**2. Seed germination conditions for the main helophyte groups for wastewater treatment**

Germany, France, the Netherlands, Switzerland, Norway, Poland, Slovenia, Lithuania, Italy, Spain, Portugal, Australia, Japan, China, India, Taiwan, South Africa, Turkey, Kenya, Uganda and México [1, 2]. A great variety of wastewaters from industries have been treated: chemical, petrochemical, textile, pulp and paper, tannery, abattoir, food processing, distillery and winery factories. Effluents from pig farms, fish farms, shrimp culture, cobalt recovery, mining or coke plants have also been managed this way. Run-offs from airports, highways, hospitals, agricul‐ tural activities or storm water have been tested as well. Finally, landfill leachate or polluted rivers have been experimentally decontaminated using constructed wetlands with vascular plants as the principal component [1, 3]. Uptake of rare earth elements (REEs) from obsolete

Different species have been used to date, all over the world. They belong to different genera and families. Frequently they are helophytes, which means plants growing in marsh partly submerged in water, so that they regrow from buds below the water surface. The helophytic plants used to build wastewater green filters in constructed wetlands have in common with these significant characteristics: (a) a rich below-ground organ, root or rhizome, as to pro‐ vide substrate for attached bacteria and oxygenation (as much as possible) of areas adjacent to the radicular apparatus; (b) a high-tolerance nutrient and organic loadings; and (c) the production of a high amount of above-ground biomass for winter insulation in cold and

From a botanical point of view, a great list of species could be used in each country, because plant biodiversity is high. Ad hoc species for each biogeographical region could be found, attending to the above-mentioned characteristics. However, in practical terms, a limited number of species have been tested by the industry. Vymazal [1] summarised the most important, and we have taken its publication as a framework. Later references [4–12] have also been consulted, and **Table 1** shows the helophytes that we have considered most

For the production and multiplication of plants in nurseries to the global market, it is necessary to standardise the operations aimed at getting living plants that can be installed in the constructed sewages. Plant reproduction can be made by vegetative way (cuttings) or by seeds (sexual way). Nowadays international trade is essential, so those systems better adapted to international transport and management are more competitive. For this reason seed multiplication systems are very interesting. Seeds are more resistant and better prepared than cuttings, to adverse environmental conditions (light, humidity, temperature), that can occur during international transport. Thus, seed technology development provides interesting tools

Various authors have highlighted that studies on seed germination of aquatic and lacustrine plants are very few [13]. This topic is a much less studied subject that other aspects of seed biology, physiology or ecology. However it has a great importance from an applied point of view. That is why we have made the following bibliographic review. It will let us summa‐

to produce in nurseries good quality plants. It is a sustainable new business.

rise and analyse the state of the art of this interesting topic.

equipment of modern technologies has also been reported [4].

180 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

temperate regions, as well as nutrient removal via harvesting [1].

relevant.

A prospective analysis of the publications issued in specialised databases allows us to know the main techniques proposed for seed germination, fitting us to the scope of (**Table 1**) helophyte species. We set out below the most relevant conditions for a successful germina‐ tion of the genera and species internationally used, excluding palms and aquatic ornamentals.




**Species Family** *Epilobium hirsutum* L. Onagraceae *Festuca arundinacea* Schreb. Poaceae *Filipendula ulmaria* (L.) Maxim. Rosaceae *Glyceria maxima* (Hartm.) Holmb. Poaceae *Gynerium sagittatum* (Aubl.) P. Beauv Poaceae *Heliconia psittacorum* L. f. Heliconiaceae *Heliconia rostrata* Ruiz and Pav. Heliconiaceae *Hemerocallis fulva* L. Xanthorrhoeaceae

182 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

*Hibiscus moscheutos* L. Malvaceae *Hymenocallis littoralis* (Jacq.) Salisbury Amaryllidaceae *Iris pseudacorus* L. Iridaceae *Iris tectorum* Maxim. Iridaceae *Iris versicolor* L. Iridaceae *Juncus effusus* L. Juncaceae *Juncus inflexus* L. Juncaceae *Juncus subsecundus* N. A. Wakef. Juncaceae *Kyllinga erecta* Schumach. Cyperaceae *Lepironia articulata* (Retz.) Domin Cyperaceae *Liatris pycnostachya* Michx. Asteraceae *Lobelia cardinalis* L. Campanulaceae *Lythrum salicaria* L. Lythraceae *Mentha spicata* L. Lamiaceae *Monochoria vaginalis* (Burm. f.) C. Presl ex Kunth Pontederiaceae

