**2.5. Carex**

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].

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

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

*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

of them are not sufficiently studied in terms of the seed technology.

**2.4. Cyperus**

Some sedges (*Carex acutiformis, Carex gracilis, Carex lacustris*) have also been employed as phytoremediators in temperate regions (East Europe) [1], with recent applications in the east of Europe countries [12]. Very few species have been studied to date, and little is known about the technologies to improve the germination of their seeds.

It is reported that *C. acutiformis* seeds should be undergone to a pre-sowing treatment consisting in maintaining them for 2–6 months at 4°C. Afterthat, they are placed of moist filter paper at germination conditions of 22/10°C, and an up to 65% germination is expected [18]. *C. gracilis* cold-wet stratification is suggested for a successful germination process (more than 80% germination). For that, seeds can be wetted with distilled water and introduced in 100-cc plastic vials, which will be sealed, wrapped in aluminium foil and stored at 4°C in a refriger‐ ator for 6 months. After that, seeds must be placed at an incubator with alternating tempera‐ tures of 22/10°C [36]. *C. lacustris* has conditionally dormant seeds which require cold stratification [37]. The species is very sensitive to storage conditions [38]. Germination temperature regimen of 27/15°C ensures (at least 50%) discrete results [37]. Better results can be obtained by using seeds produced earlierin the same growing season. They must be situated into highly humid soils. Suitable soil amendments should be added to adjust the proper organic matter content to levels found in natural sedge meadows [38].

#### **2.6. Other Cyperaceae**

*Eleocharis sphacelata, Kyllinga erecta* and *Lepironia articulata* have been locally used in Austra‐ lia, Tanzania and China [1]. For *Eleocharis sphacelata*, pretreatment, timing and water depth available for germination play an important part on the sexual multiplication of this plant, but there is still much to know about the specific requirements [39]. *Kyllinga erecta* seeds fail to germinate in darkness [40]. 85% germination has been recorded by the application of a presowing treatment (imbibing on 1% agar for 12 weeks at 5°C), a germination medium of 1% agar and germination conditions of 35/20°C and photoperiod 8 h/16 h [18]. For *Lepironia articulata* a 50% germination was achieved in seeds buried 4 months, exhumed and incubat‐ ed in anoxic/dark conditions [41].

#### **2.7. Bambusoid-like species**

Some bambusoids as *Arundo donax, Brachiaria mutica, Coix lacryma-jobi, Gynerium sagittatum, Pennisetum purpureum, Phylidrum lanuginosum, Thysanolaena maxima, Zizania caduciflora* and *Zizaniopsis bonariensis* have been utilised in Morocco, El Salvador, Costa Rica, Jamaica, Central

America, Australia, Mozambique, China and Brazil [1, 9]. But the multiplication by seeds of most of these species is barely developed.

*Arundo donax* seed germination is much better known than the rest of the bambusoid-like species. Maximum results (100% germination) are got using 1% agar medium and thermic and light conditions of 35/20°C, 8 h/16 h. Several pre-sowing treatments (cold shock −80°C, smoke and heat shock) have demonstrated not to be recommendable, especially if combined with constant temperature regimes of 20°C [18]. On the other hand, hydro-seeding has emerged as a reliable way to be employed. It consists in mixing selected seeds with colloidal substances in an aqueous solution usually along with mulch of various origins and fertilisers. Commer‐ cial names and composition are the following: Provide Verde (complete mixture of soil microorganisms), *Penicillium* sp, algae, polysaccharides, mulch (1 L ha−1), vegetal glue, organic fertiliser (4.4% N, 2.2% P2O5, 1.1% K2O, 2.1% Mg, 2.7% Ca), Envitotal-1 (total nitrogen 1.7% , organic carbon 11%, zeolites 38%, vegetal mulch mixture of cellulose 33% and vegetal glue), Envitotal-2 (total nitrogen 1.7% , organic carbon 11%, zeolites 38%, vegetal mulch mixture of cellulose 33%, soil N-fixing microorganisms 2.6%, vegetal glue), and Cellugrun (cellulose fibre mulch of 80% cellulose content, pH 7.5, bulk density 20–35 g L1-, average fibre length 1400 μm, average fibre thickness 45 μm) and an adhesive hydrocolloid compound as soil control [42]. For an excellent (100%) seed germination result in the case of *Coix lacryma-jobi*, they need to be placed in Petri dishes (11 cm in diameter) containing silica sand and 10-mL distilled waterinto a climate-controlled incubator at 25°C, for a 10 days period, with a light (10 h) and dark (14 h) cycle [43]. Alternating temperatures 35/20°C are not recommended for this species [18].

