Cent. Eur. J. Biol. • 8(1) • 2013 • 78-86 DOI: 10.2478/s11535-012-0114-4

Central European Journal of Biology

Testing achene germinationof Potamogeton praelongus Wulfen

Research Article

Romana Prausová1,*, Jana Janová1, Lenka Šafářová2

1Department of Biology, Faculty of Science, University of Hradec Králové, 500 02 Hradec Králové, Czech Republic

2East Bohemian Museum in Pardubice, 530 02 Pardubice, Czech Republic Received 14 May 2012; Accepted 14 September 2012

Abstract: The goal of this work was to determine the best method of breaking the achene dormancy in Potamogeton praelongus Wulfen. The ways of breaking achene dormancy studied in this experiment included methods of achene storage, stratification, UVA radiation, anaerobic conditions, mechanical disruption of achenes´outer layers and their chemical disruption by NaClO. Nine different treatments of achenes were combined with two methods of achene storage. Particular achene treatments and storage conditions were proven to have a significant impact on breaking dormancy. Although the highest germination rate (83.3%) was achieved when the dormancy was broken chemically by long effect of 100% concentrations of Savo detergent (containing 5% NaClO), the growth of the sprouts was subsequently inhibited due to toxic effects of Savo. Thus the most successful treatment was based on changing temperature, e.g. 2.5 months of cold storage followed by 14 days at room temperature (germination rate 32.7%). This treatment was also most similar to the natural process. Germinated achenes were also found in Petri dishes exposed to UVA radiation, anaerobic conditions and chemical disruption of the outer layers. Results of these treatments were influenced by the storage method.

Keywords: Dormancy of achenes • Potamogeton praelongus • Germination tests © Versita Sp. z o.o.

1. Introduction form special apical overwintering buds on rhizomes. Apical buds are yellow and conical (banana-shaped). Potamogeton praelongus is a rare plant all over the They are dormant storage organs that can survive and world. It is found in northern, mildly suboceanic and elongate under anoxia for a certain period, showing circumpolar regions. It grows primarily in the northern extremely strong tolerance to anaerobic conditions part of Europe and in similar latitudes in Asia and North [3]. P. praelongus reproduces almost exclusively America [1]. In the Czech Republic it is considered through vegetative reproduction. In the natural process, critically endangered and currently is only found in two achenes are probably only used for colonisation of new rather poor micropopulations in oxbows of the river sites. Achenes of P. praelongus are dark green, elliptic Orlice near Hradec Králové [2]. or obovate with a sharp dorsal keel, and their length is The vegetative season of P. praelongus starts in 4.5–5.8 mm [2]. Genus Potamogeton has a April when overwintering shoots begin to regenerate. macropodous-linear embryo [4]. It does not contain The flowering period is from late May to June. Fruiting an endosperm so food reserves are stored in a greatly shoots subsequently die and new sterile shoots grow enlarged hypocotyl [5,6]. Germination of the genus again. At the end of the vegetation season, sterile Potamogeton is limited primarily by dormancy. Its low shoots are about 0.5-0.6 m long, have living leaves germination potential can be overcome by scarification and are prepared to survive cold conditions during or stratification [7]. Passage of achenes through the the winter (Prausová unpublished data). They also digestive tract of aquatic birds may also facilitate

* E-mail: [email protected] 78 R. Prausová et al.