*Panicum maximum* Jacq. Poaceae *Panicum repens* L. Poaceae *Paspalum distichum* L. Poaceae *Pennisetum purpureum* Schumach. Poaceae *Phalaris arundinacea* L. Poaceae *Phragmites australis* (Cav.) Trin. ex Steud. Poaceae *Phragmites karka* (Retz.) Trin. ex Steud. Poaceae *Phragmites mauritianus* Kunth Poaceae *Phylidrum lanuginosum* Banks and Sol. ex Gaertn. Philydraceae *Pontederia cordata* L. Pontederiaceae *Rudbeckia hirta* L. Asteraceae

**Table 1.** Synopsis of vascular plants used for constructed greed wetlands with horizontal surface for wastewater treatment.

#### **2.1. Phragmites**

It is one of the most used genera. *Phragmites australis* (*Phragmites communis* Trin.) has been tested in Europe, Canada, Australia, many countries of Asia (except India and Nepal, where they use *Phragmites karka*) and many of Africa (except Central Africa, where it is used *Phragmites mauritianus*). In the USA and New Zealand, it has given good experimental results, but actually the utilisation of common reed has been limited because it is considered an invasive plant species [1, 14].

Seed germination of *P. australis* reaches germinative percentages up to 96–99% under the pretreatment of soaking the seeds with 0.1% KNO3, rinsing them with distilled water before they are sown on layers of Whatman grade no. 1 filter paper (pH 7) in 90-mm Petri dishes. They must be moisten for up to 10 days and maintained into an incubator, with alternating diurnal regime of 12-h daylight at 25°C and 12 h of darkness at 15°C [15]. Fourteen hours/ 25°C and 10 h/20°C regime can also be applied [16]. Watering everyday with 10 ml of 9-mM sulphide solution increases significantly the germination speed [16]. Arbuscular mycorrhizal (AM) fungal inocula of *Funneliformis mosseae* accelerate seed germination of the species and, most important, enhance growth and development of its seedlings, performing as an effi‐ cient bio-accelerator, bio-fortifier and bio-enhancer [17]. Other proposals suggest using 1% agar as a germination medium and the following light and temperature conditions: 12-h/12-h photoperiod and 33/19°C (86% germination), 12-h/12-h photoperiod and 26/16°C (93% germination) and 8-h/16-h photoperiod and 35/20°C (95% germination [18]. Chemicals have a differential effect on *P. karka* seed germination. In complete darkness, as well as in 12-h light, 12-h dark photoperiod and different temperature regimes (10/20°C, 15/25°C, 20/30°C), seed germination is significantly promoted by thiourea (10 mM), nitrate (20 mM), proline (0.1 mM), betaine (0.1 mM), GA3 (3 mM), kinetin (0.05 mM) or fusicoccin (5 μM) [19]. 5- and 10-mM ascorbic acid solutions have also given good results [20].

#### **2.2. Typha**

Cattails are very productive plants with maximum above-ground biomass values in con‐ structed wetlands. Treating systems with *Typha angustifolia, Typha capensis, Typha domingen‐ sis*, *Typha latifolia* and *Typha orientalis* have been reported in the USA, Central and South America, Asia and several European countries [1]. In Spain, floating systems with these plants have been developed as an interesting innovative green technology, QuarQ Enterprise [2] (**Figure 1**).

Typha seeds and seed heads need to be cleaned in a seed cleaner before they are sown [21]. A strong jet of distilled water can be used for this purpose. Afterwards seeds will be settled in deionised water to select the most viable ones, which sink, whereas non-viable ones float [22]. An immersion for 24 h in slightly saline water with a concentration of up to 1% ClNa and pH 6.5–7.5 has also been proposed [23]. Specialised sources of seeds are recommended by the United States Department of Agriculture (USDA) [21], who indicates typha seeds germinate readily when they are planted in clean, moist seed bed and maintained for about 2 weeks in a greenhouse in pots 1 cm under the soil surface. Greenhouse temperature should be 37 ± 3°C.

