Conservation, Propagation, Development and Utilization of Xerophytic ...

Conservation, Propagation, Development and Utilization of Xerophytic Species of the Native Greek Flora towards Commercial Floriculture E. Maloupa1, K. Grigoriadou1, K. Papanastassi1 and N. Krigas2 National Agricultural Research Foundation (N.AG.RE.F.), Laboratory of conservation and evaluation of native and floricultural species, P.C. 570 01 Thermi, Thessaloniki, P.O. Box 60 125, Greece 2 Laboratory of Systematic Botany & Phytogeography, Department of Botany, School of Biology, Aristotle University of Thessaloniki, 541 24, Greece

1

Keywords: Thymus, Teucrium, Labiatae, asexual propagation, xeriscaping Abstract Plant material originating in wild populations, collected from various areas of Greece, has been maintained at the Laboratory of Conservation and Evaluation of Native and Floricultural Species. In the frame of the conservation program of the Balkan Botanic Garden of Kroussia (BBGK) the original collections count more than 1700 accession numbers of taxa, 17% from Labiatae family. Six aromatic species (Thymus plasonii, Th. longicaulis subsp. chaubardii, Th. degenii, Teucrium divaricatum, T. polium, T. chamaedrys) were studied. Rooting of softwood cuttings was tested for all plants using various concentrations of auxins. Young individuals produced were transplanted in trays and later in bigger pots until their final transplanting at sea level as well as at 600 meters altitude in ex situ conservation area at BBGK, northern Greece. Growth and development of the plants were monitored in every stage. The rooting percentage, the number of roots and the root length were analyzed. Data were used to provide a propagation protocol for each species. Growth and development occurred rapidly and plants showed adaptability in dry climatic conditions. The six species studied, most of them Greek and/or Balkan endemics, not cultivated in other Botanic Gardens (according to Botanic Garden Conservation International database): (a) showed excellent results when propagated by softwood cuttings, (b) present an impressive effect in landscaping due to their inflorescence and/or foliage and (c) show tolerance in low-water regimes; therefore are suggested as suitable for xeriscaping (environmental friendly landscaping - rock gardens). INTRODUCTION It is estimated that up to 100,000 plants, representing more than one third of the entire world's plant species, are currently threatened or face extinction in the wild. Botanic Gardens are playing a key role in the conservation and maintenance of plants for the future. An important outcome of the 2nd World Botanic Gardens Congress in Barcelona, Spain in April 2004 was the development of a series of 20 targets for botanic gardens to be achieved by 2010, and as a contribution towards the Global Strategy for Plant Conservation (CBD, 2002). These targets briefly relate in: a) Understanding and documenting plant diversity b) Conserving plant diversity c) Using plant diversity sustainably d) Promoting education and awareness about plant diversity e) Building capacity for the conservation of plant diversity Even though Balkan Botanic Garden of Kroussia (BBGK) is a newly established garden, its objectives completely agree with the 20 targets for botanic gardens by the year 2010. The Laboratory of Conservation and Evaluation of Native and Floricultural Species and BBGK greatly contribute to the development of models with protocols for plant conservation and sustainable use, based on research and practical experience, for the rare and threatened species of Greece. Priority is given to support, promote and contribute to the integrated conservation and management of the medicinal-aromatic plants and other Proc. XXVII IHC-S5 Ornamentals, Now! Ed.-in-Chief: R.A. Criley Acta Hort. 766, ISHS 2008