germination [8]. Genetic analyses detected a low level Potamogeton praelongus plant stand and usually about of intra- and interpopulation genetic diversity in Czech 70–85% of shoots are fertile. Each fertile shoot may micropopulations (M. Kitner et al., unpublished data) bear many spikes, which depends on the number of due to clonal growth, as known with other aquatic plant branches on the shoot. Collection of spikes was carried populations [9]. Generally, species which propagate out in the major part of the plant stand (where it was asexually exhibit low levels of population genetic possible). Due to clonality of this species it is difficult to diversity [10]. Present results indicate that the genetic estimate how many plants were comprised. The number variability of micropopulations can be considerably of collected mature achenes that were subsequently increased by sexual reproduction in rescue cultures. used for germination tests was about 2800 Before Since 2003, there has been a conservation beginning the tests, the achenes were ripped out of programme in the Czech Republic aimed at preserving spikes, cleaned, washed with tap water and dried at critically endangered Potamogeton praelongus. One 21±1°C and 10–15% RH. On average, there were 20 part of the programme focuses on establishing new mature achenes, 35 maturing achenes and 35 immature micropopulations in appropriate native locations of achenes on one spike when we collected them in this species by utilizing the plant’s own generative natural sites. The maturing achenes were able to finish reproductive abilities. These abilities are strongly maturing in jars with water in the laboratory at 21±1°C. restricted by dormancy which is caused mainly by mechanical restriction due to the achene’s hard outer 2.2 Germination tests shell – the pericarp and seed coat. During 2007–2010, Dried achenes were stored using two methods. the University of Hradec Králové ran a series of Storage A (dry-stored): dry storage of achenes in germination tests focused on the investigation of paper bags at 21±1°C. breaking achene dormancy and on encouraging Storage B (dry/submerged): dry storage at 21±1°C generative reproduction of this species. A preparatory at first, then, one month before beginning the tests, survey of twenty seven treatments of achenes in achenes were submerged in water in glass jars with germination tests was described by Prausová et al. [11]. porous lids and placed in a refrigerator at 8±1°C. All This paper deals with nine treatments that are supposed germination tests were done during winter and early to successfully suppress the dormancy (mechanical spring from December to April, e.g. 4–9 months after damage of the achene’s hard outer shell, cultivation collection of achenes. in anaerobic conditions, temperature changing, UVA The goal of the germination tests was cultivation radiation, short and long effect of 5% NaClO and 2.5% of the achenes after exposing them to nine different NaClO, application of Gibberellic acid, and a control treatments that were supposed to break the dormancy. test). These nine treatments were repeated in two These nine treatments were repeated in two series which series which differed in the method of achene storage, differed in the method of achene storage, i.e., there were i.e. there were 18 different variants (treatment 1–9 with 18 different variants (treatment 1–9 with storage A, B). storage A, B). Treated achenes were cultivated in 10 cm Petri dishes There were two objectives in this experiment: How to (50 achenes/1 Petri dish) on damp filter paper, in the break the achene dormancy and which of the treatments darkness in a temperature-controlled incubator at 28°C. could work in natural sites where P. praelongus grows. During the tests, the achenes were regularly dampened Three successful methods out of the 18 were chosen to with filtered water from the oxbow near the pond Stříbrný be closer examined in further studies. Rybník (a locality where the species currently grows). Successfully germinated sprouts with 1–2 leaves were planted into a climatised growth chamber, where they 2. Experimental Procedures were allowed to grow further in 30–40 cm deep glass containers under these conditions: temperature 21°C, 2.1 Seed collection light cycle: 16 hours light, 8 hours dark [12]. Mature achenes of Potamogeton praelongus collected Eight treatments were applied to the achenes prior during July and August from the reserve population in to germination tests, one of them (No.2 – anaerobic restored backwater pools in the Protected Landscape conditions) was arranged during the whole germination Area Kokořínsko in Central Bohemia (Štampach pool test. 50°23‘43“N, 14°33‘11“E, the plant stand area about Treatments used in the experiment: 30 m2; Plešivec pool 50°32‘10“N, 14°34‘12“E, the plant 1) Scarification (mechanical damage with sandpaper) stand area about 20 m2) were used for the germination – the pericarp and much of the seed coat were removed. tests. There are about 150–200 shoots in 1 m2 of the 2) Anaerobic conditions – arranged by regularly