**Figure 1.** QuarQ Enterprise Water Technologies in Villafranco, Badajoz (Spain).

**2.1. Phragmites**

species [1, 14].

**2.2. Typha**

(**Figure 1**).

It is one of the most used genera. *Phragmites australis* (*Phragmites communis* Trin.) has been tested in Europe, Canada, Australia, many countries of Asia (except India and Nepal, where they use *Phragmites karka*) and many of Africa (except Central Africa, where it is used *Phragmites mauritianus*). In the USA and New Zealand, it has given good experimental results, but actually the utilisation of common reed has been limited because it is considered an invasive plant

184 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

Seed germination of *P. australis* reaches germinative percentages up to 96–99% under the pretreatment of soaking the seeds with 0.1% KNO3, rinsing them with distilled water before they are sown on layers of Whatman grade no. 1 filter paper (pH 7) in 90-mm Petri dishes. They must be moisten for up to 10 days and maintained into an incubator, with alternating diurnal regime of 12-h daylight at 25°C and 12 h of darkness at 15°C [15]. Fourteen hours/ 25°C and 10 h/20°C regime can also be applied [16]. Watering everyday with 10 ml of 9-mM sulphide solution increases significantly the germination speed [16]. Arbuscular mycorrhizal (AM) fungal inocula of *Funneliformis mosseae* accelerate seed germination of the species and, most important, enhance growth and development of its seedlings, performing as an effi‐ cient bio-accelerator, bio-fortifier and bio-enhancer [17]. Other proposals suggest using 1% agar as a germination medium and the following light and temperature conditions: 12-h/12-h photoperiod and 33/19°C (86% germination), 12-h/12-h photoperiod and 26/16°C (93% germination) and 8-h/16-h photoperiod and 35/20°C (95% germination [18]. Chemicals have a differential effect on *P. karka* seed germination. In complete darkness, as well as in 12-h light, 12-h dark photoperiod and different temperature regimes (10/20°C, 15/25°C, 20/30°C), seed germination is significantly promoted by thiourea (10 mM), nitrate (20 mM), proline (0.1 mM), betaine (0.1 mM), GA3 (3 mM), kinetin (0.05 mM) or fusicoccin (5 μM) [19]. 5- and 10-mM

Cattails are very productive plants with maximum above-ground biomass values in con‐ structed wetlands. Treating systems with *Typha angustifolia, Typha capensis, Typha domingen‐ sis*, *Typha latifolia* and *Typha orientalis* have been reported in the USA, Central and South America, Asia and several European countries [1]. In Spain, floating systems with these plants have been developed as an interesting innovative green technology, QuarQ Enterprise [2]

Typha seeds and seed heads need to be cleaned in a seed cleaner before they are sown [21]. A strong jet of distilled water can be used for this purpose. Afterwards seeds will be settled in deionised water to select the most viable ones, which sink, whereas non-viable ones float [22]. An immersion for 24 h in slightly saline water with a concentration of up to 1% ClNa and pH 6.5–7.5 has also been proposed [23]. Specialised sources of seeds are recommended by the United States Department of Agriculture (USDA) [21], who indicates typha seeds germinate readily when they are planted in clean, moist seed bed and maintained for about 2 weeks in a greenhouse in pots 1 cm under the soil surface. Greenhouse temperature should be 37 ± 3°C.

ascorbic acid solutions have also given good results [20].

