liquid nitrogen (N 2 ) at −196 • C. The seeds were defrosted at room temperature before the experiment.One cryoampoule of each Stipa species was used without refreezing for each experiment.

Seed Viability Testing
The seeds were carefully examined for each experiment and damaged or suspicious seeds were discarded.Seed viability was determined by the TTC (2,3,5-triphenyltetrazolium chloride) method.If seeds are viable, they are actively respiring and reducing triphenyltetrazolium chloride to red formazan.The seeds were placed in Petri dishes on moist filter paper and incubated for 48 hin the dark, at 20-25 • C. The seeds were then immersed in 0.5% TTX solution in phosphate buffer (pH = 7.5) and placed in the dark at 28 • C for 24 h.Seeds were considered viable when their tissues were stained red [53].Seed viability was determined as the ratio of stained seeds to the total number of seeds and expressed in percents.Seed viability before and after cryopreservation were determined using the TTC method.

Seed Germination Testing
Stipa seeds were stored in the dark at room temperature (20-25 • C) for three months from the time of collection until germination.For the germination control test, seeds without pre-sowing treatment were germinated in Petri dishes on moist filter paper for 14 days.Seed germination was determined as the ratio of germinated seeds to the total number of seeds and was expressed in %.

Indication of Dormancy Status
Indication of dormancy status was calculated using Offord's et al. (2004) equation: DI (DormancyIndex) = 1-(GI/VI), where GI (Germination Index)-% of germinated seeds; VI (Viability Index)-% of viable stained seeds (TTC test) A higher DI value suggests a higher probability of seed dormancy at that time.If the DI is equal to or greater than 0.4, it indicates dormancy in the seed [54].
2.6.Seed Germination after Cryopreservation 2.6.1.Experiment I (the Short-Term Effect of Cryopreservation) The seeds were divided into two groups: control and test.The control group wasstored in the dark at room temperature for six months from the start of the experiment and then stratified for 42 days.The test group was initially stored in the same way as the control group, but then cryopreserved for 14 days and then stratified for 42 days.Seeds from the control group, which had not been used before, were stored in the dark at room temperature.The remaining seeds were stored in liquid nitrogen.They were thawed as the experiments proceeded.

Experiment II (the Effect of Stratification)
The seeds were separated into four groups.The first group was stored in the dark at room temperature for eight months from the start of the experiment.The second group was also stored in the same way as the first group, but for seven months, followed by 21 days of stratification.The third group was stored in liquid nitrogen for 91 days.The fourth group was also stored in liquid nitrogen for 70 days and then stratified for 21 days.

Experiment III (the Long-Term Effect of Cryopreservation and NaOH Treatment)
The seeds were thawed after 415 days (about 14 months) of storage in liquid nitrogen.The Stipa were germinated on Petri dishes for 30 days.Ungerminated seeds were tested for viability using the TTC test.Ungerminated stained seeds were treated with NaOH.

Experiment IV (the Germination Duration)
The seeds were thawed after 1489 days (about 4 years and 1 month) of storage in liquid nitrogen.The seeds were tested for viability using the TTC test.Stratification continued for 30 days.Seed germination was carried out for 12 months.

Experiment V (the Effect of Different Stimulators)
The seeds were thawed after 1951 days (5 years and 4 months) of storage in liquid nitrogen.The seeds were separated into four groups.The seeds of the first group of were treated with H 2 O 2 , while the seeds of the second group were treated with NaOH.The seeds of the third group of were germinated with KNO 3 .The seeds of the fourth groupwere germinated with GA 3 .Unfortunately, the S. tirsa seeds were used up at that time and were not included for this experiment.

Seed Stratification
Seed stratification was carried out doors for 3-6 weeks.Seeds were planted in a soil/turf mixture in pots to a 1 cm depth.The pots were then covered with snow for the total stratification period.The air temperature was below 0 • C all the time (from −2 to −15 • C).Thus, we decided to imitate the influence of natural winter conditions on seeds, which last from 4 to 6 months in the Rostov region.At the end of stratification, the seeds were transferred to a greenhouse where they germinated at 40-60% humidity, at 20-25 • C, and 16 h illumination of 2-4 klk per day within 30 days.

