seed germination and seedling development of ...

Доклади на Българската академия на науките Comptes rendus de l’Acad´ emie bulgare des Sciences Tome 62, No 7, 2009

BIOLOGIE Botanique

SEED GERMINATION AND SEEDLING DEVELOPMENT OF THREATENED BULGARIAN ENDEMIC OXYTROPIS SPECIES (FABACEAE) Ekaterina Koˇ zuharova, Adrian-John Richards∗ (Submitted by Academician A. Atanasov on March 25, 2009)

Abstract Oxytropis urumovii J´ av. is a local endemic to the marble part of North Pirin Mts. Oxytropis kozhuharovii Pavlova, Dimitrov and Nikolova has even more restricted distribution in Pirin Mts. Both plant species reproduce by seed and have poor vegetative propagation. There is no direct human threat to these plants at the moment except hiking, but they are well localised in the wild. Due to active ski-resort development in Pirin Mts. their conservation becomes a matter of some urgency. In this study we tested the seed germination process, development of the seedlings and ex-situ seedling behaviour. Most mature test seeds would germinate within a couple of days if the seed coat was scarified. The seedlings were rather sensitive and many did not survive – the possible reasons were discussed. The seedlings of the tetraploid O. kozhuharovii developed slightly better than those of the diploid O. urumovii and during the second year after germination several seedlings bloomed. Key words: Oxytropis urumovii, Oxytropis kozhuharovii, seed germination, seedlings, Bulgaria The experimental alpine garden was created with the financial support of Ford Motor Company – Conservation and Environmental Grants 2005 and British-Bulgarian Friendship Society, grant 2006. The study was conducted with the financial support of the National Science Fund, Ministry of Education and Science, project Б – 1540/05.

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Introduction. Two alpine species of Oxytropis, O. urumovii Jav. and O. kozhurahovii Pavlova, Dimitrov and Nikolova are local endemics to the marble part of Northern Pirin Mts. O. urumovii is localised to the northern marble part of Pirin Mts., above 2000 m [1, 2 ]. Although it is not locally frequent, the population occupies a large territory part of it within a protected region, and in our judgement its status is not currently under threat. Its IUCN category is Vulnerable according to the Red Data Lists of Vascular Plants and Fungi in Bulgaria (unpubl.). The only known locality for O. kozhuharovii lies in the northernmost part of Pirin, known as the Yavorov Anticline, where it was discovered and described recently [3 ]. Plants are limited to an area of steep rough grassland on marble. About three thousand mature plants were discovered after extensive searches but they occupy a territory of only about 200 m2 . The locality is very remote and for the moment is rarely visited by man, and not heavily grazed by domesticated animals, but might be threatened by land-slips or avalanches [4 ]. Its IUCN category is Critically Endangered according to the Red Data Lists of Vascular Plants and Fungi in Bulgaria (unpubl.). The real threat is the current active development of this mountain as a huge ski resort. O. kozhuharovii is a distinctive species which must be regarded as amongst the most threatened plants in Europe. There is an urgent need to promote its regeneration. Due to the rigid seed coat leguminous seeds (family Fabaceae) are known to require specific conditions for germination. Scarification has often been used to increase the germination of legume seed (Oxytropis, Astragalus, Hedysarum, Lupinus) and the effect was often significant – resulting in up to 100% germination [3, 5–7 ]. In two rare and endemic Oxytropis of the Russian Far East, O. chankaensis and O. retusa, cryopreservation influenced not only seed viability, but also the dynamics of germination. Low temperature storage did not impair the viability of seeds, but markedly improved it compared to the control [6 ]. The seed of four Astragalus species germinated at any temperature (13 – 34 ◦ C) if they were scarified [8 ]. Peak germination percentage for Astragalus australis var. olympicus occurred at alternating temperatures (15 ◦ /25 ◦ C) and at moisture availability with low water potential (distilled water) when combined with scarification [9 ]. The aim of this preliminary study is to test the seed germination and development of the seedlings of Oxytropis urumovii and O. kozhurahovii. Raising seed in cultivation of these two rare and threatened endemics is the first step that will make possible reintroduction in nature when it is necessary. Material and methods. Seed was collected following the accepted protocol [10 ], e.g., when seed pods start cracking, the pods were harvested by hand. Some pods were collected closed. The seed of Oxytropis kozhuharovii was collected in September 2005 while that of O. urumovii was collected both in September 2005 and September 2006, all from the native populations. The seed was kept cool (4 ◦ C in the fridge but not frozen). Some seed of O. kozhuharovii was kept at 832

