Abstract: The objective of this study were to investigate present endangered status and propagation of Thermopsis turcica, which is a very unusual plant species for Turkey. T. turcica grows in a very narrow area located between the Eber and Aksehir Lakes. The field studies indicated that holding unhealthy seeds due to pest infestation and clearing new agricultural fields are two major threatened factors for T. turcica. In sterile conditions, concentrated sulfuric, hydrochloric and nitric acid pre-treatments for 0, 30, 60, 90 and 120 min used to germinate T. turcica seeds which were only obtained from small Eber populations. 99% seed germination within a few days was achieved from 120 min. sulfuric acid pretreatment; whereas the hydrochloric and nitric acid treatments did not have effect on the germination (<20%) of T. turcica seeds. In non-sterile conditions, however, a maximum 61% germination was obtained from 120 min sulfuric acid pretreated seeds. Although 20% non-sterile seedlings were survived, 86% in vitro seedlings acclimatized to green house conditions were alive after 14 weeks. This propagation technique, in vitro germination-acclimatizing to the soil, could be used to propagate endangered plant species suffering from germination and seedling development.
INTRODUCTION
There are 25 species of the genus of Thermopsis R. Br. belonging to the Thermopsideae tribe of Leguminosae (Fabaceae) family and they are found mainly spread in the mountainous and humid regions of Central Asia and North America (Dement and Mabry, 1975; Davis et al., 1988; Wojciefowski, 2003). The only endemic representative of this genus in Turkey is Thermopsis turcica Kit Tan, Vural and Kuçuköduk (Kit et al., 1983; Davis et al., 1988). T. turcica grows over a narrow area located between the southern part of Eber Lake and the south-western part of Aksehir Lake (Tan et al., 2003) and the species was classified as critically endangered CR in the Red Data Book of Turkish Plants (Ekim et al., 2000). Some morphological and anatomical specifications of T. turcica evident in the Flora of Turkey and East Aegean Islands (Davis et al., 1988) have been described as follows; densely white-villous perennial herb, with long rhizome; 35-80 cm stems erect; 3 carpellate ovaries containing 10 ovules on adaxial suture; 2-3 seeded legumes which remain closed during maturity, the seeds being 4-5x3 mm in dimension, smooth and of a pale purplish color.
Most legume seeds have a hard seed coat inhibiting water and/or oxygen intake and radicula emergency (Salisbury and Ross, 1992). Some of the methods used successfully to weaken the hard seed shells of wild plants to promote germination are mechanical (e.g., sanding and drilling holes), chemical (e.g., soaking in acid) and physical (e.g., boiling and freezing) scarifications (Rehman and Park, 2000; Kaye and Kuykendal, 2001a, b; Sy et al., 2001; Olvera-Carrillo et al., 2003). The chemical scarification of the legume seeds using concentrated acids is practical and effective germination method (Tetsuya and Takahashi, 2003); but the exposing time should be precisely adjusted to have maximum germination without damaging seed germination capacity (Sy et al., 2001).
As an alternative to protect endangered plant species with in situ methods, in vitro germination procedures are often used to obtain sterile seedlings preferably used as explant source for micropropagation of endangered plant species with low seed production, germination problems or problematic seedling development (Pence, 1999; Cerabolini et al., 2004). The objective of this study was twofold; firstly we aimed to determine the present endangered status of T. turcica species and secondly, we intent to find out the best germination and seedling development procedures using sterile and non-sterile techniques to propagate this critically endangered plant species.
MATERIALS AND METHODS
Seed collection: The seeds of T. turcica were obtained from a small plant population of less than 50 units located in the marsh area (~960 m altitude) south of Eber Lake in late August of 2004 and 2005 (Fig. 1). Relatively small amounts of seeds were collected (less than 40%) to avoid major damage to this small population. After shelling the seeds from their legumes, they were kept in glass jars in a dark room at room temperature.
Sterile seed germination: Untreated T. turcica seeds were cleaned under running water and sterilized in sodium hypochlorite solution (5%) containing a few drops of Tween-20 surfactant for 20 min. Then, the seeds were dipped into 70% ethanol for 90 sec, rinsed thrice with sterile distilled water and transferred onto an agar medium. The other seeds were immersed into concentrated sulfuric, hydrochloric or nitric acid for different periods (0, 15, 30, 60, 90 and 120 min). The scarified seeds were only rinsed three times with sterile distilled water before being transferred onto the agar medium.