*T. angustifolia* seeds can germinate with maximum success (100% germination) watering with distilled water, maintaining temperatures of 35/20°C and programming an 8-h/16-h photo‐ period. Seed scarification does not seem to be worthwhile, because 58% of germination is obtained with this pretreatment and the above-mentioned germination conditions. When using 1% agar medium and germination conditions of 33/19°C, and a 12-h/12-h photoperiod, germination can reach 85% [18]. *T. domingensis* seeds do not germinate without light [24]. Germination up to 100% is obtained [24, 25] using an environmental chamber or an outdoor shade house covered by nylon netting (light: 10,764–32,292 lx). Petri dishes with paper towels or filter paper as substrate [25] and oscillating temperatures of 32/26°C or 29/21°C for 15 days [25]. A constant temperature of 30°C and a 12-h/12-h [23] (light: 5,000 lx) or 14-h/10 h [24] light/dark photoperiod can also be used. In the latter case, 5-mm layer of water-saturated *Sphagnum* peat has been tested with good results (85±13% germinative percentage). pH peat is adjusted to 7.0 by adding calcium carbonate (7.5 g CaCO3 per pet's litre) [24]. To remove possible germination inhibitors and to retard the growth of microorganisms, seeds must be previously washed with running water and sodium hypochlorite solution (10%), commer‐ cial bleach [24], and furthermore immersed in a germination activator solution with ammoni‐ um and phosphate, pH 6.5–7 [23]. Pretreatments with 0.1% KMnO4 are also recommended [15]. Another medium quite useful to be used is 1% agar. This combined with the 8-h/16-h photoperiod can give very interesting germination results: 90–100% germination maintain‐ ing constant temperatures of 20–25°C during the germination period and 80–90% if tempera‐ ture is elevated to 30°C [18]. Alternating temperatures of 35/20°C with the same photoperiod and germinative substrate produce 100% germination as well. Changing the photoperiod to a 12-h/12-h rhythm can give good results (86% germination) at a constant temperature of 31°C, and it is excellent (98–100% germination) when a 33/19°C alternating programme is applied [18]. *T. latifolia* seed germination success depends on light, pH and alternating temperatures. It is inhibited by total obscurity and limited at low levels of pH [22] but scarcely affected by anoxic conditions [26]. Germination rates can reach 84% on mesic peat (pH 4.3), 22°C and a 16-h/6-h (day/night) photoperiod [22] and maximum rates when using 20/30°C with 12 h/12 h photo-thermoperiod [27]. Excellent results (97% germination) can be obtained in similar conditions (19/33°C, 12 h/12 h) on 1% agar germination medium [18]. Pre-sowing treatments are also recommended, moistin high humidity over waterfor 1 day at 20°C, and then removing and chipping the covering structure [18]. After that, seeds can be sown in 1% agar germina‐ tion medium and 8-h/16-h photoperiod, obtaining 78% germination at a constant tempera‐ ture of 30°C and 88–84% germination using alternating temperatures of 30/15°C and 30/20°C, respectively [18]. In pre-sowing as above-mentioned and fitting the latter photo-thermoper‐ iod (30/20°C, 8 h/16 h), 76% germination has been reported for a medium containing 1% agar + 101-mg/l potassium nitrate (KNO3) [18]. *T. orientalis* seeds germinate 100% in 1% agar and 35/20°C, 8-h/16-h photo-thermoperiod [18] (**Figure 2**).

**Figure 2.** *Typha latifolia* seeds.

#### **2.3. Scirpus**

Different species of *Scirpus* (*Schoenoplectus*) have been tested in the USA, China, Australia and New Zealand (*Scirpus acutus, Scirpus americanus, Scirpus californicus, Scirpus cyperinus, Scirpus fluviatilis, Scirpus grossus, Scirpus lacustris, Scirpus maritimus, Scirpus pungens, Scirpus sylvati‐ cus, Scirpus tabernaemontani, Scirpus validus*) [1, 5, 6]. Mexico and France have experimented with *S. validus* and *S. maritimus*, respectively. Sewages wastewaters have been frequently treated with these plants [1]. *S. grossus* has been tested for heavy metals content in Malaysia [3].

ture is elevated to 30°C [18]. Alternating temperatures of 35/20°C with the same photoperiod and germinative substrate produce 100% germination as well. Changing the photoperiod to a 12-h/12-h rhythm can give good results (86% germination) at a constant temperature of 31°C, and it is excellent (98–100% germination) when a 33/19°C alternating programme is applied [18]. *T. latifolia* seed germination success depends on light, pH and alternating temperatures. It is inhibited by total obscurity and limited at low levels of pH [22] but scarcely affected by anoxic conditions [26]. Germination rates can reach 84% on mesic peat (pH 4.3), 22°C and a 16-h/6-h (day/night) photoperiod [22] and maximum rates when using 20/30°C with 12 h/12 h photo-thermoperiod [27]. Excellent results (97% germination) can be obtained in similar conditions (19/33°C, 12 h/12 h) on 1% agar germination medium [18]. Pre-sowing treatments are also recommended, moistin high humidity over waterfor 1 day at 20°C, and then removing and chipping the covering structure [18]. After that, seeds can be sown in 1% agar germina‐ tion medium and 8-h/16-h photoperiod, obtaining 78% germination at a constant tempera‐ ture of 30°C and 88–84% germination using alternating temperatures of 30/15°C and 30/20°C, respectively [18]. In pre-sowing as above-mentioned and fitting the latter photo-thermoper‐ iod (30/20°C, 8 h/16 h), 76% germination has been reported for a medium containing 1% agar + 101-mg/l potassium nitrate (KNO3) [18]. *T. orientalis* seeds germinate 100% in 1% agar