E. Koˇzuharova, A.-J. Richards

room temperature. In 2007 were collected nine seeds from a plant O. kozhuharovii grown ex situ from seed in 2006. Several sets of seeds (20–135 at a time) kept cool (4 ◦ C in the fridge, but not frozen), or kept at room temperature, were processed for germination at a natural light – dark photoperiod. This took place in Sofia during the period 20th February – 6th May 2006, 5th March – 5th May 2007, and 12th March – 20th April 2008 at temperatures between 10–25 ◦ C; first in greenhouse conditions, and later outdoor on shaded place with south-west exposure. Scarification of the seeds was done with a needle (in 2006) or with fine sand paper (in 2007 and 2008). The seeds (n = 9) collected from individual O. kozhuharovii grown ex situ from seed in 2006 were processed for germination in 2008 with a sand paper scarification procedure. The seeds were placed in Petri dishes on wet filter paper. Seeds of both Oxytropis species were presoaked in tap water in beakers in 2006 and 2007 having in mind that scarification is also likely to damage parts of the embryo that would cause rotting later. As controls for O. kozhuharovii one seed sample (n = 50) in 2006 and one seed sample (n = 25) in 2007 , previously all kept cool, were soaked in water before germination without scarification. In addition one seed sample (n = 80) previously kept at room temperature was soaked in water but not scarified in 2007. For O. urumovii one seed sample (n = 38) collected in the previous year and kept at room temperature was soaked in water but not scarified in 2007 (Table 1). Once the seeds had germinated, the seedlings were transferred individually to plastic pots filled with a mix of 30% rough marble sand, 30% sieved humus, 30% sieved good soil, 10% perlite or with a mix of 60% silty brown soil and 40% rough marble sand. Watering was from below at first (pots stood in 3 cm tap water, Plate 1, Fig. 1). When seedlings were at the 2–6 true leaf stage, they were transported to the experimental rock garden in the foot hill of Pirin Mts. near Dobrinishte village. They were planted singly into the flower beds with fine marble gravel top-dressing (up to 1 cm size, Table 1, Plate 1, Fig. 2). The experimental rock garden was established in 2006 in hay meadow of approximately 500 m2 near the river situated at 865 m above sea level on the steepest part of a hay meadow with an exposure to north-east. The place was chosen with consideration for several factors: i) close enough to the river for watering; ii) away from potential floods; iii) moderately shaded; iv) the snow lies relatively long here protecting the plants from the spring frosts and providing a cool microclimate in summer. Results and discussion. Most mature seeds of the test plants germinated very well within a couple of days if the seed coat was scarified (Table 1). Stratification appeared to be unnecessary. Slight decrease of seed germination was observed with time (Table 1). The germination differed significantly between the samples. The germination of each seed sample O. kozhuharovii was as follows: in 2007 – Compt. rend. Acad. bulg. Sci., 62, No 7, 2009

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834 Table

1

Seed germination and development of the seedlings

Plant species

Samples

Time of experiments (seed collection)

number of seed and treat

Germination Average (min, max) [%]

Number of germinated seeds

O. kozhuharovii 2006 (leg. 2005) 2007 (leg. 2005) 2008 (leg. 2005) (leg. 2007)

300 198 105 389 9

sc sc so sc sc

99 81 (56, 100) 1 56 (2, 81) 89

297 160 1 226 8

O. urumovii

27th May

2006 4th Aug.

21th Oct.

Planting Number of seedlings [% survivals of the germinated seeds] 25 (8%) 14 11 –

–

–

–

–

–


Number of seedlings [% survivals of the germinated seeds] 14 8 6 (14%)

2006 (leg. 2005)

100 sc

99

99

2007 (leg. 2006)

49 sc 38 so

90 (83, 100) 3

44 1

–

–

2008 (leg. 2006)

38 sc

95

37

–

–

15th April

2007 24th May 16th June

10th Aug.

Planting Number of seedlings [% survivals of the germinated seeds] 10 10 10 69 (43%) – 47 1 (100%) –

–

3rd May

2 18 –

Number of seedlings [% survivals of the germinated seeds]

2008 9th 2nd June Aug.