79 Testing achene germinationof Potamogeton praelongus Wulfen

adding several drops of ethanol to Petri dishes and taping activate the germination process and was applied the lids with several layers of tape. Anaerobic conditions directly to the Petri dishes containing achenes for were maintained during the whole germination test. germination tests. This treatment simulated the conditions that achenes The experiment of gaining achenes by their passing experience in sediments on pond bottoms. through the digestive tract of aquatic birds resulted 3) Stratification (changing temperature) – achenes in a really low number of achenes. The test of their stored for 2.5 months in water in glass jars in a refrigerator germination therefore could not happen. at 8°C followed by 14 days in a room at 21±1°C. Photodocumentation of P. praelongus achenes 4) UVA radiation – irradiation of achenes in open (without or after a treatment) and their cut sections Petri dishes with UVA radiation for 30 minutes. Radiation were made on the stereomicroscope Olympus SZX18. came from an OSRAM HQV 125 UV lamp (input Results of the successful treatments were compared 125 W, output 3 W), which provides ultraviolet rays in with each other and statistically evaluated. New results 300–400 nm band. The center of the lamp was were compared with published results of other authors. positioned over the middle of the Petri dish, 13 cm above the achenes. This was the first time that the effect of 2.3 Statistical evaluation UVA radiation on germination of seeds of P. praelongus Statistical analyses were done using NCSS 2001 software was studied. [13]. Log-linear models were used for the analysis of the 5) Savo 50% (active agent 2.5% NaClO) – achenes treatment and storage impact. Contingency tables were soaked in 50% concentration of Savo for 2 hours, used for the analyses of the achenes‘ storage in the subsequently rinsed with running water. The commercial single test version. We excluded treatments No.8 (Long disinfectant and cleanser Savo contains 5% NaClO and effect of Savo 100%) and No.9 (Application of Gibberellic was used to chemically disrupt the achenes’ husks in acid) from all analyses because no achenes germinated thus breaking dormancy. in treatment No.9 and toxic effects of Savo were found 6) Short effect of Savo 100% (active agent in treatment No.8. A survey of 7 successful treatments in 5% NaClO) – achenes soaked in 100% concentration germination tests is in Table 1. of Savo for 2 hours, subsequently rinsed with running water. 7) Control test without any manipulation before 3. Results starting. 8) Long effect of Savo 100% – achenes soaked in Statistical analysis of germination through the log-linear 100% concentration of Savo for 36 hours, subsequently models showed that treatment and storage conditions rinsed with running water. have a significant impact on breaking achene dormancy 9) Application of Gibberellic acid in concentration (P<0.001). The interaction of both factors (P<0.001) is of 10–20 mg l-1. This growth hormone is supposed to also important. Using contingency tables, we examined

Number of achenes Test statistics DF P Treatment A B

1 Scarification - mechanical damage 150 150 3.055 1 0.08

2 Anaerobic environment 100 200 16.667 1 <0.001

3 Stratification - temperature changing 100 150 29.515 1 <0.001

4 UVA radiation 100 100 5.556 1 0.018

5 Savo 50% (2 hours) 100 100 4.348 1 0.037

6 Short effect of Savo 100% (2 hours) 100 100 0.272 1 0.602

7 Without any manipulation before starting test 100 100 0.244 1 0.621

8 Long effect of Savo 100% (36 hours) 100 100 excluded from the statistical test 9 Gibberellic acid 100 100

Table 1. Numbers of tested achenes used in germination tests in various treatments of achenes dormancy breaking and summary of the statistical analysis. Methods of storage: A (dry-stored) – stored in paper bags at 21±1°C exposed to the air, B (submerged/dry) – stored in paper bags at 21±1°C exposed to the air, subsequently submerged in water and stored in a refrigerator at 8±1°C for 1 month before the germination test. Germination differences between two storage groups were tested by Chi-Square test, P-values in bold are significant (P<0.05).