186 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

Different species of *Scirpus* (*Schoenoplectus*) have been tested in the USA, China, Australia and New Zealand (*Scirpus acutus, Scirpus americanus, Scirpus californicus, Scirpus cyperinus, Scirpus fluviatilis, Scirpus grossus, Scirpus lacustris, Scirpus maritimus, Scirpus pungens, Scirpus sylvati‐ cus, Scirpus tabernaemontani, Scirpus validus*) [1, 5, 6]. Mexico and France have experimented

and 35/20°C, 8-h/16-h photo-thermoperiod [18] (**Figure 2**).

**Figure 2.** *Typha latifolia* seeds.

**2.3. Scirpus**

*S. americanus* seeds need light to naturally break dormancy and germinate [28] and/or to be subjected to cold stratification (3–6°C) for 30–180 days [18]. Germination conditions of 30– 32°C, 12 h/12 h or 35/20°C are proposed [18]. When germination processes are taking place at greenhouses, it has been suggested to sow seeds in a cold frame pot standing in three centimetres of water. The seeds germinate quickly. When they are large enough to handle, they must be planted into their permanent positions in early summer [21]. Alternatively, a container (pot or flat) can be used. It should be watered from the bottom as necessary, and it should not be covered after sowing, although a light dusting of soil can be applied. If grown in outdoor beds, seeds are sown on level soil and covered with a single layer of burlap or cotton sheet. Soil dry must be avoided, shading with a window screen set 30 cm [28]. Procedures to maximise seed germination of *Scirpus acutus* have been studied in the laboratory [29]. Pregermination conditions included scarification and stratification at 4±1°C for 84 days [18, 29] while submerged in water [29]. Seeds were placed in night/day temperature regimes of 10/25°C under a 14-hour photoperiod (≈200 μmol m−2 s−1 photosynthetic photon flux density), and up to 97.5% germination was achieved [29]. At the greenhouse, similar requirements as for *S. americanus* were reported [28]. To germinate seeds of *S. californicus* in greenhouse, they must be introduced in greenhouse in 2.5 × 2.5 × 5 cm pots, 0.5 cm under the soil surface. Soil surface needs to be moisted and maintained at 35–40°C. Seeds begin to germinate after a couple of weeks. Plants are ready in 100–120 days to come out as plugs [21]. *S. cyperinus* seeds should be imbibed on agar 1% for 20 weeks at 5°C as a pre-sowing treatment that has been suggest‐ ed to have best results [18]. At greenhouses, seeds should be sown in a cold frame as soon as they are ripe in a pot standing in three centimetres of water, and they will germinate easily [21]. A loam, peat and sand wet substrate can also be used [28]. An 8-h/16-h photoperiod is a good option to obtain germinative success; using 1% agar as medium, 100%, 98%, 96% and 90% germination can be reached setting the following alternating temperatures: 35/20°C, 30/15°C or 20/5°C, 25/10°C and 40/15°C [18]. Using 1% agar + 250-mg/l gibberellic acid (GA3), with the same photoperiod, 100% germination is got at alternating temperatures 20/5°C and 93% at 25/10°C [18].

*S. fluviatilis* seeds germinate after a period of moist, cold stratification. They need to be mixed with equal amounts or more of damp sand, vermiculite or other sterile media and intro‐ duced in a plastic bag and maintained at 0–3°C for 3 months. Some seeds may sprout in the storage bag if moist stratified too long. If sprouting occurs, seeds must be immediately planted. Another method of breaking dormancy for this species at temperate climates or latitudes is to sow seeds outdoors in the fall so they may overwinter [28]. *S. lacustris* germination is report‐ ed to occur when using a pre-sowing treatment of cold stratification for 80 days and a later germination phase with alternating temperatures of 30/5°C [18]. Presoaking seeds in sodium hypochlorite and performing cold stratification under light conditions are presented for consideration as a good tool to improve germination results with this species [30]. *S. mariti‐ mus* cold stratification for 80 days is profitable before planting seeds to germinate under 30/5°C [18]. Stratification period can be reduced to 28 days if seeds are presoaked in sodium

