® 48 Ornamental Limonium Grown in Mediterranean Conditions Josefa López* • Alberto González** Departamento de Hortofruticultura, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Murcia, Spain Corresponding authors: * josefa.lopez38@carm.es ** albertot.gonzalez@carm.es Keywords: cultivation, in vitro, reproduction ABSTRACT Several species of the genus Limonium form an important part of the group of plants used for complimenting flower arrangements. An important aspect of these plants is dense multi-flowering and great inflorescences. Their tolerance to salinity and types of soil means that they are favoured for introduction into areas with a Mediterranean environmental profile. This environment supplies nearly all the thermal needs of the plant. The great demand for these different species especially for L. sinuatum and L. latifolia) has meant that large scale production has been vegetative, originating from meristems. Consequently L. sinuatum which was traditionally reproduced from seeds is now generally produced by cloning carried out in in vitro cultures. The development of new hybrids which improve their agronomic and ornamental qualities has also been a factor in using this form of reproduction. The shortening of the growth cycle due to winter cultivation demands additional husbandry practices such as training on mesh; this enables the plant to be kept erect, being gradually strengthened by the addition of adequate top fertilisation. Phytosanitary problems such as rot caused by botrytis affects the quality of production; therefore ventilation factors should be taken into account. The yellow varieties and to a lesser extent the white need to be improved in order to increase their productivity. 1. INTRODUCTION The term limonium comes from the Greek work ‘Leimon’ which means grasslands. This is probably because this plant can be found in great quantities in this environment, particularly in saline soils, preferring humid areas such as coastal salt marshes, hence its popular name ‘Sea Lavender’. It can, however, tolerate a great diversity of environments and soils, which in general constitutes its natural habitat. The genus Limonium consists of approximately 300 species of mostly herbaceous perennials and annuals, natives chiefly of salt marshes, sea cliffs and semi-desert and desert regions. This genus is well adapted to Mediterranean environmental conditions. It grows wild throughout the world as an annual or shrub plant (Waisel 1972). It is classified as native flora in many different latitudes, such as the Eurpean-Nordic, Mediterranean, Sino-Japanese groups, among others (Chapman 1977). The genus Limonium can be found throughout Spain and is the subject of many studies (Pignati 1972; Erden 1978) which relate its presence to the edaphoclimatic nature of the landscape and its geographic location. In the opinion of many experts that work with this genus, there are approximately one hundred species that grow in the Mediterranean environment and with an Atlantic influence. Different taxa can be found among Canary Island species, obviously influenced and with great resemblance to North-African species such the case of the large-leafed species L. perezii which is exploited commercially. Some 45 species can be found in the Iberian Peninsula and approximately 20 on the islands such as the Balearic Islands and Canary Island archipelagos. Among these, approximately 25 species are in danger of extinction due to the fragility of the ecosystems in which they grow. That said, one must reiterate that the plant has great resilience and is able to adapt to many different environments. The main use of this plant is ornamental, particularly as flower arrangement complements due to its inflorescence with numerous small flowers and its ability to last long in vase life. However, it also has other applications such as pot plants or in gardening as in the case of L. latifolium and L. sinuatum. In addition, as an ornamental plant it has another important feature: it has a significant place amongst the range of plants used as dried flowers, a major sub-sector with great possibilities in the future. Other species of limonium have been used for tanning and dyeing, on account of their high tannin content, principally in their roots, like as in Limonium vulgare (Uphof 1959), in Limonium carolinianum (Ahmed et al. 1999). The levels of tannin in the plant are very variable depending on the species. The species with the highest concentrations are L. gmelinii, L. latifolium, L. myrianthum, etc. The concentrations in these plants depend on environmental conditions; the optimum time for extraction being spring between the months of April and June, rather than in summer. Due to its halophytic nature, it has a great tolerance to salinity and can be used to colonize degraded and marginalised areas, as in the case of L. suffruticosum. This species, albeit low in tannins, adapts well to a saline soil. This, along with its resilience to environmental conditions López and González 497 Ornamental Limonum makes it very attractive for planting in areas that are difficult to exploit due to their high saline content. This can occur in certain depressions after flooding and subsequent evaporation which results in the formation of saline outcrops, rich in sulphates and chlorides (González et al. 1998). Its use for forage is limited owing to its low nutrient content, although there are some species which can be employed as sheep feed. There are certain species that are not edible