Solid agar composition included sucrose (20 g L-1), basal MS salts and vitamins (4.3 g L-1) (Murashige and Skoog, 1962) and agar (7 g L-1). The medium pH was adjusted to 5.7±0.1 and then autoclaved at 120°C and 2 atm for 15 min. In each treatment, only 15 seeds due to limited number available were placed into a germination box (11x11x9 cm) containing 200 mL of basal MS medium and four replicates were used for each treatment. The seeds were kept in the germination cabinet for a period of 4 weeks under GroluxTm fluorescent lamp light with 16/8 h light/dark cycle at 25±1°C. To minimize the effects within the cabinet, the places of the germination boxes were randomly changed on a daily basis. The seeds were accepted as germinated when the emerging radicula was approximately 2 mm in length (Rehman and Park, 2000). The germination data was recorded on a daily basis for 28 days.
Non-sterile seed germination: The non-sterile germination experiments were conducted during 20 Feb-31 May 2006. After 0, 15, 30, 60, 90 and 120 min exposing to only concentrated sulfuric acid, T. turcica seeds were washed a few times with dH2O. Pretreated and untreated seeds were than soaked in water for 24 h. Subsequently, the seeds were sown into soil, sand and perlite mixture (1:1:1) 1.5 cm in depth as described for Thermopsis lupinoides seeds (Tetsuya and Takahashi, 2003). Fifteen seeds were sown to 50x10x20 cm plastic pots for each treatment and 4 replicates were used for each experiment. All the non sterile germination experiments were conducted in the greenhouse conditions and the seed pots were watered (containing 1/8 MS salt and vitamins) twice a week for the first month and later once a week. The seeds were accepted as germinated by the cotyledonal emergency. The germination data was recorded on daily basis for 100 days.
Seedling developments: The sterile germination boxes were opened for five days at the 30th day of germination experiment and randomly selected 50 sterile seedlings directly transformed to soil, perlite and sand mixture (1:1:1) in greenhouse conditions. The seedling survival data of sterile and non-sterile originated plantlets was recorded on daily bases for 100 days.
Data analyses: Final germination percentage was calculated when no further germination had taken place for several days. To determine the proportion in degrees and germination percentages of arcs transformed in germination, they were subjected to analysis by Duncan test using SPSS 10 (for Windows). The formula given below was used to calculate mean germination time (MGT) as described by Chuanren et al. (2004);
MGT = Σ (nd)/N |
where n is the number of seeds germinated between scoring days; d is incubation period days and N is the total number of seeds germinated in treatment.
RESULTS
Field studies: T. turcica spreads over a 2x20 km2 (~4 000 ha) area on the banks of Eber and Aksehir Lakes (Fig. 1). The field work carried out in this restricted area revealed that T. turcica has 5 separated populations (Table 1). Four of these populations, Akbaba 1 & 2 and Kavakli 1 & 2, are close to each other and consist of a high number of various plants, however the Eber population is made up of less than 50 plants, consisting of two clone groups with 14 and 35 plants, situated at least 15 km away from the other populations.
According to our individual observations, most land demands have taken
place in the closed populations (Akbaba 1 & 2, Kavakli 1 & 2)
where T. turcica is the most widespread, so young and clonally
propagated T. turcica seedlings have covered the ploughed fields.
| Table 1: | The sizes, locations and altitudes of T. turcica populations |
| Fig. 1: | Location of the study plant species Thermopsis turcica |
Also we have recognized that T. turcica carpels remain on the
plant at least two years without seed dispersing. Furthermore, hardly
any healthy seeds were observed among the existing one or two year old
legumes in the most intensely spread populations. Surprisingly, each T.
turcica legume obtained from these populations had a drilled hole
in its hull (Fig. 2a) and was filled with granulated
or damaged seeds (Fig. 2b) black or pale purple in color
(Fig. 2c). On the contrary, all the legumes obtained
from the small Eber population contained healthy seeds and some well developed
legumes held up to 9 seeds which were brown in color (Fig.
2d, e and f).
| Table 2: | The effects of acidic scarification on germination and mean germination time (MGT) of T. turcica seeds |
| *Germination tests were undertaken on M and S agar beads at 25°C and at 16/8 h photoperiod or **in soil under greenhouse conditions. *** Values in the same line with the same letter (s) do not differ significantly (Duncan test with p<5%) | |
| Fig. 3: | Seed germination of T. turcica seeds exposed to various sulfuric acid pretreatments in non-sterile conditions |
Sterile seed germination: Since none of the seeds of other population were germinated with any treatment used in this study, sterile and non-sterile germination trials have been carried out only on the limited amount of healthy seeds obtained from the Eber population. The germination rates of untreated seeds did not exceed 10% on MS agar (Table 2). T. turcica seeds treated with hydrochloric or nitric acid for different time periods showed similar germination results with untreated seeds with the exception of nitric acid application for 120 min where 20% germination occurred. On the other hand, only 15 min sulfuric acid min pretreatment of the seeds was resulted in 23% germination (Table 2).