205

major socio-economically valuable plants (e.g., Maloupa et al., 2000, 2003, 2005) and the maintenance of associated indigenous and local knowledge around them. Greek flora is one of the richest in the world concerning aromatic and pharmaceutical plants (Polunin, 1997; Kokkini, 1994; Kokkini et al., 1989a). Unique species and special climatic conditions make the Greek and Balkan area ideal for the selection of such kind of plants. The use of these species is in high demand, as they are related to among others, ornamental, pharmaceutical and aromatic industry. The Labiatae family includes a wide variety of species valued for their uses as culinary, medicinal, aromatic, and ornamental plants. Thymus spp. are hardy creeping perennials grown as ground covers for small spaces or in containers. They are widely used in food, pharmaceutical and cosmetics industry due to their essential oils which also have antioxidant, antispasmodic and antiseptic properties (Economou et al., 1991). The concentration of the essential oils varies greatly among the species of the genus and it is found increased at the Mediterranean endemic species (Rey, 1992; Kokkini et al., 1989). The genus Teucrium comprises more than 300 species, 49 of which grow in Europe, mostly in the Mediterranean basin. Teucrium spp. have been used as medicinal herbs for over 2000 years, as diuretic, tonic, antipyretic, antispasmodic, cholagogic and many of them are used in folk medicine (Galati et al., 2000). Furthermore, they are often pruned for their green or silver foliage into garden borders or in knot gardens. Three species of the genus Thymus (Th. plasonii, Th. longicaulis subsp. chaubardii and Th. degenii) and three species of the genus Teucrium (T. divaricatum, T. polium and T. chamaedrys) were studied. Plant material was collected form its natural habitats, maintained in mother plantation, asexually propagated, cultivated at pots, evaluated in dry conditions and applied in environmentally friendly landscaping (xeriscaping). MATERIALS AND METHODS Plant Material All native plants maintained at the nursery of the Laboratory derive from natural populations as a result of botanic expeditions conducted at floristically important areas (e.g., National Parks, NATURA 2000 sites and other protected areas). For each plant, sitespecific information was kept (location, region, altitude, longitude and latitude) as well as a detailed habitat description. All plants collected, received immediate care in the nursery. They are designated as stock plants, planted at big containers or special places according to their needs, taken special treatments since they recover from transplantation shock. Thymus and Teucrium species were collected from different areas of Greece between 1997-1999 (Table 2). Asexual Propagation Research on the asexual propagation of the six species was conducted. Softwood tip cuttings of 5-7 cm were taken during winter from mother plants developed inside the greenhouse. The effect of the auxin indole-3-butyric acid (IBA) at four different concentrations (0, 2000, 4000 ppm and powder 0.066% v/v) on root formation was tested. Cuttings were placed in propagation trays in a substrate of peat (Klasmann, KTS 1) and perlite (1:3 v/v) and maintained at bottom heat benches in a plastic greenhouse. Soil temperature was kept at 18-21°C, while air temperature was 15-25°C depending on weather conditions. Relative humidity was approximately 70-85% (mist). Experiments lasted for 15 days, followed a randomized design with 50 replications per treatment, and repeated twice. At the end of that period the number of rooted cuttings, number of roots and root length was measured. Rooted was expressed as %. Analysis of variance was performed with the General Linear Model procedure (SPSS 11.0 statistical package) and mean separation with Duncan's Multiple Range Test. Significance was recorded at P≤0.05. 206