and others which contain alkaloids. These plants can be herbaceous or bushy in appearance. Their height is variable ranging from a few centimetres up to a hundred and fifty centimetres or even higher in cultivated species. In general they are perennial and rarely annuals or biannuals (Pignati 1972). The root system has no principle root. There are tubers on the roots with a diameter of up to 5 mm from which grow a large number of secondary radials. In some species such as L. carnosum (Boiss) O. Kuntze the root system is very developed reaching depths of 90 cm, as well as extending horizontally (Adul-Fatih 1975). In the case of L. vulgare Mill and L. humile Mill the roots reach 50 cm as well as ramifying laterally (Borrman 1967). The stems develop from a crown or stump on the surface of the soil. The stump is more or less circular in cross-section although some species can be polyhedral. The stems grow erect and are quite ligneous. However, in cultivated species where the stems can grow to great lengths of between 50 and 100 cm, they tend to fall to the ground and need to be supported. The stems are simple until the beginning of inflorescence. Many species have non-floriferous stems, which are of great taxonomic interest. These stems do not end in a terminal inflorescence, only having minute flowers of between 0.5 and 1 mm on their tips. The leaves are simple, normally composed of a basal rosette or, depending on the species, extended with great density along the length of the stem. The form of the limb varies considerably: from complete to lobulated, rounded or spatula in shape etc., even rolling up on itself to give the appearance of a cylinder like a cladode although arranged arborescence. This occurs with certain wild species in The Region of Murcia (Spain) such as L. insignis which grows in xerophytic environments and in very saline soils. The limb (the expanded portion of the petal) is generally longer than the petiole and usually does not differentiate itself from it. The epidermis of the leaf can vary from being hairy to waxy. The texture of the limb can vary from tough to juicy. All species of this genus have salt secretor glands in the leaves and stems enabling them to adapt to saline ecosystems by reducing the concentration of salt in the interior of the cells to tolerable levels. The pores of the glands excrete a saline solution leaving small salt deposits on the surface of the leaf. The mechanisms are described in more detail later. The inflorescence, generally a panicle corymb is situated at the end of the flowering stems, more or less thick, very ramified, erect and occasionally winged. They are green with anthocyanin tones. Their structures vary according to the species. The flower has a calyx with membranous sepals, normally coloured, occasionally dentate with small spaces between the lobes. The calyx is very persistent (more than the corolla) which makes the plant suitable as a dried flower. The corolla is normally in the shape of a short tube in which the petals are joined only at the base. The petals are five in number and have a variety of colours ranging from violet, purple, pink, yellow and white with many intermediate shades. The stamens are fused to the base of the corolla. There are five styles which are free or joined at their base and which have thread-like stigmas. The fruit develops by regular dehiscent. The majority of limoniums show dimorphism in the pollen and stigma. This is associated with self-incompatibility. The crossing of species morphologically similar has produced a series of hybrids whose physical characteristics are virtually indistinguishable, but whose agronomic performance has been enhanced. L. sinuatum are notable for their lobulated leaves and their inflorescence which is similar to L. perezii although the latter differs markedly by its complete coverage of petiolate foliage. For its arborescent inflorescence it could also be grouped with species such as caspium, latifolium, serotinum, sinensis, etc., which have certain characteristics in common with the species altaica, although the latter has fewer flowering stems without dentate leaves as well as other more specific botanical features. 1.1. Limonium sinuatum It can be an annual or biannual. It is herbaceous with erect winged stems up to 50 cm long. The leaves are pinnatifid of which there are two types: those on the base which unfold like a rosette, petiolate with blunted lobes; and lanceolate or linear lanceolate. The inflorescence in corymbs with winged branch is compact without sterile branches (Fig. 1). The spikes are dense with 3-4 flowers per small spike arranged unilaterally. The flowers have a hairless calyx and are violet in colour with a yellow or bluish corolla. 