Seed Germination Stimulators
To stimulate germination, dry seeds were immersed in 30% sodium hydroxide (NaOH) solution for 1 h or in 5% hydrogen peroxide (H 2 O 2 ) solution for 20 min.The seeds were then washed with sterile water and placed in Petri dishes on filter paper moistened with distilled water.In addition, 0.1% potassium nitrate (KNO 3 ) and 0.05% gibberellic acid (GA 3 ) (Green AgroLab, Orenburg, Russian Federation) were used to stimulate germination.GA 3 was first dissolved in 2 mL of ethanol and then added to water to the final concentration.As a control, seeds were germinated in Petri dishes on filter paper moistened with distilled water.Petri dishes with seeds were placed under conditions of stable temperature regime (20 ± 2 • C) and illumination (2 klk) at 16 h day (climatic chamber of the phytotron of IPP RAS with automatic air conditioning): Gree, PRC, and fluorescent lighting: LB-40 "OSRAM", RF.The seeds germinated for 30 days.

Statistics
Statistical analysis was performed using a one-way ANOVA test.The experimental data demonstrated a significant difference at p < 0.05.

Seed Viability Testing
Most of the Stipa seeds tested stained red in the TTC test, i.e., they were viable.After cryopreservation, no significant changes in the viability of the seeds were observed, as all the seeds were stained red after thawing.

Seed Germination Testing
For 14 days, only S. sareptana seeds (60%) germinated.Other Stipa species seeds germinated after one month.

Indication of Dormancy Status
The DI for S. sareptana seeds in this case was 0.4 minimal among Stipa studied.For the other Stipa species, the DI was equal to 1. Therefore, all the Stipa seeds were in organic dormancy.Mechanical removal of palea and lemma from caryopsis stimulated the germination of all viable embryos in our experiments.However, this operation usually resulted in catastrophic damage to the caryopsis and unfortunately did not allow for the formation of viable seedlings.Germination after the removal of seed-coats suggests physiological dormancy in Stipa seeds.

Experiment I
The first series of experiments evaluated the short-term effect of cryopreservation on seed germination.The experiments showed that the average germination level of the seeds was 40%.The DI was greater than 0.4, indicating that the seeds were in organic dormancy.The average germination increased by 19% (from 40 to 59%) after cryopreservation.The DI after cryopreservation decreased in all Stipa species (0.41).In S. sareptana and S. pulcherrima DI levels were less than 0.4, i.e., the seeds of these species were out of the dormancy (Table 2).The first experiment indicated that short-term cryopreservation increased germination and decreased DI.The second series of experiments evaluated the effect of stratification on seed germination.The effect of cryopreservation was also tested.Stratification increased the germination levels of S. sareptana, S. dasyphylla, and S. pulcherrima.However, the germination of S. adoxa was independent of stratification.The germination of S. ucrainica and S. tirsa seeds stored at room temperature did not depend on stratification, but the germination of seeds stored in liquid nitrogen increased after stratification.However, on average, stratification increased germination rates (from 14 to 23% and from 24 to 36%).More prolonged stratification is probably needed to stimulate germination in some Stipa species (Table 3).The second series of experiments also demonstrated that cryopreservation increased the germination rates of all Stipa by 13% (from 23 to 36%).S. adoxa did not germinate after 7 and 8 months of storage at room temperature, and this species was only able to germinate after cryopreservation.However, compared to the initial average levels (Table 2), germination after 70 days of cryopreservation decreased in all Stipa species except for S. sareptana (Table 3).The third series of experiments evaluated the germination of Stipa L. seeds after 14 months of storage in liquid nitrogen.After thawing, Stipa seeds, except S. sareptana, did not germinate.However, all post-cryogenic seeds stained red-brown with formazan, indicating vigorous respiration.S. ucrainica, S. tirsa, S. dasyphylla, and S. pulcherrima germinated only after NaOH treatment.S. adoxa seeds did not germinate at all. S. sareptana seeds did not require NaOH treatment as they germinated within 30 days after thawing (Table 4, Figure 2).The third experiment indicated that the germination of all Stipa seeds, except S. sareptana, decreased during long-term cryopreservation.Cryopreservation as a stress factor could possibly contribute to increasing the depth of physiological dormancy of all studied Stipa species except S. sareptana.The third series of experiments also indicated that NaOH may be used as a germination stimulant for the Stipa seeds.5).In this case, a longer germination period or a pre-treatment with stimulators is required to bring the seeds out of dormancy.Most of the seeds from the control group stored in the dark at room temperature for 19 months lost germination and did not participate in further experiments.