Growth and Development Monitoring and Establishment Ex Situ After rooting, the plants were gradually acclimatized reducing the RH 20% every week. Two transplantations followed; the first in multiple trays immediately after rooting. Plants in trays were kept in the greenhouse for their acclimatization. The second transplantation was after 30 days in 2.5 L pots and plants were placed direct under the sun as they grow at their natural environment. The substrate used was a mixture peat (Klasmann, TS 3): perlite (2:1), pH 6.5 and EC 0.91 mmhos/cm, for Thymus species and peat (Klasmann, TS 3): soil (2:1), pH 5.7 and EC 1.18 mmhos/cm, for Teucrium species. Growth and development were monitored in every stage. The height and diameter of the potted plants were measured every week starting from transplantation in pots for 4 weeks for Thymus spp. and for 10 weeks for Teucrium spp. Measurements regarded 20 plants of each species. All plant materials were finally established at a xeriscaping garden at sea level and at the BBGK in 600 m altitude. The normal adaptation of the plants in these two different environmental conditions was tested. Observations taken, regarding the normal growth and development, flowering and fructification, were compared. The possibility of adaptability in dry climatic conditions for the six species was examined. RESULTS Plant Material The collection of the Laboratory counts 92 different taxa (species and subspecies) of the family of Labiatae. Among them, 9 of the genus Thymus and 7 of the genus Teucrium, most of them endemic in Greece (31%) or in the Balkan peninsula (37.5%), (see Table 1). Six of them were considered significant for the possibility of their use as out door ornamentals and they were extensively studied (Table 2). Asexual Propagation Rooting of softwood cuttings for the three Thymus species was successfully performed under mist in a period of 15 days. Even though the high percentage of rooting was not affected by the IBA treatment, the root number and length was significantly increased by the use of 2000 or 4000 ppm IBA. Th. plasonni gave 100% of rooting with 13.1 roots/cutting and 1.5 cm root length (Table 3) and Th. longicaulis subsp. chaubardii had the same rooting percentage with 12.4 roots/cutting and 2.0 cm root length (Table 4) by the use of 4000 ppm IBA while Th. degenii showed better results by the use of 2000 ppm with 100% rooting, 13.1 roots/cutting and 1.5 cm root length. (Table 5) (Fig. 3). IBA also positively affected root formation of the Teucrium spp. cuttings. Treatment with 4000 ppm IBA of T. divaricatum (Table 6) increased rooting percentage up to 100%, root number up to 24.3 roots/cutting and root length up to 1.0 cm. Respectively, by the use of 4000 ppm IBA T. polium gave 88% rooting, with 13.1 roots/cutting and 0.9 cm root length (Table 7) but T. chamaedrys had better results with the 2000 ppm IBA treatment, increasing the rooting percentage at 83%, the no. of roots at 19.3 roots/cutting and the root length at 1.4 cm (Table 8) (Fig. 3). Growth and Development Th. plasonni proved to be the most vigorous of the three Thymus species examined. Even though it was very slowly increased in height (10.15 cm, 4 weeks after transplanted in pots), the development of its diameter was very fast reaching the 31 cm only 2 weeks after transplantation. After that period, the development has been restricted (Fig. 1). Th. longicaulis subsp. chaubardii was the least vigorous of the three. Its height remains stable approximately at 8.1 cm while its diameter was the smallest (15.15 cm) at the end of the period of 4 weeks (Fig. 1 left). Th. degenii was vigorous enough and it is the only of the three species that continuously increased gradually in height and in diameter during the period of the 4 weeks (Fig. 1 right). Among the species of the genus Teucrium tested, the T. divaricatum was the most vigorous either in height (27.77 cm, 10 weeks after transplanted in pots) or in diameter 207

(43.45 cm). Its development occurred gradually and continuously during the period of 10 weeks. T. polium showed a medium growth rate reaching the height of 20.04 cm and 35.23 cm in diameter while T. chamaedrys had the most restricted development (17.81 cm height and 23.89 cm diameter) (Fig. 2). Establishment Ex Situ All Thymus species showed very good adaptability at the xeriscaping garden at sea level and at BBGK at 600 m. They have been used as ground cover plant and proved to be very tolerant at dry climatic conditions. Plants flowered and produced seeds regularly at both places, but at 600 m flowering occurred one month later than sea level (June instead of May). Teucrium species also proved to be tolerant at dry climatic conditions. T. chamaedrys adapted without any problem at sea level and at 600 m altitude but the other two species T. polium and T. divaricatum didn’t manage to survive after the winter at 600 m. Flowering of Teucrium at 600 m was nearly one month late (late June) than in sea level (late July). DISCUSSION Labiatae family considered the most important family in the field of aromatic pharmaceutical plants. Greek endemic Labiatae species showed unique characteristics regarding the content of the essential oils and other properties related to the special climatic conditions of the area (Kokkini, 1994; Kokkini et al., 1989, 1994; Kokkini and Vokou, 1989a, b). The endemic species Thymus and Teucrium examined, except from their use at pharmaceutical and cosmetics industry proved valuable in environmentally friendly landscaping (xeriscaping) as they can be used as ground cover plants or for sort hedges. Teucrium spp. are not recommended for high altitudes. Furthermore, urban landscaping at terraces and roof gardens demands tolerant plants with limited irrigation needs. These species showed very good adaptability at water stress conditions and they could be easily cultivated in pots for such use. Literature Cited Convention on Biological Diversity (CBD). 2002. Global Strategy for Plant Conservation. CBD (eds.), Montreal, Canada. Galati, E.M., Mondello, M.R., D’Aquino, A., Miceli, N., Sanogo, R., Tzakou, O. and Monforte, A.T. 2000. Effects of Teucrium divaricatum Heldr. divaricatum decoction on experimental ulcer in rats. J. of Ethnopharmacology 72:337-342. Economou, K.D., Oreopoulou, V. and Thomopoulos, C.D. 1991. Antioxidant activity of some plant extracts of the family of Labiatae. J. American Oil Chemical Society 68:109-113. Kokkini, S. 1994. Herbs of the Labiatae. p.2342 -2348. In: R. Macrae, R. Robinson, M. Sadler and G. Fullerlove (eds.), Encyclopaedia of Food Science, Food Technology and Nutrition. Academic Press, London. Kokkini, S. and Vokou, D. 1989a. Carvacrol-rich plants in Greece. Flavour and Fragrance Journal 4:1-7. Kokkini, S. and Vokou, D. 1989b. Mentha spicata (Lamiaceae) chemotypes grown wild in Greece. Economic Botany 43:192-202. Kokkini, S., Karousou, R. and Vokou, D. 1994. Pattern of geographic variation of Origanum vulgare trichomes and essential oil content in Greece. Biochemical Systematics and Ecology 22:517-528. Kokkini, S., Vokou, D. and Karousou, R. 1989. Essential oil yield of Lamiaceae plants in Greece. p.5-12. In: S.C. Bhattacharya, N. Sen and K.L. Sethi (eds.), Essential Oils, Fragrances and Flavors. 3. Biosciences. Oxford & IBH Publishing. New Dehli. Maloupa, E., Grigoriadou, K., Zervaki, D. and Papanastasi, K. 2005. Management of the Βalkan native flora for sustainable floricultural commercial use. Acta Hort. 683:189195. 208