1.2. Limonium latifolium This belongs to the subgenus Eulimonium and in its natural state it is considered to be long-lived. In its natural ecosystem it produces stems from 30 to 60 cm long which are covered with short star-shaped hairs (Fig. 2). The leaves are broad and elliptic, blunt and attenuated at the petiole. The inflorescence is in the form of a wide, diffused panicles having a profusion of small spikes with one or two flowers in curved spikes. The flower has a white calyx and a lavender blue corolla with a philiforme stigma. It is possibly the most used species for ornamental purposes within this subgenus. The Miyoshi Company has produced interspecific hybrids by crossing L. latifolium × L. caspia. These cultivars are of great importance on the market and include ‘Saint Pierre’, ‘Beltlaard’, ‘Avignon’, ‘Charm Blue’, etc. The lower leaves are spoonbill-shaped, up to 20 × 8 cm in area, with wide visible intermediate nerves with a complete margin and totally hairless. They are arranged as a basal rosette surrounded by flowering stems. It also has cauline leaves arranged alternately up to the middle of the stem. These are sessile, with wide oval limbs which decrease in size in the shape of an apex. The two hairless almost cylindrical stems are slightly striated at their narrower parts. The inflorescence is arranged in the form of a cluster of cymes (plus) monopairs (small spikes). These begin half way up the stem and open towards the distal end. The tops are composed of 3 to 8 flowers of between 1 and 3 cm long. The flower has a violet corolla which grows up to 6 cm long. The calyx has the broad, papyrus-like margins of between 4 and 5 mm wide with subacute lobes. The bract is approximately 1 mm long. The middle and internal bracts have a wide, papyrus-like margin and are 2 and 3 mm long, respectively. In addition, this hybrid agronomically presents a higher resistance to high and low temperatures, is hardier and has a better bloom. In a Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK López and González A B C Ornamental Limonum 498 A B C D D Fig. 1 Developmental stages of L. sinuatum. (A) rosette; (B) leaf pinnate; (C) emerging corolla; (D) fully opened flower. Fig. 2 Developmental stages of L. latifolium. (A) rosette; (B) flower stalk; (C) emerMediterranean climate it can be cultivated both in the open air ging corolla; (D) fully opened flower. and in the greenhouse. There is a certain amount of parallelism in the open air and greenhouse growth cycles which is interrupted at lower temperatures (less than 15ºC) and lower luminosity. However, flower quality is superior with the protected culture, even when not taking into account the lack of risk due to climatic uncertainties. The plant can be productive throughout the year when its light and thermal needs are covered. Each plant can produce 12 stems. The stems are rigid even at high temperatures, maintaining their quality throughout the whole growth cycle. Likewise the flowers maintain their colour even at high temperatures. 2. PROPAGATION Some years ago the propagation of limonium or statice was only achieved via sexual means until the consolidation of new vegetative techniques of propagation, and the arrival of cultivars whose ornamental use justified their higher purchase price. Consequently cloning multiplication was developed for this genus using in vitro cultures. 2.1. Sexual propagation This was originally used as the means of reproducing ornamental complements of various species of the genus but particularly L. sinuatum, L. tataricum, L. bonduellii, L. suworowii, etc. (López et al. 2007). The seeds came from relatively improved sections of the population that were distributed by several commercial houses. In addition there were a number of flower-growers who collected their own seeds to be used in the following season. On the other hand its great use as dried flowers and for dyeing can compensate for its lack of some special qualities that are required for cut flowers. One gram of bare seed can contain between 450 and 500 clean seeds. Sowing can be carried out in a cold or warm greenhouse depending on the production cycle one wants to establish. The optimum temperature for germination is 20ºC (Serra 1979), although there are no problems with gradients of between 13 to 15ºC either. 2.2. Asexual propagation 2.2.1. In vitro Micropropagation protocols have been reported for Limonium species of commercial importance such as L. sinuatum or “Statice” (Hazary Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK López and González 499 Ornamental Limonum Table 1 Micropagation media for L. tartarica, L. gmelinii, 1985). Amomarco (1998) reported the possibility to start micropagation of L. cavanillesii L. otolepsis, L. latifolium, L. dumosum. from an inflorescence stem; the regeneration aptitude of micropropagated shoots of Multiplication Rooting several hybrids has been investigated by Mercuri et al. (1999). Macroelements MS ½ MS The plants used for propagation have both rosette leaves and flowering stems. Most Microelements MS MS of the buds on the stems have differentiated into flowering buds whereas those in the Vitamins MS MS rosette parts are still in the vegetative stage. The vegetative parts of the rosette are BA 0.5 mg/l IAA 0.1 mg/l 0.3 mg/l rinsed, and the older leaves are removed: 4-5 cm-long vegetative sections containing 85 mg/l NaH2PO4 axillary buds are washed in a detergent solution and rinsed under running tap water for Adenine hemisulfate 80 mg/l 30 min. surface sterilization is done by dipping the plant sections in 3 to 4% sodium Sucrose 30 g/l 30 g/l hypochlorite solution for 20 min, follow by 3 successive rinses with sterile distilled water. Agar 8 g/l 8 g/l This procedure reduced contamination about 25%. Axillary buds are stripped of their pH 5.6 5.6 MS = Murashige and Skoog; values compiled from disinfection-damaged external leaves, then planted in 25 x 100 mm culture-tubes and Ruffoni et al. (2000). held at 24°C and a photoperiod of 16 hr of 40 μmol·s-1· m-2 fluorescent light. For in vitro multiplication, internodal segments as well as various kinds of buds can be used. Regarding the behaviour of the bud according to its location in the plant, Hazary in 1985 demonstrated that the auxiliary buds produced much