Experiment IV
The fourth series of experiments evaluated the germination of Stipa seeds after four years of cryopreservation.The germination of Stipa was only observed after 90 days and that of S. adoxa after 180 days.Germination of all Stipa seeds decreased significantly (Table 5).In this case, a longer germination period or a pre-treatment with stimulators is required to bring the seeds out of dormancy.3.4.5.Experiment V The fifth series of experiments evaluated the effect of different stimulators on seed germination.As a result, we found that pre-treatment with KNO 3 and GA 3 did not cause the germination of seeds of all Stipa species except S. sareptana.These data suggest that treatment with KNO 3 and GA 3 led to the exit of the seeds from a non-deep physiological dormancy state.It may be assumed that the dormancy of seeds of all Stipa except S. sareptana was increased during cryopreservation.Pre-treatment of the seeds with H 2 O 2 increased germination levels of S. sareptana and S. dasyphylla and stimulated germination of S. ucrainica.Pre-treatment with NaOH increased germination of S. adoxa and S. pulcherrima (Table 6).The effect of these stimulators was probably species dependent.According to the summary data obtained for five years of seed cryopreservation, it can be concluded that short-term cryopreservation (14 days) stimulated their germination, but after two months of cryopreservation (70 days) germination of all Stipa seeds except S. sareptana decreased (Table 7).After four years of cryopreservation (1489 days), the dormancy of most Stipa except S. sareptana increased.Dormancy was cancelled either by stratification followed by prolonged cultivation (180-400 days) or by the use of the stimulators NaOH and H 2 O 2 (Table 7).Perhaps a combination of cold stratification for more than 40 days and seed pre-treatment with NaOH or H 2 O 2 would be an optimal stimulation of the seed germination.Previously, three studies were publishedin which the authors investigated the effect of cryopreservation on Stipa seeds.Puchalski et al. (2014) showed that 92-100% of S. pennata seeds germinated after 30 days of storage in liquid nitrogen vapour (−160 • C) [55].Levitskaya (2017b) stored S. pennata seeds at temperatures of 5 ± 1 • C (refrigerator), −20 ± 2 • C (freezer), and −196 • C (liquid nitrogen).After one month of storage, the germination rates of seeds stored at different temperatures did not decrease.After storage for 6 years, the germination of seeds stored at 5 ± 1 • C did not decrease, but the germination levels of seeds stored at −20 • C and −196 • C decreased several times [56].Shevchenko et al. (2019) stored S. capillata seeds at room temperature, at −20 • C, and at −196 • C for 18 months.Then, the seed scales were cut with a scalpel, and the seeds were put on Petri dishes and germinated at 20 • C in the dark.The experiment showed that the seeds had low germination rates, both in the control (10.46 ± 7.45%) and after low temperature storage at −20 • C (12 ± 8.18%).The germination of S. capillata seeds increased significantly to 30.28 ± 10.73% after storage in liquid nitrogen [57].
Therefore, our data are in accordance with the published observations: short-term cryopreservation for 14 days stimulated initial germination (Tables 2 and 7), as in Puchalski et al. (2014) and Levitskaya (2017b) [55,56].Cryopreservation of seeds for about 1 year (415 days) and pre-treatment with 30% NaOH reliably stimulated germination of S. sareptana and S. tirsa (Tables 4 and 7) seeds, as was reported in Shevchenko et al. (2019) [57].Cryopreservation for more than 5 years significantly reduced germination of all Stipa species except S. sareptana and made it impossible without additional stimulation (Tables 6 and 7), as was described in Levitskaya (2017b) [56].