Planting Number of seedlings [% survivals of the germinated seeds] 2 1 12

12

116 (51%) 80 4 (50%) Number of seedlings [% survivals of the germinated seeds]

11th Sept

1 12 40

5

4

3

1

1

1

1

–

–

38 (86%) 0 (0%)

20

6

4

4

4

–

–

–

–

24 (58%)

19

9

Legend: sc – scarified, so – soaked in water without scarification

56% (n = 20); 63% (n = 80); 65% (n = 20); 100% (n = 73) while in 2008 – 2% (n = 98); 61% (n = 135); 81% (n = 106). The germination of each seed sample O. urumovii was as follows: in 2007 – 83% (n = 29); 100% (n = 20); while in 2008 – 95% (n = 39). It is known that the generally accepted temperature conditions for storing the seed material (4 ◦ C) do not ensure its perfect preservation [11 ]. However, we should not explain the lower germination by this fact alone. As some pods were collected in an indehiscent state, possibly the seed was not completely ripe. Two facts support such assumption: Firstly germination of 100% (n = 73) in 2007 was achieved as a result of seed kept at room temperature for 2 years, so low temperature storage is not vital for good germination. The germination of the “ex situ” seeds collected in 2007 was 89% (n = 9). Also the lowest germination 2% (n = 98) was a result of a seed sample collected deliberately from indehiscent pods so that their seed set could be recorded. In addition it is possible that some seeds were not viable due to other reasons, e.g., inbreeding depression – further investigations are necessary. The germination of control O. kozhuharovii seeds stored cool and not scarified was 10% in 2006 (n = 50). It was even lower in 2007 – only one seed (4%) of the seed sample (n = 25) of the cool stored unscarified seed germinated within four days. None of the unscarified seeds stored at room temperature (n = 80) germinated in 2007. Only one O. urumovii seed (3%) of the control sample (n = 38) germinated in 2007 (Table 1). The observed germination behaviour in this study corresponds to germination reported for other Oxytropis species – the loss of seed coat impermeability after sulfuric acid treatment (20 min and then washed in fresh water) resulted in active seed germination (79.2% – 93.6%), indicating that seed dormancy in Oxytropis kamtschatica, O. ochotensis and O. revoluta is only of a physical type caused by the impermeability of the testa [11 ]. Oxytropis campestris var. chartacea is characterized with long seed dormancy and a long-lived natural seed bank in the soil [12 ]. Both tetraploid O. kozhuharovii and diploid O. urumovii belong to O. campestris “superaggregate” [4 ] and thus the similar results are not surprising. The first critical moment after germination was the cotyledon stage. Only about 25% of the germinated seeds survived. These survivor seedlings grew well from the start (Plate 1, Fig. 1). The stage of the third-fourth leaf was the next critical moment for them. More than a half of the surviving plants died at that stage. For some reason the connection between root and leaves was eroded in the area of the hypocotyle (this is usually a symptom of attack by a pathogen such as Pythium or Saprolegnia; mild fungicide “Radomil” was used without much success). The only O. kozhuharovii seed that germinated in 2007 without scarification grew enough to be planted in the field, but the seedling O. urumovii was a result of the same treatment rotted (Table 1). Thus the mild sand paper scarification in this case most probably was not the reason for rotting of the seedlings. Compt. rend. Acad. bulg. Sci., 62, No 7, 2009

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Plate 1. Seedlings development of Oxytropis urumovii and O. kozhurahovii: Fig. 1. Seedlings grown from seed collected from native populations; Fig. 2. Experimental rock garden – the lowest flower bed with laths for protection of too much sun; Figs 3 and 4. Seedlings grown in 2006, 2007 and 2008; Fig. 5. The flowers of O. kozhurahovii; Fig. 6. Offspring of O. kozhuharovii plant grown ex situ from seed in 2006