The highest germination values (>94%) were obtained with 90/120 min sulfuric acid pretreatment of T. turcica seeds in sterile conditions. The Mean Germination Time (MGT) of the intact and hydrochloric acid pretreated seeds was approximately 7 days and nitric acid slightly decreased the MGT to 5.8 days (Table 2). On the other hand, 60-120 min sulfuric acid scarification sharply reduced the average germination time to almost half as compared to the intact seeds.
Non-sterile seed germination: Unlike the germination results of
sterile conditions, none of the untreated and 15 min sulfuric acid treated
T. turcica seeds germinated in greenhouse within 14 weeks (Fig.
3). The increased acidic scarification time was induced the germinations
and the maximum germination (61.66%) was obtained from 120 min pretreated
group.
| Fig. 4: | Survival percentages of T. turcica seedlings
obtained from; ( |
We have calculated the GMT values as almost 27 days for 30, 60 and 90 min pretreated groups; in fact some germination were recorded at the 84th day of the experiment initiation in 90 min pretreated group. However, the average germination time was 13 days for 120 min pretreated group.
Seedling survivals: Totally, 86 of 360 seeds were germinated and 80% of the plantlets were dead in non-sterile conditions after 100 days (Fig. 4). Most of the seedling mortalities were observed between 20-60 days of the experiment. On the other hand, only 7 sterile seedlings were lost during the acclimatization process, so 86% of acclimatized T. turcica seedlings were surviving after 14 weeks.
DISCUSSION
Based on our field observations, T. turcica has some ecological advantages such as not grazing by domestic animals, not used for medical purposes and vegetating via their rhizomes. On the other hand, there are some important factors placing this endangered plant species into a more critical position. Because of the favorable climate and land structure and also due to the fact that Eber and Aksehir Lakes have receded over 5 km during the last decades (Tan et al., 2003), efforts to clear more land for agricultural purposes in the natural habitat of this plant species has become a preliminary concern of the local farmers and this situation manifests itself with radical measures taken by the local people. Kit et al. (1983) and Davis et al. (1988) previously described the habitat of the T. turcica as marsh, pasture and muddy field land but presently T. turcica populations, except Eber, are completely in the agricultural fields. Therefore, newly vegetated T. turcica seedlings are mainly reproduced vegetating via their rhizomes on ploughed fields, because Akbaba 1 & 2 and Kavakli 1 & 2 populations are also suffering from insect attacks which inhibits the natural seed dispersion. In contrast, all T. turcica legumes of the small Eber population contained healthy seeds. This might be due to the fact that they are at least 15 km away from the other populations and naturally protected against insect attacks.
A sharp reduction in the number of the present populations and propagation via rhizomes possibly causes reduction in genetic variation among the T. turcica populations. Ellstrand and Elam (1993) argued that genetic drift, inbreeding and gene flow were likely to put rare plants and small populations at genetic risks. Barrett and Khon (1991) claimed that the loss of variation is thought to reduce the ability of populations to adapt to changing environments and increase their susceptibility to pest and disease pressures which might be a good description for the present endangered status of T. turcica populations.
The germination results indicated that T. turcica seeds have a strong physical dormancy caused by the hard seed coat which prevents seed germination without any pretreatment as reported for other legume species (Rehman and Park, 2000; Cerabolini et al., 2004; Kaye and Kuykendall, 2001b; Aparicio and Guisande, 1997). In the present study, 120 min sulfuric acid scarification is enough to obtain 98% germination in a few days in sterile conditions. Moreover, germination in sterile conditions and subsequently acclimatizing the sterile seedlings to the non-sterile conditions was found as effective and practical method for the propagation of this rare plant species as compared to the propagation in only greenhouse conditions. The reason of low germination and seedling survival in green house conditions remains to be investigated whether they are due to lower embryo fitness (Jarne and Charlesworth, 1993) or to eco-physiological or nutritional causes (Aparicio and Guisande, 1996). It was reported that some rare plant species seeds strictly need high nutrients and/or phytohormones to germinate, for instance, Cerabolini et al. (2004) showed the germination of Physoplexis comosa seeds only in sterile conditions. Fay (1992) and Pence (1999) argued that appropriate techniques must be selected based on the requirements of each species, particularly rare and/or endemic species. Therefore, high nutrient requirement might be an important factor for germination and early seedling development as removing the hard seed coat to have successful propagation strategy for T. turcica. It may be possible to reintroduce populations of T. turcica through direct seeding of habitats and trough transplanting sterile origin seedlings. In conclusion, the present study demonstrates that ex situ propagation of T. turcica is possible from seeds.
ACKNOWLEDGMENT
The authors wish to thank Afyon Kocatepe University Research Fund (Project No: 042.FENED.04) for supporting this study financially.