Effect of Cryopreservation on Seed Germination
It is known that cryopreservation has different effects on the germination of seeds of even closely related plant species.For example, Nikishina et al. (2007) found that after 30 days of cryopreservation, seeds of Dactylorhiza maculate and Platanthera bifolia (L.) Rich.showed decreased germination, whereas for seeds of D. fuchsii (Druce) Soo, D. incarnata, and D. baltica (Klinge) Orlova stimulated germination was seen [58].Liquid nitrogen treatment causes cracks to form in the seed-coat.As a result, the palisade layer of cells underlying the outer cuticle is disrupted and the seed-coat becomes more water permeable.Disruption of the seed-coat results in faster bulking and activation of intracellular metabolism [59].It is also possible that cryopreservation, as a stress factor, may activate redox processes in the embryo and thus stimulate germination.In some cultivated species, a stimulating effect of cryopreservation has been observed, associated with the induction of enzyme complex activity [60].In our experiments, after 70 days of cryopreservation, germination of all Stipa species except S. sareptana was reduced compared to the initial germination (Table 7).After 415 days of cryopreservation, germination was found only in seeds of S. sareptana that did not undergo additional stimulation (Table 4).At the same time, all the seeds were alive because they had been stained with TTX.After 1489 days of cryopreservation, all Stipa seeds germinated, butonly after 180 days (Table 5).Thus, the decrease in germination after cryopreservation progressed over time as a result of cumulative stress rather than initial stress.A similar phenomenon has been observed previously [41,61].Walters et al., (2005) suggested that the mechanical structure of biopolymers is transformed when seeds are desiccated below critical moisture content.At cryogenic temperatures, intense intracellular molecular mobility is observed in over-desiccated seeds compared to more humid seeds.On this basis, it has been suggested that the combination of extreme desiccation and extreme cooling may result in anomalous temperature responses in aging kinetics [42].This hypothesis is supported by Chmielarz's (2009) research on the cryopreservation of Prunus avium L. seeds.The study found that seeds with more than 17% free water did not germinate, and those with less than 7% free water germinated less than 20%, while those with 9 to 16.9% free water showed maximum germination levels of more than 20% [62].Levitskaya [43,61,63] conducted studies on the cryopreservation of seeds with different types of dormancy.The authors concluded that storage at freezing temperatures would be optimal for orthodox seeds of species growing in drought or frosty climates.These may be non-dormant seeds and seeds with organic dormancy of different types other than physiological.Seeds with deep physiological dormancy, adapted to storage in humid conditions, may be unstable to extreme drying and storage at freezing temperatures [43].Using five Campanula species, Levitskaya (2015) revealed that the deeper the seed dormancy, the faster they aged at ultra-low temperatures.To explain this, the author suggested that seeds with and without physiological dormancy have different mechanisms involved in viability [63].Perhaps, seeds with morphological and morphophysiological dormancy, as well as not-dormant seeds, survive in a dry state due to the general inhibition of metabolism as a consequence of the low hydration.They have evolved sHSPs (small heat shock proteins) and LEA-proteins (late embryogenesis abundant proteins), which are synthesized at late stages of seed maturation and are responsible for cell resistance to dehydration and dry storage, including these conditions at low temperatures.Seeds with physiological dormancy are able to maintain viability for a long time in a humid, hydrated state by repairing cytological damage through specific metabolism [64].The cellular structure of such seeds is likely to change less during seed maturation and desiccation.It may be assumed that the development of seeds with physiological dormancy is terminated at an earlier stage, when low amounts of sHSPs and LEA, or less than the whole functional spectrum of these proteins, are synthesized [43].Therefore, before cryopreservation, it is necessary to find out whether seeds are in dormancy, to determine the type of dormancy, to select the methods of dormancy removal, and the conditions of seed germination.