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The preliminary observations indicated a low tolerance to the environment provided for the seedlings. Obviously a complex of factors acted – soil water balance, insufficient/excess of soil moisture, heat, dust and gas air pollution suppressed the seedlings as the early stages took place in Sofia. The low survival of the seedlings may also be genetically determined. The situation with Oxytropis urumovii was rather similar only the seedlings did slightly better (Table 1). The survivors were planted in a flower bed in the foot hill of Pirin Mts. (Plate 1). About half of them lived through the summer (Table 1). Once planted in the field in the mountain foothills both young seedlings and adult plants (grown previous years) of O. urumovii and O. kozhuharovii were vulnerable and many did not survive (Table 1). For comparison the alpine Oxytropis nigrescens from south-west Yukon tolerated the removal of 58–61% of summer leaf production under natural conditions [13 ]. Only O. kozhuharovii bloomed ex situ while O. urumovii produced only leaves (Plate 1, Fig. 5). Two plants of O. kozhuharovii bloomed in 2007 (the next summer after sowing the seed, with 7 and 8 flowering stems respectively). One of them died until the next spring but the other bloomed again in 2008 (with 5 flowering stems). Also three plants grown in 2007 bloomed in 2008 (with 3, 4 and 6 flowering stems). The average number of flowers per head was 8±2.06. The presence of a great number of empty pods (67% in 2007 and 68% in 2008) indicate the lack of ability for spontaneous pollination. In 2007 both plants had fruits. In 2008 the fruits were produced by only one of the five flowering plants, which may mean that the flowers are self-incompatible. In addition the flowering period of “plant 2006” was a week or two earlier than the “plants 2007” although there were open flowers of at least two plants at a time. Conclusion. This paper is just “another brick in the wall” of the knowledge about seed germination and development of the seedlings. Recently seed banks are designed to store duplicates of seeds from seed collections around the globe. If seeds are lost, e.g., as a result of natural disasters or human activity, the seed collections may be reestablished using seeds. There is a local seed bank in Bulgaria (in Sadovo) for seeds of crops and wild medicinal plants. The Svalbard Global Seed Vault, which is established in the permafrost in the mountains of Svalbard, is the other hudge seed collection that preserves crop seeds from all over the world [14 ]. The Millennium Seed Bank Project (MSBP) is the largest ex situ conservation project ever conceived and it preserves seeds of wild plants [15 ]. For all these projects very important point is the knowledge about the peculiarities of germination and seedlings development of each particular plant species. The genus 836


← Plate 1. Fig. 7. The most distinctive feature of O. kozhurahovii, the indumentum of the calyx, particularly the apex, which is very densely covered with long white hairs is kept in the seedlings grown ex situ

Oxytropis has about 300 species distributed through the northern hemisphere. There are several research publications on some germination specifics of distant relatives of the studied species [6, 11, 16 ]. The closest relatives that have been studied from germination point of view belong to the North American members of the Oxytropis campestris “superaggregate” [17–19 ]. For the first time the local Pirin endemics Oxytropis. urumovii and O. kozhurahovii are studied from germination point of view. Having in mind that most mature test seeds of the studied plants germinated within a couple of days if the seed coat was scarified but the seedlings were rather sensitive and many did not survive we consider appropriate in the future to apply the in vitro method. It has been proved to work well for other rare plants [20 ]. Acknowledgements. Special gratitude is expressed to P. D. Schlorhaufer for the kind sharing of germination and gardening experience and to Assoc. Prof. D. Kozuharov, M. Bonkin and I. Bonkin for their help in the field work.

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[12 ] Chung M., G. Gelembiuk, T. J. Givnish. Molecular Ecology, 13 2004, No 12, 3657–3673. [13 ] Mcintire E. J. B., D. S. Hik. Journal of Ecology, 90, 2002, 348–359. [14 ] http://www.regjeringen.no/en/dep/lmd/campain/ svalbard-global-seed-vault.html?id=462220 [15 ] http://www.kew.org/msbp/index.htm [16 ] Delaney R. H., R. H. Abernethy, D. W. Johnson. Crop Sci., 26, 1986, 161–165. [17 ] Chung M., G. Gelembiuk, T. J. Givnish. Molecular Ecology, 13, 2004, No 12, 3657–3673. [18 ] http://www.onrockgarden.com/guide/seedo1.htm [19 ] http://midwest.fws.gov/endangered [20 ] Djilianov D., G. Genova, D. Parvanova, N. Zapryanova, T. Konstantinova, A. Atanassov. Plant Cell, Tissue and Organ Culture, 80, 2005, 115–118.

Department of Pharmacognosy and Botany Faculty of Pharmacy Medical University of Sofia 2, Dunav Str. 1000 Sofia, Bulgaria e-mail: ina [email protected]

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School of Biology University of Newcastle UK ∗