more uniform plants than when using adventitious buds, although with the latter the production rate was higher. Ruffoni et al. (2002) demonstrated that plant regeneration is possible in several Limonium genotypes. The use of immature floral scape explants permitted to avoid contamination problems. Green, immature inflorescence branches were cut into fragments, surface sterilized in an aqueous hypochlorite solution and rinsed twice. The media that were used are detailed in Table 1. The material was cultured in a 16 h photoperiod PPFD 28 μmol·s-1·m-2 and temperature of 23°C. 2.2.2. Somatic embryogenesis Plant regeneration via somatic embryogenesis is an efficient route to propagate genetically uniform plants for conventional breeding and genetic transformation. Shoot regeneration via organogenesis was achieved in L. perezii F.T Hubb. (Kunitake and Mii 1990), L. perigirnum R.A. Dyer (Seelye et al. 1994), and L. altaica Mill. (Jeong et al. 2001). Rathinasabapathi et al. (2001) developed a protocol to induce somatic embryogenesis from 5-day old cotyledon and hypocotyl explants. The explants were cultured on agar-solidified Murashige and Skoog (1962) medium supplemented with 3% (w/v) sucrose, 1 mg/l 2,4-D and 0.1 mg/l kinetin. Globular embryogenic calluses developed in two weeks and differentiated into cotyledonary stage embryos. Experiments are in progress to optimize in vitro embryo maturation to the seedling stage. The above was the first report of somatic embryogenesis in L. latifolium. Other studies shown that the induction of somatic embryogenesis from cotyledon explants in a modified MS medium is possible in three species: L. aureum, L. latifolium and L. sinuatum. A rapid and efficient protocol was developed to induce somatic embryogenesis, which consisted of relatively low levels of 2,4-D to induce embryogenic callus from cotyledon explants and MS based medium with kinetin for somatic embryo maturation (Aly et al. 2002). 2.2.3. Genetic transformation Leaf explants of the sterile hybrid L116 (L. otolepis x L. latifolium) were inoculated with Agrobacterium tumefaciens LBA4404 harbouring the binary vector pBin19 containing a T-DNA fragment encompassing rolA, B and C genes of A. rhizogenes Ri plasmid (pRi1855), and the transformed plants showed ornamental traits such as dwarfness and early flowering which are highly desirable (Mercuri et al. 2001). 3. EDAPHOCLIMATIC REQUIREMENTS Studies of the behaviour of this genus in the environment have mainly focused on L. sinuatum and a number of other morphologically similar species such as L. suworowii, etc. This opened the way for its use as an ornamental plant which was hindered, however, by the emergence of genetic advances and the taste for other new species. Studies have been made of the adaptation of cultivars to lucrative production calendars focusing on yield, the ascertainment of its precocity and the establishment of the productive period. 3.1. Temperature The limonium of Mediterranean origin flowers naturally from spring to summer. Obviously, these plants in their wild state grow spontaneously and generally produce only one bloom a year. In the case of L. sinuatum, when the crop is produced from seeds and flowering is required in winter (making it more exportable), it is necessary to previously satisfy the vernalization needs of the plant so that is able to induce flowers. In addition, these needs can only be addressed by vegetative means, starting from two real leaves which can not be obtained when these treatments are carried out in the seed form. In this case sowing is previously carried out in trays, and when the phenological conditions are right the cold treatment is initiated. The optimum temperature is between 11 and 12°C (Shillo 1976) having established that temperatures of around 2°C are not sufficient and up to 8°C (a temperature at which floriferous primordia are supposed to begin to form) vernalization can take place but with less positive results. The period of vernalization lasts for approximately 8 weeks after which the plants are ready for transplanting. It appears that the period of vernalization can be shortened when transplanting is carried out in spring which has moderate temperatures; while the period should not be shortened if cultivation takes place in autumn. The plantation responded well to high daytime temperatures that tended to become more moderate at night. With night time temperatures ranging from 13 to 21°C differences of seven days were found with the earliness of production (Semeniuk and Krizek 1973) of different cultivars of L. sinuatum, which is more or less thermosensitive. These differences are often found in this species and are reflected in their flowering. The plant can survive temperatures as low as -3 and -4°C and later functions normally (González et al. 1998). Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK López and González 500 Ornamental Limonum According to Alrson (1993), who insists on the importance of the varietal element to obtain early production of L. sinuatum, believes that cultivation must be carried out within a range of average temperatures of 16 to 18°C during the day and around 10 to 13°C at night. 3.2. Lighting The intensity of supplementary illumination can be low, photoperiod of 12 hours PPFD of 50 mmol·s-1·m-2 by incandescent lamps measured at the plant canopy. The lighted horizontal areas are very affected by the distance between the plant and light source. Below these levels the plant does not flower, even if light is applied for 24 hours. The required photoperiod for the plant was established by some researchers (Semeniuk and Krizek 1972) at 16 hours. 