Effect of Dry Storage and Cold Stratification on Stipa Seed Germination
At the beginning of our experiment, the seeds were stored in the dark at room temperature for three months after their collection.Only S. sareptana germinated after 14 days and had a DI of 0.6.The other Stipa seeds germinated after one month and had a DI of 1.Therefore, they had a deeper physiological dormancy than S. sareptana from the beginning of the experiment.All Stipa seeds were in the state of organic dormancy [53].According to Nikolaeva (1985), other species of Stipa: S. bigeniculata D. K. Hughes, S. nitida V. S. Summerhayes et S. E. Hubbard, and S. viridula Trin., do not have a deep physiological dormancy [65].According to Hu et al. (2014), S. bungeana also has a non-deep physiological dormancy [66].Since we were not able to find any information, we assumed the same non-deep physiological dormancy for the Stipa we studied.This type of dormancy is due to the low growth activity of the embryo which, combined with the poor gas permeability of the seed-coat, provides a dual or physiological mechanism for inhibiting germination [67,68].To test this, we cut seed scales.This stimulated the germination of all living embryos, confirming our suggestion about non-deep physiological dormancy of Stipa seeds studied.
In addition to damaging the scales, dry storage for several months, short cold stratification, or treatment of the seeds with phytohormones gibberellins (GA 3 , GA 4+7 ) and cytokinins (kinetin, zeatin, benzylaminopurin) are recommended to break out of the non-deep physiological dormancy [69].According to the data available in the literature, germination of Stipa under laboratory conditions may vary considerably.For example, Ronnenberg et al. (2008) reported that the field germination levels of S. krylovii was 3%, S. glareosa-0.6%, and S. gobica-0.1%.In addition, the three-year-old seeds were germinatingin the field on the third year after very heavy rain.The laboratory germination of freshly collected seeds of all three species studied at 20/10 • C was 90%.Dry storage of seeds for six months did not change germination levels.Cold stratification increased the germination only of S. krylovii [70].Hu et al. (2014) reported that the highest germination rates under laboratory conditions were 25% for fresh S. bungeana seeds in the dark at 20 • C.After storage of the seeds in the dark for six months at 5 • C, germination increased from 25% to 63%, and after storage of the seeds at 20 • C, germination increased from 25% to 69% [66].Zhang et al. (2017) observed that the laboratory germination of fresh S. bungeana seeds in 2015 ranged from 13.7% to 41.2% at 10/20 • C, 17.4% to 73.4% at 15/25 • C, and 0% to 0.7% at 20/30 • C.After six months of dry storage, the average germination of S. bungeana seeds from the eight populations increased.However, germination varied between populations.In some populations, germination rates decreased [71].Krichen et al. (2017) reported that the germination under laboratory conditions of S. tenacissima seeds at 20 • C was 50%.After cold stratification, the germination of S. tenacissima seeds increased from 50% to 70% depending on the length of stratification [72].Nozdrina et al. (2021) found that germination of S. capillata seeds in the laboratory was 16%, and of S. pennata L.-4%.After stratification, the germination of S. capillata L. increased almost 3-fold and reached 45%, whereas the germination of S. pennata L. did not change [73].In the above examples, the effect of cold stratification on germination of Stipa seeds is ambiguous, and probably depends on the conditions and duration of stratification and the nature of species used.The relatively low germination rate of wild species may be attributed to the seeds' ability to germinate at varying times due to the presence of distinct types of organic dormancy.As a result, a seed bank is formed in the soils, which serves as a backup in times of difficult growing years [69].Furthermore, germination depends on weather conditions in the year of reproduction, population growth conditions, i.e., seed formation conditions, seed germination conditions, and seed genetics [56,71,74].
In our second experiment without pre-treatment (stratification and stimulators), after eight months of storage at room temperature in dark, seed germination rates varied from 0% to 55% (average 14%) (Table 3).The best germination was observed in S. sareptana, while seeds of S. adoxa and S. pulcherrima did not germinate (Table 3).Most of the seeds of S. sareptana were probably in a non-deep physiological dormancy, while the seeds of the other Stipa investigated had a deeper physiological dormancy.In the first experiment, after six months of storage at room temperature in the dark and 42 days of stratification, seed germination rates ranged from 21% to 57% and averaged 40% (Table 2).In the second experiment, after eight months of storage at room temperature in the dark and 21 days of stratification, seed germination varied from 0% to 79%, with an average of 23% (Table 3).After stratification, the germination of S. sareptana, S. dasyphylla, and S. pulcherrima increased.However, the germination of S. ucrainica and S. tirsa did not change, and S. adoxa did not germinate (Table 3).The decrease in average germination in the second experiment compared to the first was most likely due to the reduction in stratification by half.The cold stratification usually promotes seed emergence from physiological dormancy, and the deeper the dormancy, the longer the stratification period should be [68,69].
On the one hand, stratification can soften the seed-coat and improve its permeability.On the other hand, the process of stratification changes the physiological parameters of the seed: enzyme activity increases, hormonal balance changes, nucleic acids are synthesized, and all metabolic processes are activated.At low temperatures, the initial changes necessary for the synthesis of gibberellins also take place [69].As cold stratification stimulates the synthesis of endogenous gibberellins, treatment of seeds with exogenous gibberellins may replace cold stratification, as well as dry storage and light, to emerge from non-deep physiological dormancy [75].