Maloupa, E., Zervaki, D., Grigoriadou, K. and Papanastasi, K. 2003. The development of native plant collection nursery in the Kroussia Balkan Botanic Garden. Eurogard III proceedings, Robbrecht and Bogaerts (eds.) p.15-20. Maloupa, E., Zervaki, D. and Marnasidis, A. 2000. Introduction of the Mediterranean native species Thymus mastichina, Lotus cytisoides, Lavandula stoechas, Centranthus rubber, Limonium pectinatum and Limonium sinense into commercial floriculture. Acta Hort. 541:57-65. Polunin, O. 1997. Flowers of Greece and the Balkans; a field guide. Oxford University Press. Ray, Ch. 1992. Selection of thyme (Thymus vulgaris L.) for extreme areas. Acta Hort. 306:66-70.

Tables

Table 1. Species of the genus Thymus and Teucrium conserved at Balkan Botanic Garden of Kroussia.

Species Thymus holosericeus Thymus longicaulis subsp. longicaulis Thymus longicaulis subsp. chaubardii Thymus plasonii Thymus praecox subsp. jankae Thymus sibthorpii Thymus striatus Thymus thracicus Thymus degenii Teucrium arduini Teucrium aroanium Teucrium chamaedrys Teucrium divaricatum Teucrium flavum subsp. hellenicum Teucrium halacsyanum Teucrium polium

Endemism Local Greek endemic Mediterranean Balkan subendemic Local Greek endemic Balkan subendemic Balkan subendemic Mediterranean Local Balkan endemic Local Balkan endemic Local Balkan endemic Local Greek endemic Mediterranean East Mediterranean Greek endemic Local Greek endemic Mediterranean

Conservation in other Botanic Gardens No Yes No No No Yes Yes No No Yes Yes Yes Yes No Yes Yes

209

Table 2. General collection information regarding the Thymus and Teucrium species. Species

Year

No. of plants collected

Thymus longicaulis subsp. chaubardii Thymus plasonii Thymus degenii Teucrium divaricatum Teucrium polium

1998 3 1997 7 1999 2 1999 Seeds and cuttings 1999 10

Teucrium chamaedrys

1999

5

Area Kilkis, northern Greece (Natura 2000 site) Asvestoxori, northern Greece Mt. Athos, northern Greece (Natura 2000 site) Crete island, southern Greece Mt. Athos, northern Greece (Natura 2000 site) Mt. Falakro, northern Greece (Natura 2000 site)

Table 3. Rooting results of Thymus plasonii after 15 days at mist. Treatment (IBA) Control 2000 ppm 4000 ppm 0.066% powder

Thymus plasonii Rooting Root number (%) 96 a 7.5 a 98 a 13.0 b 100 a 13.1 b 98 a 9.7 c

Root length (cm) 1.9 a 1.8 a 1.5 b 1.9 a

Different letters within columns indicate significant differences (P≤0.05), Duncan's Multiple Range Test.