3.3. Relative humidity The vegetative material used for ornamental purposes is in general the result of interspecific crossing between species with not always the same needs. As a result the characteristics of this material can vary. How the plant responds to hydric and hydrometric conditions depends mostly on the vegetative material used and the proper application of cultivation technology in accordance with the plants’ needs. Different irrigation systems such as sprinkling, drip, etc., provide a good distribution of water depending on the vegetative state of the plant. Suitable greenhouse infrastructure such as a good ventilation system: central, mixed, etc., provides greater aeration and better control of the levels of humidity relative to the ambient. A good distribution of plants such as a traversal layout of the rows of cultivation when the longitudinal axis of the greenhouse is a lot longer than the transversal axis provides better circulation of air, avoiding excessive humidity. A good understanding of the vegetative growth of the species enables better planning to achieve an optimum density of plants, so that when adult, the plants do not form barriers of vegetation which can provide perfect breeding grounds for diseases. 3.4. Soil Limonium is available or crop management and yield response in natural soil (Paparozzi et al. 1998; Zizzo et al. 2000). With the same systematics described for humidity it can be deduced that the species can tolerate a great variety of soils, although preferring a light texture from sandy to slightly clayey. Certain wild species, such as L. insignis for example, grow perfectly well in clay soils. A possible reason for this might be the structure the soil, providing good aeration or because of adequate drainage. The optimum level of pH in the soil is believed to be between 6 and 7. Regarding salinity, average levels are considered optimum, although the plant is in fact resistant to high levels of salt. Trials carried out by Zizzo et al. (2003) show that Limonium can also be grown in soiless culture with inorganic substrates, facilitating the maintenance of healthy plants at a good rate. 3.5. Salinity One important feature of the Plumbaginacea family, which includes the genus Limonium, are glands on the green tissue, leaves and flowering stems which secrete saline solutions. These glands were first described by Volkens in 1884 and subsequently by De Fraine in 1916. They are glandular trichomes made up of multi-cell glands and contain a base of apical secretory cells. Secretion activity takes places at several layers of cells deep. The structure and physiology of Limoniun and other salt glands have been studied (Hill and Hill 1973; Campbell et al. 1974; Hill and Hill 1976; Gunning 1977; Baumeister and Ziffus 1981). Studies of the plasmalemma invaginations, such as those which occur with wall protuberances, have been proposed to function as areas where the movement of solutes and water is osmotically coupled to and dependent upon ion pumping (Diamond and Bossert 1977). The surfaces of leaves of Limonium indicate that a variety of elements like as sodium, magnesium, silica, sulphur, phosphorus, chloride, potassium and calcium are secreted by the glands (Faraday and Thomson 1986a, 1986b). The secretion of salts by the salts glands of Limonium is dependent upon and active transmembrane efflux ions across the plasmalemma of the gland secretory cells (Faraday and Thomson 1986a, 1986b; Vassilyev et al. 1990). The higher capacity to compartmentalise salts in the vacuole, and the better regulation of toxic ions by excretion through salt glands permits them to tolerate higher levels of salinity than in cultivated species (Morales et al. 2001). 4. PLANTING In general we can establish two seasons for planting, one in spring and the other in autumn. This is the case for both open air and greenhouse cultivation. Spring planting takes place from May to June with the expectation of producing a harvest in autumn which generally makes use of the majority of flowering stems produced from the onset of harvest. Autumn planting is carried out exclusively in a greenhouse from October onwards. This second phase which is aimed a commencement of production for the end of winter, needs vegetative growth during the cold months to be at an optimum. Therefore all the flowering stems that appear before the target date for flowering are eliminated. In this way the vegetative period is extended extraordinarily around five to six months. 4.1. Density Although in general, similar frameworks are employed, in all species there are factors that can influence how the plants are laid out, for example, if the species is planted in the open air or in a protected environment. In certain cases the different vegetative growths make it inadvisable to have high densities of plants, as this can bring about competition between the plants and provoke mechanisms which can have a harmful effect on them. This was demonstrated in Murcia using equal densities of the species perezii, altaica and latifolia in greenhouse cultivation (González et al. 1998). It was shown that with densities of 4.25 plants/m² both altaica and latifolia grew well. Even when the population was increased to Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK López and González 501 Ornamental Limonum 6.25 plants/m² for altaica it still grew