Effect of GA 3 on Stipa Germination
According to Naylor and Simpson (1961), gibberellins have a dual effect.Firstly, at low concentrations (0.5 mg/L), they stimulate embryo metabolism.Secondly, at much higher concentrations (50 mg/L), they stimulate endosperm enzymes and promote the activity of hydrolytic enzymes in the aleurone layer of barley seeds [76].The main function in starch hydrolysis in the endosperm is performed by αand β-amylases.The α-amylases hydrolyze starch into oligosaccharides.β-amylases convert oligosaccharides into maltose [77].αamylase gene expression is regulated by the gibberellin-dependent transcription factor GAMyb [78].As a result, gibberellins secreted by the embryo initiate the expression of α-amylase genes in the aleurone layer.This leads to the lysis of starch granules in the endosperm and provides the young seedling with food substances [79].Thus, GAs are widely used to break the non-deep physiological dormancy of seeds [80].Hu et al. (2014) found that treatment of fresh S. bungeana seeds with GA 3 increased germination by 11% (from 25% to 36%) [66].In our experiments, germination of S. sareptana seeds increased by 25% (from 35% to 70%) after treatment with GA 3 (Table 6), while other Stipa seeds did not germinate at all.According to Nikolaeva et al. (1999), GA 3 , as a rule, has no effect on intact seeds with deep dormancy, even after partial stratification [69].This confirms our assumption that S. sareptana has a non-deep physiological dormancy, while other Stipa species have deeper physiological dormancy.

Effect of KNO 3 on Stipa Germination
Nitrate is a prevalent inorganic ion in soils.It has the ability to decrease seed dormancy and promote seed germination [65,80].Previously, potassium nitrate has been widely used to break the dormancy of cereal seeds [81].Nitrate promotes germination by binding NINlike protein 8 to the CYP707A2 promoter and activating its expression, thus reducing ABA levels after germination [82].Nitrate treatment (KNO 3 ) enhances seed quality potentially, and most likely, due to the fact that K + at optimum concentration in KNO 3 is used as a catalyst to enhance the metabolic activities of adenosine triphosphatase (ATPase) and nicotinamide dinucleotide (NAD).Additionally, K + promotes biosynthesis and the regulation of auxin activity in seeds [83].According to Roberts and Smith, (1977) the non-deep physiological dormancy of seeds may be disturbed by nitrite and nitrate (more often KNO 3 ) as hydrogen acceptors [84].Hu et al. (2014) found that treatment of fresh S. bungeana seeds with 1 mM KNO 3 for 14 days increased germination by 19% (from 25% to 44%) [66].In our own experiments, germination of S. sareptana seeds increased by 18% (from 35% to 63%) after KNO 3 treatment, while other Stipa seeds did not germinate under these conditions (Table 6).This also confirms our assumption that S. sareptana has a non-deep physiological dormancy, while other Stipa species have deeper physiological dormancy.

Effect of NaOH on Stipa Germination
Seed treatment with strong acids or alkalis promotes scarification, i.e., damage to the seed-coat for access to oxygen and water [65].If seed dormancy is associated with synthesis of inhibitors by the seed-coat, NaOH may promote the washing of these inhibitors from the embryo covering tissues [66].Hu et al. (2014) found that the treatment of fresh S. bungeana seeds with 30% NaOH for 20, 40, and 60 min increased germination rates from 25% to 45%; and 63% and 82%, respectively [66].In our own experiments, only NaOH treatment stimulated seed germination of S. ucrainica, S. tirsa, S. dasyphylla, and S. pulcherrima (Table 4).In the following experiments, germination of all Stipa seeds, except S. ucrainica, was observed after NaOH treatment (Table 6).

Effect of H 2 O 2 on Stipa Germination
Peroxide, as well as other inorganic reagents, is able to effectively modify the permeability of the seed-coat by scarification and thus break dormancy.Furthermore, H 2 O 2 acts as a respiratory inhibitor, while simultaneously promoting the pentose phosphate pathway, which plays an important role in seed germination [84].The activation of certain respiratory inhibitors may promote the germination of dormant seeds by suppressing cytochrome oxidase or catalase activity, leading to increased NADPH oxidase activity and, consequently, the induction of the pentose phosphate pathway.These resultson the reoxidation of NAD-PHxH, as suggested by Roberts and Smith, could be important for dormancy breaking.However, the function of NADPH in seed germination remains unclear.It is believed that NADPH plays a significant role in reductive reactions [84].In the current study, all Stipa seeds, except for S. pulcherrima, germinated after treatment with H 2 O 2 (Table 6).
It is possible that pre-treatment stimulators act in a species-dependent manner.In the case of the Stipa seeds studied, prolonged stratification (more than 40 days) combined with H 2 O 2 or NaOH pretreatment would be the optimal to stimulate seed germination (Table 7).