Table 4. Rooting results of Thymus longicaulis subsp. chaubardii after 15 days at mist. Thymus longicaulis subsp. chaubardii Treatment Rooting Root number Root length (IBA) (%) (cm) Control 94 a 10.1 a 1.9 a 2000 ppm 100 b 10.9 a 1.9 a 4000 ppm 100 b 12.4 b 2.0 a 0.066% powder 98 ab 10.1 a 1.7 a Different letters within columns indicate significant differences (P≤0.05), Duncan's Multiple Range Test.

210

Table 5. Rooting results of Thymus degenii after 15 days at mist. Treatment (IBA) Control 2000 ppm 4000 ppm 0.066% powder

Thymus degenii Rooting Root number (%) 100 a 6.3 a 100 a 13.1 b 100 a 14.7 b 100 a 14.1 b

Root length (cm) 2.1 a 1.8 b 1.7 bc 1.5 c


Table 6. Rooting results of Teucrium divaricatum after 15 days at mist. Treatment (IBA) Control 2000 ppm 4000 ppm 0.066% powder

Teucrium divaricatum Rooting Root number (%) 92 a 13.7 a 96 ab 28.7 c 100 b 24.3 b 98 ab 14.8 a

Root length (cm) 0.9 a 0.9 a 1.0 a 0.9 a


Table 7. Rooting results of Teucrium polium after 15 days at mist. Treatment (IBA) Control 2000 ppm 4000 ppm 0.066% powder

Teucrium polium Rooting Root number (%) 58 a 6a 70 a 15.7 b 88 b 13.1 b 60 a 6.9 a

Root length (cm) 1.0 a 1.0 a 0.9 a 1.1 a


Table 8. Rooting results of Teucrium chamaedrys after 15 days at mist. Treatment (IBA) Control 2000 ppm 4000 ppm 0.066% powder

Teucrium chamaedrys Rooting Root number (%) 75 a 7.7 a 83 a 19.3 b 85 a 17.4 b 78 a 10.5 a

Root length (cm) 1.3 a 1.4 a 1.0 b 1.4 a


211

Figurese

Thymus longicaulis

Thymus degenii

Thymus plasonii y p

12

35

10

30

plant diameter (cm)

plant height (cm)

Thymus plasonii

8 6 4 2

1

2

3

g

Thymus degenii

25 20 15 10 5

weeks in pots

0

Thymus longicaulis y

0 1

4

2

3

4

weeks in pots

weeks pots weeks weeks in inin pots pots

weeks in pots

Fig. 1. Plant height (left) and diameter (right) development of Thymus spp. 4 weeks after transplanted in pots.

Teucrium chamaedrys

Teucrium divaricatum

Teucrium polium

Teucrium chamaedrys

Teucrium divaricatum

Teucrium polium

30

50

25

40

pla nt dia m e te r (c m )

plant height (cm)

Teucrium polium

20 15 10 5

30 20 10 0

0 1

2

3

4

5

6

in pots weeksweeks in pots

7

8

9

10

1

2

3

4

5

6

7

8

9

10

in pots weeksweeks in pots

Fig. 2. Plant height (left) and diameter (right) development of Teucrium spp. 10 weeks after transplanted in pots.

212

Thymus plasonii

Control

2000 ppm

4000 ppm

0.066%powder

Control

Teucriumdivaricatum

2000 ppm

4000 ppm

2000 ppm

4000 ppm

0.066%powder

Teucriumpolium

Control Control

Thymus degenii

Thymus longicaulis subsp. chaubardii

2000 ppm

4000 ppm

Control

2000 ppm

4000 ppm

0.066%powder

Teucrium chamaedrys

0.066%powder

Control

2000 ppm

4000 ppm

0.066%powder

0.066%powder

Fig. 3. Rooting of Thymus and Teucrium spp.

213

214