well, but for latifolia this density was excessive and its development was mediocre. Whereas with perezii it was observed that at 4.25 plants/m² the vegetative growth of the plants overwhelmed them, but at 3.25 plants/m² the crop grew fully and was very productive. The final results of this trial could not be published as the crop was severely attacked by Tomato Spotted Wilt Virus. One should bear in mind that floral production of altaica and latifolia is concentrated in the basal foliar rosette from which all its floriferous primordia grow. In the case of perezii, its growth is more arborescent forming several rosettes. These branchlets unfold centrifugally bearing the floriferous stems separately, forming a much more robust plant which spreads out and colonizes all the useable land. For this reason it is not advisable to use any species at random without knowing how they behave in practice, and especially, as in the case of perezii, if its continual production all through the year begins with continual vegetative growth. 4.2. Inductive action of gibberellins Based on the hypothesis that when the amount of gibberellins in the plant reaches certain levels they stimulates flowering, two cultivars of L. sinuatum strains ‘Iceberg’ and ‘Midnight Blue’ were tested with different concentrations of GA3. This was undertaken out of season to achieve greater earliness and increase yield (Wilfret and Green 1975). It was found that concentrations of around 100 ppm did not have any effect. The optimum dose was 500 ppm. Higher doses of more than 1,000 ppm harmed the bloom and produced foliar chlorosis. GA3 was applied in the leaves 110 days after sowing, promoting new, straighter and longer leaves, greater precocity and an increase in flowering under environmental conditions in which the trial was carried out. Subsequently a study was undertaken to ascertain the optimum time of treatment (Wilfret and Raulston 1975). The best results were obtained when GA3 was applied 87 to 101 days after sowing. If it was applied after this period, it did not have any effect, although this may be due to a shorting of the remaining time period. Varietal dependence was demonstrated plus the (above mentioned) relative effectiveness of the gibberellins in relation to greater or reduced light exposure, which was all linked with the need for comparatively cold night time temperatures. The treatments with GA3 increased plant height and spread of plant, number of flowering stems per plant, the length of flowering stem and the size of panicle. GA3 accelerated flowering i.e. reduced the mean flowering time and increased the duration of flowering. The fresh weight of cut flowering stem increased in response to GA3 treatments (Rajesh et al. 2002). The treatment should be carried out preferably at the end of the day, when temperatures fall to avoid leaf burn, aiming the pulverization at the centre of the plant. In addition, there is a technique to improve the quality of the bloom which consists of the application of pulverizations with gibberellins in low concentrations (10 ppm), mixed with a foliar fertilizer rich in phosphate at a concentration of 0.1 per 100, applied during the productive period. This technique is not generally used. 5. IRRIGATION Most of the species of the genus Limonium are not demanding on Table 2 Absorption of nutrients for a L. sinuatum cultivar ‘Midnight’ (adopted from water. Oviedo 1988). Accumulation Age It is advisable to sprinkle during the phase of vegetative (Days) K N S Ca P Mg growth when the basal leaf rosette is formed. This should be 75 334 137 31 11 15 8 Leaves maintained with the appropriate environmental conditions for the 137 2.105 1.293 184 157 95 92 plant for between two and three weeks after transplant. In addition 168 2.260 1.156 246 148 83 86 drip irrigation should be used during the entire period of cultivation. 197 1.184 709 109 138 39 88 228 841 426 51 63 21 58 The density of the sprinklers depends on how far they are placed 267 1.280 789 154 91 89 50 above the ground - the higher they are, the fewer the sprinklers Stem 168 264 174 33 11 16 6 that are needed. A sprinkler set at 1.8 metres above the crop can 197 841 637 69 61 43 32 water 9 m². Apart from supplying a portion of the plants’ water 197 177 74 115 228 22.248 1.069 requirements, sprinkling reduces ambient temperature and the risk 267 4.699 1.994 356 337 178 271 Flowers 168 31 25 7 2 2 1 of plant loss due to hydric stress by decreasing the transpiration of 197 154 124 17 9 11 5 the plant when the root system still has not established itself in the 228 524 380 62 39 36 24 cultivation bed. Sprinkling is recommended once or twice a day 267 1.005 762 152 113 96 61 during winter plantations and three to five times a day with spring 334 137 31 11 15 8 Total plant 75 transplants. 