80 R. Prausová et al.

35

30

25

20

15

10 Germination rate (%) rate Germination 5

0 1234567 Treatment

Figure 1. Results of 7 successful treatments of germination tests. Differences in achene storage were tested through the Contingency tables, P-values in bold are significant (P<0.05). Dormancy interruption methods: 1 – mechanical damage (P=0.08), 2 – anaerobic environment (P<0.001), 3 – temperature cycling (P<0.001), 4 – UV radiation (P=0.018), 5 – Savo 50% (2 hours, P=0.037), 6 – Savo 100% (2 hours, P=0.602), 7 – control test without any manipulation before starting (P=0.621). Methods of storage: black column – stored in paper bags at temperature 21±1°C exposed to air, white column – stored in paper bags at temperature 21±1°C, exposed to air, subsequently submerged in water and stored in a refrigerator 8±1°C for 1 month before germination test. the effect of particular storage methods on the achenes’ NaClO, respectively). No inhibition of germination or germination. In four treatments (2, 3, 4, and 6 – anaerobic subsequent growth was observed. Germination rates conditions, stratification, UVA radiation and short effect were higher with dry-stored achenes in treatments 5A of Savo 100%) we found a statistically significant impact (12%) and 6A (9%) than with dry/submerged achenes of storage (Table 1). Only the first 7 treatments were in 5B and 6B, where germination rates were 4 and 7%, successful (i.e. seeds germinated and sprouts grew respectively. further). Figure 1 shows the results of these successful Achenes in the control group without any treatment germination tests. germinated in both types of storage (7A 8%, 7B 10%). Germination rate in treatment 1 (mechanical Treatments 3B, 2B and 4B were the most damage using sandpaper on the pericarp and seed successful methods that also simulated the natural coat) was lower than in the control test without any growing conditions of the achenes. Treatment 3B was manipulation before starting. Dry-stored achenes in temperature stratification of dry/submerged achenes treatment 1A showed lower germination rate (3.3%) with 32.7% germination. Treatments 2B and 4B than the dry/submerged ones in treatment 1B (8%). (anaerobic conditions and exposition to UVA radiation, In treatment 2, while exposed to anaerobic conditions, both with dry/submerged achenes) germinated at achenes achieved germination rates of 15% in 15%. treatment 2B, dry-stored ones (treatment 2A) did not The treatment 8B with achenes soaked in Savo germinate at all. The most successful treatment and 100% for 36 hours resulted in a high percentage of closest approximation to the natural process was germination (83.3%). However, the growth of the treatment 3B (32.7%) – temperature changing with sprouts was subsequently inhibited due to the toxic storage B. Germination rates in treatment 3A were 4%. effects of Savo. The detergent completely removed the Germinated achenes were also found in Petri dishes pericarp and seed coat, but also affected the embryo. exposed to UVA radiation for 30 minutes (treatment Most of the sprouts ended their growth at about 1.3 cm 4). Achenes from storage B achieved germination length. Treatment 8A was not carried out after having rates of 15% (treatment 4B), and dry-stored ones 5% seen the toxic effects of long exposition to Savo (treatment 4A). 100%. Treatment No.9 (application of Gibberellic acid) Successful germination was also seen in treatments resulted in no germination at all. 5 and 6, where dormancy breaking was achieved Differences in achenes’ disruption due to particular by the use of Savo detergent in concentrations 50% Savo’s concentrations and exposure times, due to and 100% for 2 hours, respectively (i.e. 2.5% and 5% endozoochory and mechanical damage, are shown

81 Testing achene germinationof Potamogeton praelongus Wulfen

Figure 2. Cut section of long-leaved pondweed achene – protection Figure 3. Cut section of long-leaved pondweed achene – disruption of embryo by pericarp and seed coat. of embryo by 100% concentration of Savo applied for 4 hours.

in the stereo photomicrographs (stereomicroscope Olympus SZX18) in Figures 2–7. Control achenes without any manipulations before starting tests are shown in Figure 2. The pictures clearly show that the achenes are protected by both the pericarp and seed coat (Figure 2), and after removal of these layers, damage to the embryo is possible. In our germination tests, damage to the embryo was seen as early as 4 hours after soaking in 100% Savo (Figure 3). The embryos of achenes affected by 50% Savo for 2 hours weren’t damaged (Figure 4).