hypochlorite and kept under natural light conditions [30]. Mechanical pretreatment of the seeds to evade physical dormancy has also been stated [31]. *S. pungens*, opposite to the abovementioned Scirpus species, does not seem to respond so positively to the mechanical scarifi‐ cation (by squeezing with tweezers) orthe stratification in cold water, although further studies on this taxon must be performed [32]. *S. sylvaticus* germination works very well (80% germi‐ nation), imbibing for 56 days the seeds on 1% agar at 6°C and maintaining them afterwards on a 1% agar medium and a thermo-photoperiod of 33/19°C, 12 h/12 h [18]. Even better results (89% germination) can be obtained replacing the pretreatment by adding to the 1% agar, gibberellic acid (GA3) 250 mg/l [18]. *S. tabernaemontani* pre-sowing treatments have been proposed: (a) cold stratification for 80 days [18], (b) imbibing the seeds for 56 days in 1% agar at 5°C [18] and (c) introducing them for 42 days into a sodium hypochlorite solution under low temperatures and natural light conditions [30]. During the germination period, fluctuat‐ ing temperatures are recommendable [30], 30/5°C [17] and 35/20°C [30]. In the latter condi‐ tions, the use of 1% agar as medium and a photoperiod of 8 h/16 h assert germinative percentages up to 84% germination [18]. *S. validus* seeds germinate after a period of [28] moist cold stratification of 180 days [18]. They need light to naturally break dormancy [27] and germination thermic conditions of 30–32°C [18]. They can easily be sown in flat which will be watered from the bottom as necessary. A light dusting of soil can be applied slightly, cover‐ ing the seeds. If they are grown in outdoor beds, they can be sown on level soil, covering it with a single layer of burlap or cotton sheet, shading it with a window screen (set 30 cm) [28].

#### **2.4. Cyperus**

Several species from *Cyperus* genus (*Cyperus alterniflorus, Cyperus articulatus, Cyperus dubius, Cyperus esculentus, Cyperus flabelliformis, Cyperus grandis, Cyperus immensus, Cyperus involucra‐ tus, Cyperus isocladus, Cyperus malaccensis*) have been tested in Asia (China, Thailand), America (Nicaragua, Brazil), Africa (Kenya) and New Zealand among other countries [1]. Some species, such as *C. alterniflorus*, have recently been used in a pilot scale in Italy [7]. Most of them are not sufficiently studied in terms of the seed technology.

*C. alterniflorus* germinates 100% utilising 1% agar medium and alternating temperatures of 25/10°C or 35/20°C and a photoperiod of 8 h/16 h [18]. *C. articulatus* seed germination is easy going if a photoperiod of 8 h/16 h is fixed and a basic 1% agar medium is used for the process. No pretreatments are required. Maximum results are obtained at different alternating temperatures: 100% at 40/25°C, 98–100% at 35/20°C, 96% at 30/15°C and 88% at 25/10°C. By adding gibberellic acid (GA3) 250 mg/l to the agar medium, we can obtain 100% germina‐ tion at 30/15°C, 98% at 40/25°C, 96% at 25/10°C and 92% at 35/20°C [18]. *C. dubius* can be successfully germinated (76%–85%) sowing the seeds in 1% agar medium, an 8-h/16-h photoperiod and alternating temperatures of 35/20°C and 30/15°C, respectively [18]. *C. esculentus* germination of the seeds is significantly influenced by both light and temperature. It is highest at 35°C, and poor germination was observed at other temperatures (27 and 45°C). The plant growth regulators enhance the seed germination and radical length to a different degree [33]. *C. flabelliformis* germination percentage can reach 100% when using 1% agar as medium and temperature and light conditions of 35/20°C, 8 h/16 h. Cold-wet stratifications as

pretreatments should not be planned because they reduce germination to 80% [18]. *C. involucratus* can be propagated by seeds in temperate latitudes at 18 to 21°C in spring in constantly moist seed compost [34]. *C. malaccensis* germinative process is mediated by arbuscular mycorrhizal colonisation, which is influenced at the same time by pH and moisture [35].