137 2.150 1.293 184 157 95 92 168 2.682 1.452 311 170 111 98 Drip irrigation was carried out using black polyethylene 197 2.277 1.581 211 218 101 129 emitters-drip hoses. The tubes had a 10 mm internal and 12 mm 228 6.016 2.021 332 298 139 204 external diameter, in which interlinear emitters were placed, with a 267 7.226 3.879 711 573 386 398 discharge rate of 2-4 L/h. The emitters were placed at intervals of between 2-3 per linear metre, depending on the soil conditions. One tube was used per row of plants or for each pair, generally maintaining a distance of 50 cm between each line of tubes. An example of the absorption of nutrients for a L. sinuatum cultivar ‘Midnight’ is show in Table 2 (Oviedo 1988) with a plant density of 5 plants/m². Among the systems of coverage fertilization used in the Region of Murcia the following give good results: Before flowering: 4 g/m² of ammonium nitrate at 33.5 per 100; 3 g/m² of monoamonium phosphate at 12 and 61 per 100; 3 g/m² of potassium nitrate at 13 and 46 per 100. After flowering: 2.5 g/m² of ammonium nitrate; 2 g/m² of monoamonium phosphate; 4.5 g/m² of potassium nitrate. The fertilizer is delivered in two applications, attempting to alternate fertirrigation with water irrigation containing no additives. In addition the fertilizer should be enriched once a month with: 5 g/m² of magnesium nitrate at 20-4.5 per 100 of nitrogen and magnesium respectively; 1.5 g/m² of iron chelate; 10 g/m² of calcium nitrate at 30.5-28 per 100 of nitrogen and calcium respectively; and another application of 10 g/m² of potassium sulphate at 50 per 100 of potassium. Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK López and González 502 Ornamental Limonum 6. HARVESTING It has been demonstrated that the flowering stem continues to open its flowers if immediately after being cut it is put into a solution of gibberellic acid, 30 mg/l, with silver nitrate, 30 ppm, at a temperature of 12°C (González et al. 1998). 6.1. Postharvest Once the floriferous stems have been harvested, it is recommended that they be put in water with a simple disinfectant such as sodium hypochlorite at 1 part per 100. They should be placed in a dry and cool protected environment. In this way the stems become ‘vitalized’ which can take between 4 to 6 hours at a temperature of between 18 and 22°C depending on their consistency. The postharvest display life of Limonium cv. ‘Fantasia’, ‘Misty Blue’ and ‘Saint Pierre’ is also reduced by the short length of time flowers are open after harvest (Doi and Reid 1995). The water uptake of these stems declined rapidly over the first 5 days from harvest and was only slightly increased by sucrose vase solution treatments. However, the sucrose treatments increased the length of time that flowers were open from 4 to 17 days, and the longevity of individual flowers from 2.7 to 4.1 days. In another Limonium species, L. sinuatum, flower bud opening also ceases soon after harvest. However, sucrose vase-solution treatments did not extend the period over which flower buds opened (Steinitz and Cohen 1982). Adverse water relations of cut stems can occur via a number of mechanisms that cause vascular blockages. These include bacterial growth, air embolisms, and deposition of mucilage, gums and resin in the xylem conduits (van Doom 1997). Antimicrobial compounds are used to reduce stem blockages caused by microbial growth. Wetting agents can also reduce water relation problems of cut stems by bypassing bacterially induced blockages of the xylem vessels or by dissolving air embolisms (Durkin 1980; Jones et al. 1993a, 1993b). The floriferous stems can be submitted to more complex treatments for their conservation. This is undertaken by the addition of: sugars as sources of energy; silver nitrate and 8-hydoxiquinolene as germicides; silver thiosulphate as inhibitor of ethylene and cytoquinine as correctors of chlorosis of the leaves and stem. In this way the stems can be preserved for 3 to 4 weeks if they are kept at a temperature of 2-3°C (González et al. 1998). 6.2. Yield Around 35 stems per plant with annuals; whereas with pluriannuals the yield is approximately 5 to 15 stems in the first year, 10 to 30 stems in the second year and 20 to 30 stems in the third. REFERENCES Aly MAM, Rathinasabapathi B, Bhalsod S (2002) Somatic embryogenesis in member of Plumbaginaceae ornamental statice Limonium and sea thrift Armeria maritima. HortScience 37, 1122-1123 Ahmed KM, Kandil FE, Mabry TJ (1999) An anticancer tannin and other phenolics from Limonium axaillare (Fam. Plumbaginaceae). Asian Journal of Chemistry 11, 261-263 Amomarco JB, Ibañez MR (1998) Micropopagation of Limonium cavanillessi Erben, a threatened statice, from inflorescence stem. Plant Growth Regulation 24, 49-54 Baumeister W, Ziffus G (1981) Salzsekretion durch die salzdrusen von Armeria maritime L. Zeitschift für Pflanzenphysiologie 102, 273-278 Borrman L (1967) Biological flora of the British Isles Limonium vulgare Mill and L. humile Mill. Journal of Ecology 55, 221-232 Campell N, Thomson WW (1974) The apoplastic pathway of transport to salt glands. Journal of Experimental Botany 25, 61-69 Chapman V (1977) Net Coastal Ecosystems, Elsevier, NY De Fraine E (1916) The morphology and anatomy of the genus Statice as represented at Blakerrey point Part. Annals of Botany 30, 239-282 Diamond JM and Bossert WH (1967) Standing-gradient osmotic flow: A mechanism for coupling of water and solute transport in epithelia. Journal of General Physiology 50, 20612083 Doi M, Reid MS (1995) Sucrose improves the postharvest life of cut flowers of a hybrid Limonium. HortScience 30, 1058-1060 Durkin DJ (1980) Factors effecting hydration of cut flowers. Acta Horticulturae 113, 109-117 Erben M (1978) Die cattung Limonium in SW Mediterranean raum. Mitt. Bott. Stoatssamml München 14, 361-631 Faraday CD, Thomson WW (1986a) Morphometric analisys of Limonium salts glands in relñation to ion efflux. Journal of Experimental Botany 37 (177), 471-481 Faraday CD, Thomson WW (1986b) Functional aspects of the salt glands of the Plumbaginaceae. Journal of Experimental Botany 37 (181), 1129-1135 González A, Bañón S, Fernández JA (1998) Cultivos Ornamentales para Complementos del Ramo de Flor, Mundi