4. Discussion

Figure 4. Cut section of long-leaved pondweed achene – Some authors [14] claim that sexuality in aquatic plants preservation of embryo, damage to pericarp and seed is a holdover and the trend is towards asexuality as a coat by 50% concentration of Savo applied for 2 hours. means of preserving adapted genotypes in a stable aquatic environment. Genetic analyses (M. Kitner et al., at 1–3°C for 2–3 months. Muenscher [15] reported unpublished data) have discovered a low level of overall 0% germination of other wide-leaved pondweed genetic variability in Czech micropopulations. Current P. crispus seeds stored in dry conditions. The same efforts to preserve the species in the Czech Republic (0% germination) results were shown by Wehrmeister use generative reproduction to restore new genetically [16] with P. crispus seeds stored for over a year in wet variable populations in original historic locations where conditions. Germination rate of P. crispus seeds from the species died out due to human activity, such lakes in South Africa was 0.001% in tests under natural as water pollution, inappropriate regulatory actions conditions [17]. Teltscherová and Hejný [18] report on watercourses, culturing herbivorous fish in river 30–40% germination of P. crispus seeds collected from channels, etc. Germination of the genus Potamogeton ponds in South Bohemia under stratification, scarification, can be influenced primarily by physiological dormancy and application of HCl together with Pancreatin (similar interruptible by stratification and by mechanical barriers to the passage of achenes through the digestive tract of interruptible by scarification [7]. Muencher [15] studied birds). Laboratory germination of seeds from 10 British seed germination in 21 species of Potamogeton. species of Potamogeton [19] showed that maximum The germination rates were 0–94%. P. praelongus germination ranged from 13% (P. polygonifolius) to 90% germinated successfully (germination rate 18%) in (P. berchtoldii). Germination was mainly influenced by one series only where seeds were stored in water cold stratification, changing temperatures, oxygen and