Pearl millet (*Pennisetum glaucum*) seeds need to be washed previously with liquid soap solution and bavistin (fungicide) for 3 min and rinsed twice with deionised water [44]. Then they must be placed in Petri dishes (90 mm in diameter), containing one moisted piece of filter paper Whatman no. 1, and after that they have to be covered and maintained on a lab bench at room temperature (30 ± 5°C) [44]. An incubator programme of 30/19°C 16 h/8 h has also been successfully used [45]. Better results (100% germination) can be offered if the germination medium is 1% agar and the conditions are constant temperatures of 21°C or 26°C and photoperiod 12 h/12 h or 20°C and photoperiod 8 h/16 h [18]. With the same medium, 80% germination is obtained fixing 33/19°C, 12 h/12 h, and 75% germination with 25°C, 8 h/16 h [18]. Maximum success (100%) is aimed by removing the seed coat and performing the germina‐ tive process at 25°C and an alternating light regime of 8 h/16 h [18]. Finally, in selected lines of this species (`MS 841A', `MS 841B' and `D 23'), some pretreatments have been investigated to ensure at least 75% germination. Thus storage containers (cloth bags, polylined cloth bags), storage environments (ambient, controlled) and seed dressings (carbendazim, captan, thiram, *Trichoderma viride* and *Pseudomonas fluorescens*) were tested. The germination of seeds stored under controlled conditions (82.25%) (temperature 20°C and relative humidity 40%) and in polylined bags (76.62%) was significantly higher than seeds stored under ambient condition (66.43%) and in cloth bags (72.05%). Treatment with bioagent *Trichoderma viride* gave a germination percentage of 77.37%, *Pseudomonas fluorescens* treatment 59.58% and untreated control 74.27%. The incidence of seed mycoflora was 32.39, 28.26 and 29.14% in seeds of 'MS 841A', 'MS 841B' and 'D 23', respectively. Seeds stored under controlled conditions germinat‐ ed 35.31%, under ambient condition (24.55%) and maintained in cloth bags 31.21% and in polylined cloth bags 28.66%. The incidence of contamination with seed mycoflora was 40.24% in the untreated control, 6.90% in thiram treatment, 11.65% in captan treatment, 34.07% in *Trichoderma viride* treatment, 39.46% in carbendazim treatment and 47.28% *Pseudomonas fluorescens* treatment [46].

#### **2.8. Non-cane-like Poaceae**

America, Australia, Mozambique, China and Brazil [1, 9]. But the multiplication by seeds of

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

*Arundo donax* seed germination is much better known than the rest of the bambusoid-like species. Maximum results (100% germination) are got using 1% agar medium and thermic and light conditions of 35/20°C, 8 h/16 h. Several pre-sowing treatments (cold shock −80°C, smoke and heat shock) have demonstrated not to be recommendable, especially if combined with constant temperature regimes of 20°C [18]. On the other hand, hydro-seeding has emerged as a reliable way to be employed. It consists in mixing selected seeds with colloidal substances in an aqueous solution usually along with mulch of various origins and fertilisers. Commer‐ cial names and composition are the following: Provide Verde (complete mixture of soil microorganisms), *Penicillium* sp, algae, polysaccharides, mulch (1 L ha−1), vegetal glue, organic fertiliser (4.4% N, 2.2% P2O5, 1.1% K2O, 2.1% Mg, 2.7% Ca), Envitotal-1 (total nitrogen 1.7% , organic carbon 11%, zeolites 38%, vegetal mulch mixture of cellulose 33% and vegetal glue), Envitotal-2 (total nitrogen 1.7% , organic carbon 11%, zeolites 38%, vegetal mulch mixture of cellulose 33%, soil N-fixing microorganisms 2.6%, vegetal glue), and Cellugrun (cellulose fibre mulch of 80% cellulose content, pH 7.5, bulk density 20–35 g L1-, average fibre length 1400 μm, average fibre thickness 45 μm) and an adhesive hydrocolloid compound as soil control [42]. For an excellent (100%) seed germination result in the case of *Coix lacryma-jobi*, they need to be placed in Petri dishes (11 cm in diameter) containing silica sand and 10-mL distilled waterinto a climate-controlled incubator at 25°C, for a 10 days period, with a light (10 h) and dark (14 h) cycle [43]. Alternating temperatures 35/20°C are not recommended for this species [18].

Pearl millet (*Pennisetum glaucum*) seeds need to be washed previously with liquid soap solution and bavistin (fungicide) for 3 min and rinsed twice with deionised water [44]. Then they must be placed in Petri dishes (90 mm in diameter), containing one moisted piece of filter paper Whatman no. 1, and after that they have to be covered and maintained on a lab bench at room temperature (30 ± 5°C) [44]. An incubator programme of 30/19°C 16 h/8 h has also been successfully used [45]. Better results (100% germination) can be offered if the germination medium is 1% agar and the conditions are constant temperatures of 21°C or 26°C and photoperiod 12 h/12 h or 20°C and photoperiod 8 h/16 h [18]. With the same medium, 80% germination is obtained fixing 33/19°C, 12 h/12 h, and 75% germination with 25°C, 8 h/16 h [18]. Maximum success (100%) is aimed by removing the seed coat and performing the germina‐ tive process at 25°C and an alternating light regime of 8 h/16 h [18]. Finally, in selected lines of this species (`MS 841A', `MS 841B' and `D 23'), some pretreatments have been investigated to ensure at least 75% germination. Thus storage containers (cloth bags, polylined cloth bags), storage environments (ambient, controlled) and seed dressings (carbendazim, captan, thiram, *Trichoderma viride* and *Pseudomonas fluorescens*) were tested. The germination of seeds stored under controlled conditions (82.25%) (temperature 20°C and relative humidity 40%) and in polylined bags (76.62%) was significantly higher than seeds stored under ambient condition (66.43%) and in cloth bags (72.05%). Treatment with bioagent *Trichoderma viride* gave a germination percentage of 77.37%, *Pseudomonas fluorescens* treatment 59.58% and untreated control 74.27%. The incidence of seed mycoflora was 32.39, 28.26 and 29.14% in seeds of 'MS 841A', 'MS 841B' and 'D 23', respectively. Seeds stored under controlled conditions germinat‐

most of these species is barely developed.

In this group we have included species as *Echinochloa polystachya, Festuca arundinacea, Glyceria maxima, Panicum maximum, Panicum repens, Paspalum distichum, Phalaris arundinacea, Sorghum halepense, Spartina alterniflora, Spartina argentinensis, Spartina densiflora, Spartina maritima, Spartina pectinata* and *Stenotaphrum secundatum*, which are reported to be employed in Ecuador, Alabama, Kentucky and Washington (USA), New Zealand, Germany, Czech Republic, Jordan, Italy and Portugal [1]. Although there is some information on the seed technology of these taxa, many of them stillrequire further development of proper methodologies adapted to each species.

Some interesting improvements have been made with *Festuca arundinacea*, for example. To ameliorate seed germination performance of it, hydropriming can be advised. Good results can be obtained by maintaining them in a germinator at 15 to 25°C for a period of 8 h of darkness and 16 h of light with a light intensity of 38 μmol m−2 s−1 provided by cool-white fluorescent lamps [47]. *Glyceria maxima* seed technology for germination has been more profusely tested. 100% germination has been obtained by sowing in 1% agar medium and maintaining one of the following regimes: 20°C, 8 h/16 h; 15°C, 8 h/16 h; 25/10°C, 8 h/16 h and 23/9°C, 12 h/12 h [18]. Using the same medium 95–96% germination was obtained when employing 21°C, 12 h/ 12 h, and 25°C, 8 h/16 h, respectively, and 85% germination if the conditions were 21/11°C, 12 h/12 h [18]. The latter result has been improved (94% germination) repeating the condi‐ tions but pretreating the seeds by imbibition on agar 1% for 4 weeks at 2°C [18]. Several tests adding 101-mg/l potassium nitrate to the agar medium did not give extremely advisable results, neither imbibing the seeds on agar 1% agar for 8 weeks at 6°C [18]. Opposite to this, an imbibition on 1% agar for 1 day at 20°C and then for 2 days at 4°C (germination condi‐ tions of 20/15°C, 12 h/12 h) has been reasonably recommended for it ensures 90% germina‐ tion [18]. This species can also be germinated on damp filter paper into a Petri dish covered with a colourless plastic to allow light penetration and to prevent rapid moisture loss. They will be introduced in a grow chamber with a temperature range of 21–23°C with a 12-h/12-h (light/dark) cycle. If cold stratification at 4°C for 8 weeks has been implemented, more than 80% germination is reported [48].

For *Panicum maximum* recommended method for best (100%) seed germination is to sow them in 1% agar medium and introduce them in a room chamber at a constant temperature of 21°C and 12 h/12 h or a combined cycle 35/20°C, 8 h/16 h [18]. In the latter case, seeds must previously be immersed in 10% Domestos solution for 5 min [18]. Other scarifying proce‐ dures (shallow incisions, mechanical removing) decrease germination in this species [49] from percentages to 92%, 85%, 78%, 72% or even 40% [18]. If the available light cycle is 12 h/12 h, the former conditions should be maintained; increasing temperature to 26°C lightly decreas‐

es the percentage of germination to 90%, and changing to alternating 33/19°C reduces it to 75% or 80% (in the particular case that a solution of 101-mg/l potassium nitrate has been added to the agar medium) [18]. *Panicum repens* germinates 80% if seed coats are removed and seeds are germinated on 1% agar medium and photothermic conditions are 35/20°C, 8 h/16 h [18]. Some authors have pointed out [50] that *Phalaris arundinacea* germination does not occur in the dark [50] and that this species is photoperiod insensitive in the range of 12–16 hours. Upon them, the highest germination percentages (up to 80) can be obtained under white light and red light (11.0 μmol s−1 m−2) and up to 40 with high red: far-red ratios [50]. On the other hand, 96–98% germination has been reported [18], scarifying the seeds and sowing them in 1% agar at germination conditions of 23/9°C, 12 h/12 h [18], and 90% at an 8-h/16-h photoperiod and 20°C [18]. Without removing the coat, 90% germination can be reached on 1% agar medium, fixing the incubator conditions at 25/10°C, 8 h/16 h [18], and 80% by adding to the agar a solution of 101-mg/l potassium nitrate and choosing the cycle 23/9°C, 12 h/12 h [18].

*Sorghum halepense* germinates perfectly (100% germination) on 1% agar medium at a con‐ stant temperature of 26°C and a day/light rhythm of 12 h/12 h or at an alternating 35/20°C, 8 h/ 16 h, if seed coat has previously been removed [18]. *S. halepense* has neutral photoblastic seeds and presents mechanical-type dormancy [51]. Different sorts of scarification have been proposed: sandpaper [51], scalpel, pericarp excision from along proximal to distal ridge above embryo and previous imbibition on 1% agar for 18 weeks at 20°C, or at 25/10°C, or for 8 weeks at 6°C [18]. In the 12-h/12-h light regime, alternating temperatures (23/9°C) reduced germination to 80%, so it seems better to maintain it constant at the cited value (26°C) [18]. If an 8-h/16-h photoperiod is of our convenience, we should consider that the thermic alterna‐ tion of 25/10°C lowers germination to 89%, even for scarified seeds [18]. As well fixing temperatures to 20°C or 30°C brings germination percentages down to 85%, despite the scarifying [18]. Adding 101-mg/l potassium nitrate to the agar medium has not improved the germination results in many tests made in these conditions. It is not a best choice, for it may get down germination even to 78% [18]. *Spartina alterniflora* germination of the seeds is not affected by light or dark, and the optimal temperature 16/26°C (night/day) gives a germina‐ tion rate >90% [52]. Salinity does not reduce seed germination of this halophilous species [53] when it does not exceed to 450-mM NaCl [54]. Improved smooth cordgrass cultivars of easy germination, 'St. Bernard' (LA12-101) (Reg. No. CV-268, PI 665014), 'Las Palomas' (LA12-102) (Reg. No. CV-269, PI 665015) and 'Lafourche' (LA12-103) (Reg. No. CV-270, PI 665016), are available for implementation [55]. Good results have been obtained in climate chambers at 25°C constant temperature or alternating 20/30°C, much better [56]. In the case of the close taxa *S. pectinata*, 91% germination has been reported scarifying the seed before sowing and doing it on 1% agar at 25/10°C, 8-h/16-h conditions [18].

#### **2.9. Juncoid species**

We include here plants belonging to Juncaceae (*Juncus effusus, Juncus inflexus, Juncus subse‐ cundus*), Juncaginaceae (*Triglochin procerum*) and *Hemerocallis fulva* (Xanthorrhoeaceae) that have been utilised for wastewatertreatmentin Australia, Kentucky (USA), Portugal, Germany, Canada, Slovenia and Spain [1, 10].

*Juncus effusus* seeds need light, moisture and heat for germination. To decrease the time the seed takes to sprout, two methods have been proposed: soaking the seeds [21] and imbibing them on 1% agar for 8 weeks at 2°C [18]. To grow seeds in greenhouses, they will be placed on soil surface, pressing lightly to assure good soil contact, not covering them and keeping the soil moist. Greenhouse should be kept hot (32–38°C). Seeds begin to germinate in approxi‐ mately 1 week. Soil moisture has to be maintained until plants are to be transplanted [21]. In incubators, to ensure 97% germination, the medium suggested is 1% agar + 250-mg/l gibber‐ ellic acid (GA3). Germination conditions are alternating temperatures and photoperiod of 23/9°C, 12 h/12 h. If no scarification is made and no GAE is used, germination may be reduced to 91% (35/20°C), 8 h/16 h, or even 88% (30°C, 8/16) [18]. *J. inflexus* seed germination is a challenge, and the best published results about their seed germination reach just 96%. Basic 1% agar has been repeatedly used as germination medium. Germination cycles of 23/9°C, 12 h/ 12 h, and 35/20°C, 8 h/16 h, let to reach 95%. With the same photoperiod and medium, changes in the thermic programme decrease the germinative success to 88% (25/15°C, 8 h/16 h), 86% (25°C, 8 h/16 h) and 70% (25/15°C, 8 h/16 h), respectively. Moving the light plan to a 12-h/12 h cycle and the temperatures to 23/9°C has reduced the germination to 50% [18]. Pretreat‐ ments have also been tested. Chipping with scalpel has given good results (94–96% germination) when combined with adequate photothermic programmes: 26°C, 12 h/12 h. Other proposals have been to water the seeds in high humidity over water for 1 day at 20°C and then imbibing on 1% agar for 8 weeks at 5°C. In this case 85% germination has been got by applying the photothermic routine 20/10°C, 8 h/16 h, and 95% by just changing the photoperiod to 12 h/12 h. It does not seem so recommendable to use another pre-sowing treatments published, vg. 1% on agarimbibition for 8 weeks at 5°C at 20/10°C, 8 h/16 h, or agar imbibition during 6 weeks at 20/10°C, 8 h/16 h, using 25°C, 8 h/16 h, and 25/10°C, 8 h/16 h, Respectively, bacause they let to reach 88% and 75%. On this occasion, even using GA3 gibberellic acid (250 mg/l) as a complement of the agar medium [18]. Finally to germinate *J. subsecundus* seeds, 1% agar medium is suggested, maintaining 20°C, 8 h/16 h, as to reach to 75% germination [18].

#### **2.10. Flowering ornamental monocots**

es the percentage of germination to 90%, and changing to alternating 33/19°C reduces it to 75% or 80% (in the particular case that a solution of 101-mg/l potassium nitrate has been added to the agar medium) [18]. *Panicum repens* germinates 80% if seed coats are removed and seeds are germinated on 1% agar medium and photothermic conditions are 35/20°C, 8 h/16 h [18]. Some authors have pointed out [50] that *Phalaris arundinacea* germination does not occur in the dark [50] and that this species is photoperiod insensitive in the range of 12–16 hours. Upon them, the highest germination percentages (up to 80) can be obtained under white light and red light (11.0 μmol s−1 m−2) and up to 40 with high red: far-red ratios [50]. On the other hand, 96–98% germination has been reported [18], scarifying the seeds and sowing them in 1% agar at germination conditions of 23/9°C, 12 h/12 h [18], and 90% at an 8-h/16-h photoperiod and 20°C [18]. Without removing the coat, 90% germination can be reached on 1% agar medium, fixing the incubator conditions at 25/10°C, 8 h/16 h [18], and 80% by adding to the agar a

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

solution of 101-mg/l potassium nitrate and choosing the cycle 23/9°C, 12 h/12 h [18].

it on 1% agar at 25/10°C, 8-h/16-h conditions [18].

**2.9. Juncoid species**

Canada, Slovenia and Spain [1, 10].

*Sorghum halepense* germinates perfectly (100% germination) on 1% agar medium at a con‐ stant temperature of 26°C and a day/light rhythm of 12 h/12 h or at an alternating 35/20°C, 8 h/ 16 h, if seed coat has previously been removed [18]. *S. halepense* has neutral photoblastic seeds and presents mechanical-type dormancy [51]. Different sorts of scarification have been proposed: sandpaper [51], scalpel, pericarp excision from along proximal to distal ridge above embryo and previous imbibition on 1% agar for 18 weeks at 20°C, or at 25/10°C, or for 8 weeks at 6°C [18]. In the 12-h/12-h light regime, alternating temperatures (23/9°C) reduced germination to 80%, so it seems better to maintain it constant at the cited value (26°C) [18]. If an 8-h/16-h photoperiod is of our convenience, we should consider that the thermic alterna‐ tion of 25/10°C lowers germination to 89%, even for scarified seeds [18]. As well fixing temperatures to 20°C or 30°C brings germination percentages down to 85%, despite the scarifying [18]. Adding 101-mg/l potassium nitrate to the agar medium has not improved the germination results in many tests made in these conditions. It is not a best choice, for it may get down germination even to 78% [18]. *Spartina alterniflora* germination of the seeds is not affected by light or dark, and the optimal temperature 16/26°C (night/day) gives a germina‐ tion rate >90% [52]. Salinity does not reduce seed germination of this halophilous species [53] when it does not exceed to 450-mM NaCl [54]. Improved smooth cordgrass cultivars of easy germination, 'St. Bernard' (LA12-101) (Reg. No. CV-268, PI 665014), 'Las Palomas' (LA12-102) (Reg. No. CV-269, PI 665015) and 'Lafourche' (LA12-103) (Reg. No. CV-270, PI 665016), are available for implementation [55]. Good results have been obtained in climate chambers at 25°C constant temperature or alternating 20/30°C, much better [56]. In the case of the close taxa *S. pectinata*, 91% germination has been reported scarifying the seed before sowing and doing

We include here plants belonging to Juncaceae (*Juncus effusus, Juncus inflexus, Juncus subse‐ cundus*), Juncaginaceae (*Triglochin procerum*) and *Hemerocallis fulva* (Xanthorrhoeaceae) that have been utilised for wastewatertreatmentin Australia, Kentucky (USA), Portugal, Germany, Decorative perennial herbs are easily available and can grow well under local climatic conditions. Among monocotyledons, many species have been used, especially for on-site treatments where aesthetic or look of the place is an important factor. Some of those species are *Acorus calamus, Hymenocallis littoralis, Colocasia esculenta, Canna glauca, Canna indica, Canna x generalis, Heliconia psittacorum, Heliconia rostrata, Iris pseudacorus, Iris tectorum, Iris versicolor* and *Thalia geniculata*. There are reports of their use in Tanzania, Ohio, Kentucky (USA), Mozambique, Brazil, Mexico, Colombia, El Salvador, Czech Republic, Estonia and Portugal [1, 7, 8].

*Acorus calamus* is an emergent macrophyte with great potential for use in wetland restora‐ tion in North America. Seed germination occurs only in full light at 15/25°C or 20/35°C and seeds fully submerged [7]. In temperate climates, they also can be planted in a greenhouse during the fall or winter. A 5-cm deep tray filled with an organic-soil mix can be used. Seeds

should be scattered sparsely on the surface and pressed firmly into the soil, burying them no further than 0.3-cm deep. Soil needs to be moist to saturation. Seed does not require stratifi‐ cation and germinates in less than 2 weeks. When plants get enough size, they must be transplanted [21]. *Colocasia esculenta* germ plasm can be conserved as seed for at least 2 years at constant 5°C and −20°C when seed moisture content is reduced to 10–12% and at ambien‐ tal room temperature (21.5–34.4°C, mean 27.2°C) when seed moisture content is reduced to 7.3% [57]. *Canna indica* is an herbaceous species with ornamental and medicinal value, having seeds with a hard seed coat. Seed germination is good at 10–40°C, being the optimal temper‐ ature range between 13.84 and 34.41°C, determined by the enthalpy of activation [58]. To open the imbibition lid, a raised incubation temperature of 50°C during 24 hours in wet surround‐ ings is enough. This hydration induces germination. As a result, the integumentary part of the seed coat softens, making it possible forthe germinalroot to emerge from the seed. This waterregulating mechanism, combining an impermeable palisade layer and imbibition lid, is a unique feature of the Cannaceae. The seed coat is mainly of chalazal origin, and the main mechanical layeris formed by the exotesta. Otherfamilies of the Zingiberales, in contrast, open by an operculum formed by all seed coat layers. Moreover, the seed coat in those families is of integumentary origin, and the main mechanical layer is formed by the exo- and/or endo‐ testa [59]. *Heliconia rostrata* seed germination is well known as difficult and problematic, because the embryo is not yet well differenced when the seed matures, and it has a hard testa which does not allow waterto get into it. It can take from 3 months to 3 years [60]. *Iris versicolor* seeds germinate 58% in the greenhouse using cold stratification; this is storing seeds in wet paper towels at 4–5°C for 3–4 weeks [61]. *Iris pseudacorus* germinates 80–86% in an incubator at 12-h/12-h light regime and 30/20°C alternating temperatures or constant temperature of 26°C. In the latter case, seeds must be pretreated by immersion in 10% Domestos solution for 5 min and then imbibed on 1% agar for 8 weeks at 6°C, and then the seed is scarified (covering structure removed and seed coat chipped). After that, a solution of 250-mg/l gibberellic acid (GA3) must be added to the 1% agar medium.

#### **2.11. Megaforbics and other groups**

We have included here species as *Asclepias incarnata, Hibiscus moscheutos, Filipendula ulmaria, Liatris pycnostachya, Lobelia cardinalis, Lythrum salicaria, Mentha spicata, Rudbeckia hirta* and *Silphium perfoliatum* [1] that have been tested in Ohio, Kentucky and Minnesota (USA).

To germinate *Asclepias incarnate* seeds, they must be placed into plastic bags filled with moist perlite or vermiculite and stored for 4–12 weeks in a cold (1–3°C) place. Good germination results have been reported without stratification by soaking the seed twice in 87°C for 12 hours. Germination trays can also be used, filling the cells with a commercial seedling mixture or a mix of *Sphagnum* peat moss and vermiculite and moistening them very well. Seeds should be gently pressed into the soil, three seeds per cell, and covered with a very thin layer of soil. It is recommended to keep the soil moist during germination by spraying or misting. Ambient temperatures should remain between 18 and 23°C. This species requires light for germination [21]. *Filipendula ulmaria* is successfully germinated (95%) using 1% agar as germination substrate and incubator conditions of 25/10°C, 8 h/16 h, or 23/9°C, 12 h/12 h

[18]. *Liatris pycnostachya* germinates in 1% agar medium with maximum percentages at a lightalternating cycle of 8 h/16 h, maintaining a constant temperature of 15°C and 93% if temper‐ ature is elevated to 20% [18]. Stratification is needed to obtain efficient germination in this genus. Benzyladenine and thiourea are considered good agents for breaking dormancy. Fixing the germination chamber at 20°C and stratification at 4°C for 10 weeks produced 98% germination. Similar good results were obtained with the application of the following pretreatments: aqueous solutions of benzyladenine at 10 or 100 mg/l are applied to blotter paper; dry seeds treated for 3 minutes in benzyladenine at 0 to 1126 mg/l and dissolved in Acetone; a 3-minute acetone permeation of seeds with benzyladenine at 225 or 1127 mg/l; or seeds immersed in thiourea at 0.76 or 7.61 mg/l for 24 hours. Nevertheless gibberellic acid GA3 at 1, 10 or 100 mg/l in H2O did not show significant efficiency [62]. *Lobelia cardinalis* seeds will germinate without cold stratification, but they need light. For this reason they can be sown in a flat with a damp fine grade peat light mix. If we keep the flats moist and under lights or in a greenhouse, they should green up in a few weeks. Afterwards they can be transplanted in 4–6 weeks into individual pots [21]. At an incubator, 92% germination is reported for the germination medium 1% agar and the germination conditions 15°C, 8 h/16 h [18]. *Lythrum salicaria* pre-sowing treatments have been fully described. Imbibition on 1% agar for 8 weeks at 6°C, 5°C and 2°C and further cultivation on 1% agar medium have given excellent results: 100% germination (21/11°C, 12 h/12 h, or 20/10°C, 8 h/16 h) and 95% germination (21/11°C, 12 h/12 h). Similar results have been reported even without that pre-sowing treatments but fixing the specific photothermic programmes of 35/20°C, 8 h/16 h (100%), or 20°C, 8 h/16 h (95%). Lower levels (84%) are obtained at 25/10°C, 8 h/16 h. The addition of 250-mg/l gibber‐ ellic acid (GA3) to the agar germination medium does not seem to have a clearly positive effect, although 92% germination is reached when using 12 h/12 h and 21/11°C and 88% if applying 8 h/16 h and 20/10°C [18]. *Mentha* spp. cultivation has been studied from different perspec‐ tives. Recent studies have explored the influence of high-frequency pulsatile electromagnet‐ ic fields and ultrasound pulsatile fields to improve mint seed germination. Further studies are needed, but there are promising first results [63]. In *Mentha spicata*, optimal temperature for seed germination is 30°C. It could be affected under different light qualities and full dark‐ ness conditions. White light is the most conductive to seed germination, but full darkness is the least conductive. After soaking treatment with GA 3 0.25% KNO3, seed germination percentage is significantly promoted [64]. *Rudbeckia hirta* can be easily multiplied by seeds cultivating them on 1% agar medium in an incubator at 21°C, 12/12 (100% germination) or at 10°C, 15°C and 25°C and 8-h/16-h light cycle (84%, 89%, 89%, respectively) [18]. Finally *Silphium perfoliatum* seeds germinate 79% in 1% agar, alternating temperatures of 25/10°C and photoperiod 8 h/16 h [18].