The Ability to Cryopreservation Is Species-Dependent
It is interesting to note that S. sareptana, the most widespread of the Stipa species studied, showed high germination rates and the best cryopreservation ability.Habitats of S. sareptana have been recorded in a widespread area from the south of the Nizhny Novgorod region from Russia to Tajikistan and from the eastern border of Kazakhstan to the west of the Rostov region of Russia.According to Lavrenko (1993), S. sareptana is the dominant formation in the deserted steppes of the Zavolzhsko-Kazakstan province [85].S. sareptana communities occupy large areas in almost all habitat types and are landscape-scale in this province, especially east of the Ural River [85,86].According to Tsvelev (2012), S. sareptana belongs to the Leiostipa section, is related to S. capillata [87], and is a xerophilous dense turfgrass [86].The other Stipa species studied in this work belong to the Eurasian section Stipa, are related to S. pennata [87], and are mesoxerophilic turfgrasses native to meadows and true steppes [86].It is probably that the characteristics that differentiate S. sareptana from other Stipa species, such as its greatest drought tolerance, lowest seed weight with the least amount of free water in it (unpublished data), and non-deep physiological dormancy, contribute to its survival under more extreme and stressful conditions and, therefore, correlate with its greatest resistance to cryostress.

Conclusions
As a result of the conducted research, the authors came to the following conclusions: all Stipa seeds studied were viable and had different germination rates depending on the species.S. sareptana had a non-deep physiological dormancy, while the other Stipa species studied had a deeper dormancy at the start.As germination of about half of the Stipa species seeds was stimulated by short-term cryopreservation and removal of the seed-coats, the seeds were in a non-deep physiological dormancy.Since germination of S. sareptana seeds was stimulated by KNO 3 and GA 3 , its seeds were in a non-deep physiological dormancy.As germination of other Stipa species was not stimulated by KNO 3 and GA 3 , their germination decreased over time; perhaps cryopreservation increased the depth of their physiological dormancy.It is possible that cryopreservation may have a negative effect on the germination of some orthodox seeds.NaOH treatment contributed to germination of S. adoxa and S. pulcherrima seeds after 5 years of cryopreservation.H 2 O 2 treatment contributed to germination of S. sareptana, S. ucrainica and S. dasyphylla seeds after 5 years of cryopreservation.
We believe it is necessary to continue research focused on the conservation of Stipa seeds by studying the seeds collected at different years, and also test the seeds with various moisture contents and stored at different temperatures.Further research is needed to minimize germination loss during seed storage.It would also be interesting to find out why some orthodox seeds are sensitive to cryopreservation and how this can be overcome.

Figure 1 .
Figure 1.Seed collection site of the Stipa samples.

Figure 1 .
Figure 1.Seed collection site of the Stipa samples.

Life 2023 , 19 Figure 2 .
Figure 2.After 14 months of storage in liquid nitrogen, S. sareptana seeds were thawed and germinated within 30 days.3.4.4.Experiment IV The fourth series of experiments evaluated the germination of Stipa seeds after four years of cryopreservation.The germination of Stipa was only observed after 90 days and that of S. adoxa after 180 days.Germination of all Stipa seeds decreased significantly (Table5).In this case, a longer germination period or a pre-treatment with stimulators is required to bring the seeds out of dormancy.

Figure 2 .
Figure 2.After 14 months of storage in liquid nitrogen, S. sareptana seeds were thawed and germinated within 30 days.

Table 1 .
Coordinates of the collection site of the Stipa samples.

Table 1 .
Coordinates of the collection site of the Stipa samples.

Table 2 .
Effect of short-term cryopreservation on germination and DI levels of Stipa seeds.

Table 3 .
Effect of stratification and cryopreservation on germination of Stipa seeds.

Table 4 .
Effect of pre-treatment with NaOH on germination of Stipa seeds.

Table 5 .
Effect of germination duration of Stipa seeds.

Table 6 .
Effect of GA 3 , KNO 3 , NaOH, and H 2 O 2 stimulators on germination levels of Stipa seeds.

Table 7 .
Summary: factors (duration of cryopreservation, presence and duration of stratification, treatment with stimulators, and duration of germination) affecting the germination of Stipa seeds.