82 R. Prausová et al.

light conditions. Cold stratification was very important test without any manipulation before starting. This could for germination of P. lucens and P. perfoliatus which be caused by a low grade of hand-made damage and by belong to the group of wide-leaved pondweeds like contamination with microorganisms. P. praelongus. Those three species grow in similar Statistical analyses of our germination tests from the sites with comparable conditions. They occur in slow- years 2007–2010 confirmed that experimental methods flowing rivers and streams, lakes and sandpits with of breaking dormancy do affect germination, as do relatively high water transparency. Their stalks with storage conditions. Application of the growth hormone alternate leaves are submerged and they overwinter Gibberellic acid (GA3), which was supposed to interrupt due to rhizomes and apical overwintering buds. Hay the embryo’s dormancy, produced no germination. Thus et al. [19] found maximum levels of germination with our results confirmed the conclusions of published P. lucens at 30% and P. perfoliatus at 65%. The low germination tests conducted on different species germination rates of our tests may have been caused by of pondweed [18,23] that the inability of pondweed contamination of microorganisms in Petri dishes during achenes to germinate is not caused by the dormancy of the tests. However, contamination is an unavoidable part the embryo, but by the mechanical pericarp barrier of the of the natural process occurring on the bottom of rivers, seeds and achenes which blocks out water. Comparisons lakes or oxbows. Waisel [20] reported that seeds may of our test treatments showed that chemical damage be important in dispersal of wide-leaved pondweeds to the pericarp and seed coat were more effective (e.g. P. alpinus, P. lucens, P. perfoliatus, P. praelongus) than mechanical damage using sandpaper [24]. but not in the development of standing local crop. Anyway, results from the application of Savo showed Genetic analysis (M. Kitner et al., unpublished data) that the damage must be carefully controlled, so that confirmed that Czech micropopulations ofP. praelongus it does not proceed further into the embryo. Optimal prefer vegetative reproduction. It can be a strategy that damage to the seed coats is achieved at 100% Savo favors well adapted genotypes in sites with specific concentration for 2 hours, or a weaker concentration conditions. Other wide-leaved pondweed P. alpinus, for longer. Good germination rates (without damage to which lives together with P. praelongus in the last Czech the embryo) were also seen with 50% Savo solution natural population in the oxbow of the river Orlice, has for 2 hours. It is assumed that the passage of achenes a very similar morphology to P. praelongus but prefers through the digestive tract of aquatic birds is a path to streams, has natant leaves and creates terrestrial aid germination, since the achene husks are damaged forms. Brux et al. [21] studied growth and reproduction by abrasion in the digestive tract [8]. High germination of P. alpinus. It germinated easily in 2 to 4 weeks after (60%) of Potamogeton natans achenes was found after beginning the germination test. In comparison with a passage through the digestive tract of domestic ducks growth of germinating turions (e.g. overwintering buds [25]. The damage degree of an achene of P. praelongus on rhizomes), the germinating seeds were very small retrieved after passing through the digestive tract of a and slow growing. They did not reach the water surface domestic duck corresponds with that one achieved by in that year but turions did [21]. using 50% Savo concentration for 2 hours or mechanical Anaerobic conditions were very necessary for the damage with sandpaper. Unfortunately, we were not germination of P. berchtoldii and P. pusillus that grow in shallow waters [19]. Teltscherová and Hejný [18] report better germination rates when the achene husks were damaged with the aid of microorganisms in decaying water than by mechanical disruption of the pericarp and seed coat with scalpels. Likewise, our tests showed better germination rates in an anaerobic environment (treatment 2) than in the case of mechanical damage with sandpaper (treatment 1). Fischer [22] states that a chemical stimulus caused by fermentation and decomposition of organic material in ponds is required for germination of Potamogeton seeds. Scarification [19], e.g. mechanical abrasion of pericarp and testa, also improved germination of pondweed seeds (with several species it increased to about 80%). Our results show that the germination rate of achenes that were mechanically Figure 5. Long-leaved pondweed achene after passage through damaged using sandpaper was lower than in the control the digestive tract of a domestic duck.

83 Testing achene germinationof Potamogeton praelongus Wulfen

Figure 6. Long-leaved pondweed achene after application of 50% Figure 7. Long-leaved pondweed achene after mechanical damage concentration of Savo for 2 hours. using sandpaper.

able to get enough achenes exposed to endozoochory This confirms the claim that the greatest sensitivity to UV for our tests. radiation occurs after the achene begins to absorb water The most successful treatment of our germination [27]. Subsequent growth of the sprouts was not tracked tests without using chemicals was the temperature individually, so it is not possible to confirm or refuse variation: 2.5 months in a refrigerator/14 days at room retardation of the plants‘ growth. temperature which simulates the natural seasonal Our germination test results showed higher variation experienced by an achene after leaving germination of achenes stored in paper bags at 21±1°C, its parent plant. The husks are damaged by these exposed to air, then subsequently submerged in water conditions, cracking the shell and allowing water to get for 1 month before the germination test except for into the embryo. An interesting finding connected to this chemical disruption of husks. But also achenes stored treatment was a significantly higher germination rate in in dry conditions were able to germinate (except for achenes simultaneously exposed to humidity variations, anaerobic conditions). i.e. stored dry, then submerged in water for 1 month Guppy [25] states that seeds of Potamogeton keep before beginning the germination tests. their ability to germinate for several months of a dry Another successful treatment of the germination period. His germination tests of Potamogeton natans test, during which achenes were exposed to UVA for were successful after a 3-month dry period, conversely 30 minutes, leads to the conclusion that achenes could all achenes died after slow 30 months’ drying time. survive in terrestrial conditions, where they dry out and are Germination tests carried out in this study showed that immediately exposed to UV radiation. During subsequent for breaking the dormancy of P. praelongus achenes it is increased humidity they would germinate. Increased important to remove or at least to disrupt the outer layers of germination rates upon UV exposure were documented the achene and of the seed, so that water can penetrate to in other studies [26-28], during which UVB radiation was the embryo. Then, under suitable temperature conditions, used in the 280–320 nm wavelength band. The more seeds begin to germinate. Previous results [11,12] energetic UVB radiation significantly impacts the outer showed that germination is more successful under higher surface of plant cells, and increases and accelerates and balanced temperature (with minimum fluctuation). germination, but subsequently causes undesirable Unlike in other germination tests of Potamogeton species shortening of the hypocotyledonous stem, forming thicker [15-19,21,23], UVA radiation and application of Savo and curlier root balls [26]. Seeds are most sensitive to UV detergent were used as methods to break dormancy. radiation after they begin to absorb water. For example, The most effective method of breaking dormancy was in tests on Phacelia tanacetifolia, this period began changing temperature, which is common in natural 13–17 hours after the beginning of water absorption [27]. conditions. Alongside, another near-natural method is In our germination tests of Potamogeton praelongus, we germination under anaerobic conditions. Temporary used UVA radiation, which is more energetic than visible anaerobic conditions are common in P. praelongus light, but less so than UVB. UVA radiation produced biotope in the last Czech localities (muddy bottom of significantly higher germination rate (15%) in achenes an oxbow or a pool with accumulated organic litterfall submerged in water for 1 month before starting the tests. from bank stands). On the base of these findings,

84 R. Prausová et al.

we can suppose that supporting and reinforcing the conditioned boxes and their transfer into artificial water natural process is probably the most effective way to pools to be grown. Then the sprouts will be planted in achieve more germination of P. praelongus achenes. selected natural localities within the rescue programme Next tests P. praelongus germination will be aimed at [24]. methods that correspond with the natural process, i.e. germination in water (water taken from current localities of P. praelongus or substrates used for sterile tissue Acknowledgements cultures, etc.). We intend to continue our germination tests with achenes submerged in about 3–5 cm water The authors would like to thank J. Špaček for or physiological medium column. This experiment is photodocumentation of P. praelongus achenes and their thought to have less contamination of microorganisms cut sections. We would like also to thank M. Jůzová for and to be closer to natural conditions in the current English language corrections. The study was supported sites of P. praelongus. Further attention will be focused by the funds awarded by EHP/Norway and the Ministry on methods of subsequent cultivation of sprouts in air- of Environment of the Czech Republic.

References

[1] Vöge M., Development of Potamogeton [11] Prausová R., Janová J., Adamec L., Rescue of praelongus in Grossensee lake near Hamburg the critically endangered long stalked pondweed [Die Entwicklung von Potamogeton praelongus im (Potamogeton praelongus) in the Czech Republic, Grossensee bei Hamburg], Tuexenia, Göttingen, Acta Biologica Slovenica, 2011, 54, 43–54 1992, 12, 275–284 (in German) [12] Janová J., Long stalked pondweed (Potamogeton [2] Kaplan Z., Potamogeton L. – rdest, In: Chrtek J. Jr., praelongus WULFEN) in the Czech Republic [Rdest Kaplan Z., Štěpánková J. (Eds), Květena České dlouholistý (Potamogeton praelongus WULFEN) v republiky 8. Vyd. 1., Academia, Prague, 2010 České republice], MSc thesis, University Hradec [3] Koizumi Y., Hara Y., Yazaki Y., Sakano K., Ishizawa Králové, Czech Republic, 2010, (in Czech) K., Involvement of plasma membrane H+-ATPase [13] Hintze J., NCSS 2001 – Number Cruncher in anoxic elongation of stems in pondweed Statistical System, Kaysville, Utah, USA, 2001 (Potamogeton distinctus) turions, New Phytologist., [14] Less D.H., Breeding systems, population structure, 2011, 190, 421–430 and evolution in hydrophilous angiosperms, Ann [4] Martin A.C., The comparative internal morphology Mo Bot Gard, 1988, 75, 819–835 of seeds, Am. Midl. Nat., 1946, 36, 513-660 [15] Muenscher W.C., The germination of seeds of [5] Goebel K., Organography of plants, Part II, Potamogeton, Ann. Bot., 1936, 50, 805–821 Clarendon Press, Oxford, 1905 [16] Wehrmeister J.R., An ecological life history of [6] Dahlgren R.M.T., Clifford H.T., The Monocotyledons the pondweed Potamogeton crispus L. in North A Comperative Study, Academic Press, New York, America, MSc thesis, Ohio State University, 1982 Columbus, Ohio, 1978 [7] BaskinC.C., Baskin J.M., Seeds: Ecology, [17] Rodgers K.H., Breen C.M., Growth and reproduction biogeography, and evolution of dormancy and of Potamogeton crispus in a South African lake, J. germination, Academic Press, San Diego, 1998 Ecol., 1980, 68, 561–571 [8] Santamaria L., Charalambidou I., Figuerola J., [18] Teltscherová L., Hejný S., The Germination of Green A.J., Effect of passage through duck gut Some Potamogeton Species from South Bohemian on germination of fennel pondweed seeds, Arch Fishponds, Folia Geobot. Phytotax, 1973, 8, 231– Hydrobiol, 2002, 156, 11–22 239 [9] Waycott M., Assessment of genetic variation and [19] Hay F., Probert R., Dawson M., Laboratory clonality in the seagrass Posidonia australis using germination of seeds from 10 British species of RAPD and allozyme analysis, Mar. Ecol. Prog. Potamogeton, Aquat Bot, 2008, 88, 353–357 Ser., 1995, 116, 289–295 [20] Waisel Y., Seasonal activity and reproductive [10] Loveless M.D., Hamrick J.L., Ecological behaviour of some submerged hydrophytes in determinants of genetic structure in plant Israel, Hydrobiologia, 1971, 12, 219–227. populations, Ann. Rev. Ecol. Syst., 1984, 15, 65– [21] Brux H., Toeskino D., Wiegleb G., Growth and 69 reproduction of Potamogeton alpinus, Arch

85 Testing achene germinationof Potamogeton praelongus Wulfen

Hadrobiol. Beih. Ergebn. Limnol., 1987, 27, 115– [25] Guppy H.B., Germination of seed of aquatic plants, 127 Proc. Royal Phys. Soc. Edinb., 1894–1897, 13, [22] Fischer A., Hydrogen and hydroxyl such conditions 344–360 germination [Wasserstoff- und hydroxylionen als [26] Farokh P., Mahmoodzadeh H., Satari T.N., Keimungsreize], Ber. Deutsch. Bot. Gesells., 1907, Response of Seed Germination of Safflower to 15, 108-122, (in German) UV-B radiation, RJES, 2010, 4, 70–74 [23] Crocker W., Germination of Seeds of Water Plants, [27] Schulz M.R., Klein R.M., Effects of visible Bot. Gaz., 1907, 375–380 and ultraviolet radiation on the germination of [24] Prausová R., Adamec L., Kitner M., Pásek K., Phacelia tanacetifolia, Amer. Jour. Bot., 1963, Dvořák V., Janová J., et al., Rescue of the long 50, 430–434 stalked pondweed (Potamogeton praelongus) in [28] Tosserams M., Bolink E., Rozema J., The effect the Czech Republic [Záchrana rdestu dlouholistého of anhanced ultraviolet-B radiation on germination (Potamogeton praelongus) v České republice], and seedling development of plant species Final report, Agency for Nature Protection of the occurring in a dune grassland ecosystem, Plant Czech Republic, Prague, 2010, (in Czech) Ecol, 1997, 128, 139–147

86