Prensa (Ed), Madrid Grieve A (1984) Modern Herbals, Penguin, London Gunning BES (1977) Transfer cells and their roles in transport of solutes in plants. Science Progress 64, 539-568 Hazary A, Leshem B, Cohen A, Rabinowitch HD (1985) In vitro propagation of statice as an aid to breeding. HortScience 20, 361-362 Hill AE, Hill BS (1973) The Limonium salt gland: a biophysical and structural study. International Review of Cytology 35, 299-319 Hill AE, Hill BS (1976) Mineral ions. In: Baker DA, Hall JL (Eds) Transport in Plant Cells and Tissues, North-Holland, Amsterdam, pp 335-376 Jeong JH, Murthy HN, Paek Y (2001) High frequency adventitious shoot induction and plant regeneration from leaves of statice. Plant Cell, Tissue and Organ Culture 65, 123-128 Jones RB, Faragher JD, van Doorn WG (1993a) Water relations of cut flowering branches of Thryptomene calycina (Lindl.) Stapf. (Myrtaceae). Postharvest Biology and Technology 3, 57-67 Jones RB, Serek M, Reid MS (1993b) Pulsing with Triton X-100 improves hydration and vase-life of cut sunflowers (Helianthus annuus L.). HortScience 28, 1178-1179 Kunitake H, Mii M (1990) Plant regeneration from cell culture-derived protoplast of statice (Limonium perezii Hubbard). Plant Science 70, 115-120 Lewis DH, Borst NK (1993) Gibberellic acid promotes flower bud opening on detached flower stalks of statice (Limonium sinuatum L.). HortScience 17, 903-904 López J, Romero M, González A, Guerrero L (2007) Complementos Ornamentales de Verde y Flor: Especies de Interés para la Región de Murcia, Serie Técnica y de Estudios. Consejería de Agricultura y Agua, Región de Murcia, Spain, 216 pp Mercuri A, Antonetti M, Burchi G, Bianchini C, Pasqualetto PL, Schiva T (1999) Manipolazione in vitro di Limonium. Colture Protette 1, 89-93 Mercuri A, Bruna S, Benedetti L, Burchi G, Schiva T (2001) Agrobacterium-mediated transformation of Limonium spp.: modification of plant architecture. Italus-Hortus 8, 43-49 Mercuri A, Bruna S, Benedetti L, Burchi G, Schiva T (2002) Modification of plant architecture in Limonium spp. induced by rol genes. Plant Cell, Tissue and Organ Culture 65, 247253 Morales MA, Torrecillas A, Rodríguez P, Alarcón JJ, Sánchez-Blanco MJ (2001) Effects of salinity on growth and leaf water relations of two genotypes of Limonium sp. Acta Horticulturae 541, 419-423 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiologia Plantarum 15, 473-497 Oviedo I (1988) Algunos aspectos sobre la biología de Limonium sinuatum (L.) Millar. MSc Thesis, SEP Escuela de Biología, Universidad de Costa Rica Paparozzi ET, Hattermann HM (1988) Fertilizer applications on field-grown statice. HortScience 1, 157-160 Pignati S (1972) Limonium Miller. Flora Europea 3, Cambridge University Press Rajesh T, Gupta YC, Sharma YD, Dhiman SR, Pathania NS (2002) Floriculture research trends in India. Proceedings of a National Symposium on Indian Floriculture in the New Millennium, Lal-Bagh, Bangalore, pp 200-202 Rathinasabapathi B, Aly MAM (2001) Somatic embryogenesis in Limonium latifolium (SM.) O. Kuntze, Plumbaginaceae. Proceedings of the Florida State Horticultural Society 113, 170-172 Ruffoni B, Fabbrico G, Semeria C, Dalla Guda C, Volpi L (2000) Shoot differentiation from somatic tissues of ornamental Limonium species. Acta Horticulturae 541, 289-294 Seelye J, Maddocks DJ, Burge GK, Morgan ER (1994) Shoot regeneration from leaf discs of Limonium perigrinum using thidiazuron. New Zealand Journal of Crop and Horticultural Science 22, 23-29 Semeniuk P, Krizek T (1972) Long days and cool night temperature increase flowering of greenhouse grown Limonium cultivars. HortScience 7, 293 Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK López and González 503 Ornamental Limonum Semeniuk P, Krizek T (1973) Influence of germination and growing temperature on flowering of six cultivars of annual statice (Limonium cv.). Journal of the American Society of Horticultural Science 98, 140-142 Serra G (1979) Aspetti tecnici della coltivazione dello Statice. Colture Protette 8, 43-46 Shillo R (1976) Control of flower initiation and development of statice by temperature and day length. Acta Horticulturae 64, 197-203 Steinitz B, Cohen A (1982) Gibberellic acid promotes flower bud opening on detached flower stalks of statice (Limonium sinuatum L.). HortScience 17, 903-904 Uphof JC (1959) Dictionary of Economic Plants, Weinheim Van Doorn WG (1997) Water relations of cut flowers. Horticultural Reviews 18, 1-85 Vassileyev AE, Stepanova AA (1990) The ultrastructure of ion-secreting and non-secreting salt glands of Limonium platyphyllum. Journal of Experimental Botany 222, 41-46 Volkens G (1884) Die kalkdrusen der Plumbagineen Berichte der Deustchen. Botanischen Cesellschaft 2, 334-342 Waisel Y (1972) Biology of Halophytes, Academic Press, NY Wilfret G, Green J (1975) Optimum gibberellic acid (GA3) concentration to accelerate flowering and increase yield of statice. Proceedings of the Florida State Horticultural Society 80, 527-530 Wilfret G, Raulston J (1975) Acceleration of flowering of statice (Limonium sinuatum Mill.) by gibberellic acid (GA3). HortScience 10, 37-38 Zizzo GV, Constantino G, Fascella G, Agnello S (2000) Confronto tra diverse cultivar di Limonium sinuatum (L.) Mill. Allevate in pienaria in ciclo estivo-autunnale. Flortecnica 5, 4344 Zizzo GV, Fascella G, Constantino G, Agnello S (2003) First evaluation of Limonium suitability for soilless cultivation. Acta Horticulturae 614, 235-239 Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK