Application of plant growth-regulators, a simple technique for improving the establishment success of plant cuttings in coastal dune restoration
Elena Balestri*, Flavia Vallerini, Alberto Castelli, Claudio Lardicci Dipartimento di Biologia, University of Pisa, via Derna 1, 56126 Pisa, Italy
* Corresponding author. Tel: + 39 050 2211442; Fax + 39 050 2211410 E-mail addresses: [email protected] (E. Balestri),
[email protected] (F. Vallerini),
[email protected] (A. Castelli),
ABSTRACT
Exogenous application of plant growth regulators (PGRs) may be an effective technique for increasing the rooting ability and the growth of vegetative fragments (cuttings) of plants used in dune restoration programs. Various concentrations (0, 50 and 100 mg-1) of two auxins, alpha-naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA), and two cytokinins, 6-furfurylaminopurine (Kinetin) and 6-benzylaminopurine (BAP), were applied separately to cuttings of two widely used species for restoration, Ammophila arenaria and Sporobuls virginicus. Root development and production of new buds in cuttings were examined under laboratory conditions one month after application. Cuttings were also examined one year after transplanting into a sandy substrate under natural conditions, to test for possible long term effects of PGRs on plant establishment success and growth. The response of the two study species to PGRs differed substantially. In A. arenaria the auxin NAA at 100 mg l-1 reduced the time for root initiation and increased the rooting capacity of cuttings, while the cytokinin Kinetin at 50 mg l-1 facilitated root growth. No auxin had effect on rooting or growth of S. virginicus cuttings, but treatment with 100 mg l-1 Kinetin resulted in higher rooting success than control. One year after planting, the cuttings of A. arenaria treated with 100 mg l-1 NAA showed a higher establishment success (90% vs. 55%) and produced more culms and longer roots than control; those treated with cytokinins did not differ in the establishment success from control, but had longer roots, more culms and rhizomes. On the other hand, the cuttings of S. virginicus treated with 100 mg l-1 Kinetin showed a higher establishment success (75% vs. 35%) and had more culms than control. Therefore, in restoration activities that involved A. arenaria a pre-treatment of cuttings with NAA would be beneficial, as it allows the production of a higher number of well-developed plants with high survival potential and greater area cover. Instead, a
pre-treatment of cuttings of S. virginicus with Kinetin would be useful to achieve more acceptable plant survival rates. This easy and low cost-effective technique may be extended to other dune plant species and applied on a large scale to improve the chance of dune restoration success.
1. Introduction
Coastal dunes are under threat of extensive erosion in most parts of the world (Bird, 1996; Feagin et al., 2005), particularly in Europe (Géhu, 1985; Salman and Strating, 1992; De Lillis, 1998; Gómez-Pina et al., 2002; Brown and McLachlan, 2002; van der Meulen et al., 2004; Defeo et al., 2009; Maun and Fahselt, 2009) where costal dunes are listed as priority natural habitats (EEC, Habitats Directive 1992), whose preservation requires conservation and management actions to be adopted by Member States (O'Briain, 2005). Because of the fundamental ecosystem services provided by sand dunes to nearby urban areas, including protection from costal erosion (Schlacher et al., 2007; Defeo et al., 2009; Gutiérrez et al., 2010), efforts have been made worldwide, especially in the United States, Europe, South Africa and Australia, for rescuing to damaged dunes at least a minimum of the form and function inherent in pristine habitats (Woodhouse, 1982; Salman and Strating, 1992; AGENC, 1994; De Lillis, 1997; Caniglia et al. 1998; Tinelli et al., 1998; Hertling and Lubke, 2000; Greipsson, 2002; Gómez-Pina et al., 2002; Bovina et al., 2003; Rozé and Lemauviel, 2004; Clements et al., 2010; Escaray et al., 2010). Sand fencing in conjunction with the planting of perennial indigenous species is the most successful and cost-effective restoration practice to reconstruct or stabilize coastal dunes (Gómez-Pina, 1999; Pickart, 1990; van der Salm and Unal, 2001; Doody, 2001; Benavent Olmos et al., 2004; De Lillis et al., 2004). There are two primary sources of planting material for dune restoration: cuttings (culms, rhizomes or shoots) obtained by thinning established stands, and seeds. The planting of cuttings has specific advantages over seed sowing for large scale programs. Cuttings may be taken theoretically year-round from natural stocks,
while seeds are available for a relatively short period and for some species there is not always sufficient supply of seeds, and therefore seedlings (Woodhouse, 1982; Huiskes and Harper, 1979; Gomes Neto et al., 2006). In addition, plants regenerated from cuttings are genetic clones of their parent stocks, thus they can be re-introduced to the original native habitat without altering the genetic integrity of populations. Although potentially efficient, this system is limited in practice by the inability of cuttings of some species to develop a well-structured root system rapidly after planting (Craig et al., 1981; Rozema et al., 1985; Hesp, 1991; Maun, 1997; Thetford and Miller, 2002; Gomes Neto et al., 2006). Rapid root regeneration is crucial for restoration of full nutrient and water absorption capacity of plants, particularly in environments such as coastal dunes characterized by low moisture and nutrient supply, and substrate instability (Barbour et al., 1985; Zhang, 1996; Gilbert et al., 2008). The application of fertilizes is often recommended to enhance dune plant establishment success, but this practice may be not beneficial before the cuttings had formed roots (van der Putten and van Gulik, 1987; van der Putten, 1990; Willis and Hester, 2008). Another method that is likely improve the chance for planting success consists of maintaining cuttings in a nursery for one-two years before transfer to the field (Woodhouse, 1982). However, this strategy is time-consuming and increases the costs of restoration projects. Therefore, there is interest in developing cost-effective techniques for rapid initiation of roots and proliferation of shoots in cuttings, as well as establishing plants from a minimum amount of starting plant material via in vitro culture (Balestri et al., 2001; Park et al., 2004; Valero-Aracama et al., 2007; Panayotova et al., 2008).
Formation of roots and shoots is under hormonal control, and exogenous application of plants growth regulators (PGRs), such as auxins and cytokinins, is frequently used in vegetative propagation to improve rooting and transplant quality in a wide range of terrestrial and aquatic species (Blakesley, 1994; Terrados Muñoz, 1995; Nadeem et al., 2000; Balestri and Bertini,
2003; Balestri and Lardicci, 2006; Butola and Badola, 2007). Few attempts have been made to examine the role of PGRs, or to determine the optimum PGR for rooting in dune plants (Craig et al., 1981; Alpert et al., 2002; Thetford and Miller, 2002; Park et al., 2004). Available data indicate that for some species application of an auxin is not essential for rooting, but it improves root quality measures (i.e., number and length of roots) in cuttings (Craig et al., 1981; Thetford and Miller, 2002).
The objective of the present study was to examine whether a pre-treatment of dune plant cuttings with PGRs increased rooting and plant development, and if so, whether the use of treated cuttings improved the success of dune restoration programs. To test these hypotheses, two perennial clonal grasses of worldwide distribution in sand dunes, Ammophila arenaria (L.) Link., Sporobuls virginicus Kunth, were used. Because of the ability to stabilize large areas of blowing sand and build foredunes, the planting of bundles of culms of A. arenaria is widely used to stabilize dunes in Europe, United States and South Africa (Woodhouse, 1982; Huiskes, 1979; van der Meulen et al., 1989; van der Putten, 1990; Vestegaard and Hansen, 1992; Sanjaume and Pardo, 1992; van der Laan et al., 1997; De Lillis et al., 2004; Rozé and Lemauviel, 2004). S. virginicus is considered a valuable candidate for projects which mainly require stabilization of seaward slopes (Converio et al., 2007; Escaray et al., 2010). Specifically, we examined under laboratory conditions the influence of four PGRs (two auxins and two cytokinins), exogenously applied at various concentrations, on the regeneration ability and growth of cuttings. The performance, in terms of plant establishment success and growth, of the cuttings was also monitored for one year after transplanting into pots maintained under natural conditions in order to test for possible long term effects of treatments. We expected that auxins would maximise root initiation capacity and growth, and cytokinins would mainly stimulate
vegetative development (shoot formation and branching) of cuttings, allowing treated plants to cover and stabilize larger areas of substrate than untreated plants.
2. Material and methods
1.1. Plant material
Plant material was collected from natural plant populations growing along a dune system at Rosignano Solvay (Italy) in spring (late May 2007) when plants were actively growing. The collection of this material was done manually, with special care to not damage parent plants. After collection, the plant material was placed in plastic bags and transported to the laboratory. To minimize the impact on existing populations a minimum plant material was collected as source of cuttings. Cuttings of Ammophila arenaria consisted of single vegetative culms; the apical tips of culms were cut to a length of about 15 cm to provide material of uniform size. Cuttings of Sporobolus virginicus consisted of single non-branched vegetative culms, 10-15 cm in high, with intact apical meristem.
1.2. Rooting and growth responses of cuttings
The effect of two auxins, alpha-naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA), and two cytokinins, 6-furfurylaminopurine (Kinetin) and 6-benzylaminopurine (BAP), on regeneration (root development and growth) of cuttings was tested in laboratory. The PGRs (100 mg) were each dissolved in 2 ml of distilled water and ethanol 90° (50% v/v) the day of treatment to minimize the loss of strength through photo-oxidation and bacterial decomposition to prepare stock solutions. Final solutions of 50 and 100 mg l-1 of each PGR were prepared by
serial dilution with tap water from the respective stock solutions and used as treatments. The bases (1-2 cm long) of cuttings were immersed in 100 ml final solutions of PGRs for 60 sec, followed by 20 min of air drying. Untreated cuttings were immersed for the same period in the distilled water and ethanol solution (50% v/v) diluted with tap water (100 ml) to serve as control. Treatments were applied to two groups, each of ten cuttings for species. There were 9 treatments for each species, with a total of 540 cuttings. After treatment, the cuttings were inserted vertically into moistened rock wool blocks (5 cm height) and placed randomly in rooting beds (multi-cell containers, 20 x 40 x 10 cm, commercially available tray units used to grow seedlings and cuttings of plants). Two separate rooting beds (experimental units), each containing ten cuttings, were used for each treatment and species. The beds were randomly arranged in a growth chamber and maintained at 24 (± 0.5) °C, relative humidity of 60-80%. Illumination was with cool white fluorescent tubes providing approximately 35 µmol m–2s–1 during a 12-h photoperiod. The cuttings were kept humid by spraying them with tap water throughout the experimental period. Cuttings were gently taken off from the blocks at interval of 3-4 days for 35 days and replanted soon after data collection in their respective block. For each treatment and species, root development was scored as rooting percentage, number of roots per rooted cutting and length of longest root. The mean rooting time in a given treatment and species was calculated as,
where tr = mean rooting time,
k i 1
= maximum number of rooted cuttings in the treatment,
ni = non-accumulated number of cuttings that rooted during the interval ti – ti –1, and ti = time
i k i i i r n t n t 1 . .between the start of the experiment and the ith observation, applying the concept of mean time used for germination by Labouriau and Osborn (1984). Because both rooting and germination are biological processes distributed in binomial fashion, the statistical treatment of the mean time of germination is applicable to the rooting process (number of rooted cuttings achieved during a certain time interval in relation to the maximum number of rooted cuttings). The number of new buds and rhizomes in survived cuttings was also measured.
2.3. Performance of rooted cuttings
At the end of the experiment described above (July 2007), all cuttings were extracted from blocks and transplanted into pots (30 cm in diameter), filled with a mixture of sand from natural stands and commercial peat (1:1). There was a single cutting per pot. The pots were maintained in a full-sun location near a beach of Rosignano Solvay (Italy) for 12 months to test for possible long term effects of PGRs on plant growth and establishment success. The cuttings were planted in pots, rather than directly in sand, since it is difficult to avoid breakage of the roots and the rhizome systems in A. arenaria during excavation from sand dunes (Hobbs et al., 1983) for measurements. In July 2008, plants were carefully extracted from the pots. As the effective stabilization success of a dune by planting is related to the number of shoots and size of the horizontal rhizome system produced by plants (Hobbs et al., 1983), the number of newly produced culms and buds, and the number and length of new underground rhizomes which grow plagiotropically (runners) were recorded for each plant. Since it was difficulty to determine the number of roots produced by each plant, only the length of the longest root was measured. As the roots of these species mainly grow horizontally (van der Putten, 1989), the length of roots may be an indicator of the ability of plants to spread and exploit resources from the substrate.
Finally, the establishment success was calculated as proportion of the initial number of cuttings (20) from each treatment that survived at the end of the experiment. Only non-destructive measures were taken, as the plants were used in a mitigation project, legally authorized, aimed at stabilizing a dune section reconstructed artificially at the planting site (Balestri, personal communication).
2.4. Statistical analysis
To test for differences in the mean rooting time among control and treatments, a t–test for independent grouping variables was performed for each species. One-way analysis of variance (ANOVA) with PGR treatment as fixed factor was used to assess differences among control and the auxin or the cytokinin treatments for each species. A separate analysis was performed for each of the variables analysed in each species (rooting percentage, budding percentage, root number, length of roots, number of culms plus buds and number of runners per cutting). Data were partitioned into two time periods (initial and final root emergence period), and each period was analysed separately. Because the relatively low number of cuttings that rooted in some treatments, data were pooled across experimental units. Since the temporal pattern of rooting differed between species, A. arenaria data were analysed 7 and 14 days after PGR application, while for S. virginicus the analyses were performed at 14 and 30 days. When the ANOVA detected significant differences, post-hoc comparison Dunnet’s test was used to test for differences between means. Before running the analyses the data were checked for the ANOVA assumption of normality and heteroscedasticity (Sokal and Rohlf, 1995). Data relative to the number of roots produced by A. arenaria cuttings treated with cytokinins after 14 days were log-transformed before the ANOVA to remove the evident heteroscedasticity of the variance
(Sokal and Rohlf, 1995). Data relative to the number of culms (plus buds) produced by A. arenaria cuttings treated with auxins and those produced by S. virginicus treated with cytokinins one year after planting were also log-transformed. Data transformation did not reduce the sensitivity of the analyses, leaving the transformed means of different samples in the same rank order of untrasformed means. Finally, Chi-square test was used to test for the effect of PGRs on the establishment success.
3. Results
3.1. Rooting and growth responses of cuttings
Root initiation in A. arenaria was evident at the first week after application of PGRs (Fig.
1). Most of the cuttings treated with the auxin 100 mg l-1 NAA rooted within 7 days; the rooting
percentage achieved with 100 mg l-1 NAA was higher as compared to untreated control (F(4,5) = 8.12, P = 0.02; Table 1). No differences in rooting were observed among cytokinins and control (F(4, 5) = 1.2, P = 0.41; Table 1). The mean number of roots per cutting (Fig 1) ranged from 0.8 (± 0.3) to 2.3 (± 0.4) and was similar among treatments and control (F(4,45) = 0.55, P =
0.69 for auxins and F(3,20) = 0.59, P = 0.62 for cytokinins); the 100 mg l-1 BAP treatment was excluded from the analysis because of the low number of cuttings that rooted at this time. Roots
of cuttings treated with 100 mg l-1NAA were significantly longer (F(4,45) = 3.2, P = 0.01) than those of control (0.98 ± 0.1 cm vs. 0.5 ± 0.1 cm; Fig. 2), while no differences were detected among cytokinins and control (F(3,20) = 0.43, P = 0.73; Fig. 2). A number of cuttings (5-30%)
had produced at least a new bud after 7 days; the number of buds per cutting (Fig. 3) was
significantly lower in cuttings treated with 100 mg l-1 Kinetin than control (F(4,70) = 2.76, P =
0.03), while no significant differences were observed among auxins and control (F(4,80) = 0.24, P = 0.91). Rooting increased up to 2 weeks after treatment (Fig 1); the mean rooting time of cuttings treated with 100 mg l-1 NAA was significantly shorter as compared to control, while no differences were observed among cytokinins and control (Table 1). At the end of the rooting period (at week 2), the percentage of rooted cuttings did not differ significant among treatments and control (Table 1; F(4,5) = 1.65, P = 0.29 for auxins and F(4,5) = 2.5, P = 0.17 for cytokinins). The mean number of roots in rooted cuttings (Fig. 1) varied between 1.3 (± 0.2) and 3.6 (± 0.9): no significant differences among treatments were detected (F(4,45) = 0.32, P = 0.85) among auxins and control, while the number of roots in cuttings treated with the cytokinin BAP (50 mg
l-1) was significantly lower than control (F(4,25) = 3.1, P = 0.03). The mean length of roots varied among treatments, ranging from 1.3 ± 0.3 to 2.9 ± 0.4 cm (Fig. 2); no significant differences were detected among auxins and control (F(4,45) = 1.68, P = 0.17), while the roots of
cuttings treated with the cytokinin Kinetin (50 mg l-1) were longer than control (F(4,25) = 3.5, P = 0.02). No differences in the frequency of budding (Table 1) were evident among treatments (F(4,5) = 0.20, P = 0.92 for auxins and F(4,5) = 0.87, P = 0.54 for cytokinins). The mean number of buds per cutting (Fig. 3) ranged from 1.3 (± 0.1) to 2.2 (± 0.2); not all cuttings with a new bud had also produced visible roots at this time. No significant differences in the total number of culms (plus buds) were detected among treatments (F(4,80) = 0.76, P = 0.54 for auxins and
In cuttings of S. virginicus root initiation was not evident for up 11 days, and mean number of cuttings rooted within the firsts 2 weeks (Table 1) was low (Fig. 4), ranging from 0 and 25 % (± 5). No differences were observed in rooting among control and auxins (F(4,5) = 1.2, P = 0.41), while the rooting percentage was higher in cuttings treated with the cytokinin Kinetin at
100 mg l-1 (F(4,5) = 6.61, P = 0.03). Because of the low number of rooted cuttings, the number of roots (Fig. 4), root length (Fig. 5) and the number of new buds per cutting (Fig. 6) were not analysed at this time. Cuttings continued to form roots up to 30 days after treatment (Fig. 4), and the mean rooting time was similar in all treatments (Table 1). After 30 days, significant differences in rooting percentage were still evident among cuttings treated with 100 mg l-1 Kinetin and control (Table 1; F(4,5) = 5.22, P = 0.04) and no significant differences in rooting among auxins and control were detected (Table 1; F(4,5) = 0.65, P = 0.65). The mean number of roots per rooted cutting (Fig. 4) ranged from 2.2 (± 0.2) to 3.4 (± 0.3) and no significant differences among treatments and control were observed (F(4,35) = 0.68, P = 0.60 for auxins and F(4,30) = 0.38, P = 0.81 for cytokinins). The length of roots varied between 2.2 (± 0.3) and 3.1 (± 0.2) cm (Fig. 5) and there were no significant differences among treatments and control (F(4,35)
= 0.26, P = 0.9 for auxins and F(4,30) = 0.38, P = 0.80 for cytokinins). The mean frequency of budding varied from 35% and 75% (Table 1), and no differences were evident among treatments and control (F(4,5) = 0.68, P = 0.63 for auxins and (F(4,5) = 3.06, P = 0.12 for cytokinins). The number of new culms (plus buds) per cutting was similar among treatments (Fig. 6) and control (F(4,40) = 0.48, P = 0.76 for auxins and F(4,40) = 0.35, P = 0.83 for cytokinins). Finally, no rhizome production was observed in the two species during the observation period.
3.2. Performance of rooted cuttings
After transplanting, cuttings of A. arenaria e S. virginicus grew rapidly. At the end of the experiment, the mean length of the root system of A. arenaria varied from 9.7 (± 0.3) cm and 24.3 (± 0.7 ) cm (Table 2); the root system of plants treated with auxins (F(4,40) = 131.44, P = 0.00001) or citokinins (F (4,20) = 19.57, P = 0.000001) was significantly longer than that of control (Fig. 7). All survived plants produced new culms and about half of them (48.8%) had runners up to 82 cm in length (Fig 7). The mean number of new culms (plus buds) per plant was highly variable (Table 2), ranging from 4.2 (± 1.6) to 37.6 (± 9.1). The number of culms was significantly higher in plants treated with auxins (except that in those treated with 50 mg l-1 NAA; F(4,40) = 6.17, P = 0.0005) or cytokinins (F(4,20) = 6.19, P = 0.002) when contrasted with control. The mean number of runners produced per plant (Table 2) varied between 0.4 (± 0.2) and 3.8 (± 0.8); cuttings treated with cytokinins had a higher number of runners than those of control (F(4,20) = 4.6, P = 0.008) while no significant differences were detected for cuttings treated with auxins and control (F(4,40) = 0.76, P = 0.55). The establishment success of cuttings treated with the higher concentration of NAA (100 mg l -1) was greater than that of control, while no differences were observed among the others treatments and control (Table 2).
In cuttings of S. virginicus the mean length of the root system was highly variable, ranging from 12 (± 0.9) cm and 21.1 (± 1.6) cm (Table 3), and the ANOVA did not detect significant differences among cytokinins and control (F(4,20) = 1.61, P = 0.21) or auxins and control
(F(4,20) = 0.87, P = 0.49). All plants produced new culms and 29% of them had runners up to 35.3 cm in length. The mean number of new culms (plus buds) was highly variable (Table 3),
ranging from 5.2 (± 1.2) to 17.4 (± 4.3). Cuttings treated with the cytokinin Kinetin (50 mg l-1) tended to produce more culms (F(4,20) = 3.17, P = 0.03) although the Dunnet test did not discriminate between control and treatments; no differences among auxins and control cuttings were detected (F(4,20) = 0.90, P = 0.48). Up to 2 runners were produced per plant (Table 3) and no differences in the mean number of runners among auxins and control (F(4,20) = 1.18, P = 0.34) or cytokinins and control (F(4,20) = 0.56, P = 0.69) were detected. Finally, the
establishment success of cuttings treated with 100 mg l-1 of Kinetin was greater than that of control, while no differences were evident among the others treatments and control (Table 3).
4. Discussion
In this study the exogenous application of auxins to cuttings of A. arenaria and S. virginicus was aimed at maximising root initiation capacity and growth, while cytokinins were used to stimulate vegetative development (shoot formation and branching), in order to increase the chance of success in dune restoration programs. The results of the laboratory experiment revealed considerable variations in the pattern of rooting initiation and rooting potential of cuttings among the two study species. Cuttings of A. arenaria showed a shorter time for root initiation and better rooting capacity without the application of PGRs than S. virginicus. The species also differed substantially in their responsiveness to PGRs. As concerns root
development, the auxin NAA at 100 mg l-1 was effective in stimulating rooting in A. arenaria, resulting in a doubling rate of rooting as compared to control within only 7 days, and the
cytokinin Kinetin at 100 mg l-1 was active in enhancing the rooting frequency of S. virginicus. The positive effects of the auxins recorded here on root development was in accordance with that observed in other species from different environments (Cho, 1985; McCown, 1988; Cho and Soh, 1989; Drew et al., 1991), while the effects of cytokinin was surprisingly as this process is likely generally inhibited, or delayed, by exogenous cytokinins (Harris and Hart, 1964; Bollmark and Eliasson, 1986; Pierik, 1987; McCown, 1988; Van Staden and Harty, 1988; De Klerk et al., 1995). To our knowledge, there are only some reports that cytokinins, alone or in combination with auxins, promoted root formation (Fabijan et al., 1981; Bollmark and Eliasson, 1986; Vincent et al., 1992; Remeshree et al., 1994). No data are available on the effect of cytokinins in other dune plants. As concerns vegetative development, no species was apparently influenced by cytokinins or auxins, at least in the first month after PGR application. This appeared be due to the initial limited number of nodes (one-two nodes) in cuttings and a longer time required for initiating new buds, rather than to a lack of responsiveness. In fact, the transplanting experiment demonstrated that application of some PGRs was beneficial for cuttings on long term. In particular, the treatments found more effective in promoting rooting in
laboratory (100 mg l-1NAA for A. arenaria and 100 mg l-1 Kinetin for S. virginicus) resulted also in greater plant survival success in sandy substrate. Moreover, in A. arenaria all the other
tested auxins (except that NAA at 50 mg l-1) and cytokinins had positive effects on vegetative development. Cytokinins resulted also particularly active in stimulating the production of new runners in survived cuttings.
This is the first report demonstrating the feasibility of using exogenous application of PGRs to promote rooting and growth of cuttings, and to enhance the establishment success of coastal dune plants. This rapid and easy technique may be of great interest for large scale restoration
activities, providing potentially a number of advantages compared to traditional techniques such as direct planting of cuttings or nursery grown plants. By using the appropriate PGR the survival rate of planted cuttings would be expected to be ca. two fold higher as compared to controls. In addition, the larger size achieved by plants would more effectively stabilize sand and provide greater substrate cover. Finally, the production of a larger and deeper root system would allow plants to better survive in unstable dunes. The initial cost for producing a rooted plant by using PGRs may be relatively low (ca. 0.20 $ for commercially available hormones and rooting beds) and could easily be justified for use in large scale projects by greater survival and quicker cover provided by treated plants. Recent studies (Thetford et al., 2005) indicated that the maintenance of dune plants in nursery is a major cost in planting projects, ca. 2.25 $ for plant material alone. In addition, replacement of dead plants in restoration projects increases the cost of planting, it is time-consuming and increases the impact of collection on donor populations.
Application of this technique could be extended to other dune species. As the response of plants to PGRs may vary considerable, further studies are needed to determine the more appropriate PGR for each species. The method (exposure period to PGRs and experimental conditions) established in this study is open to further refinement. Since plant development is the result of the concurrent actions of various hormones interacting in different ways (Leopold and Noodén, 1984), further improvement of plant performance would be probably achievable using a combination of auxins and cytokinins. Studies would also investigate whether cuttings treated with PGRs can be planted directly in the field in order to further reduce the operative time and the production costs.
Planting of cuttings from established dune plants is a practice often used in restoration efforts to stabilize and reconstruct damaged sand dunes. The long time required to cuttings to develop a well-structured root system and new branches may limit the success of restoration. Our results demonstrate that root development and growth of cuttings from A. arenaria, S. virginicus may be influenced by manipulating appropriate PGRs. Specifically, application of NAA at high concentration may be useful in promoting rooting in A. arenaria cuttings, and on long term in stimulating root growth and shoot development. Instead, the cytokinins, Kinetin and BAP, may be effective in promoting vegetative development, particularly runner production. On the other hand, application of the cytokinin Kinetin at higher concentration appears to be indispensable for achieving an acceptable rooting percentage in S. virginicus cuttings. If extrapolated to field conditions, the enhanced growth responses of cuttings to some PGRs observed here could result in a higher probability of success of transplants of A. arenaria and S. virginicus. Thus, PGR application could provide a simple and relatively un-expensive tool for fast propagation and improvement of the planting success in large scale dune restoration projects.
Acknowledgements
We are grateful to the Corpo Forestale dello Stato (Cecina, Italy) for general assistance, and to Solvay Chimica Italy (Rosignano Solvay; Italy) farm that permitted the maintenance of plants in the field. The study was conducted in the framework of a mitigation project, legally authorized, funded by Rosaelectra S.p.A. of Rosignano Solvay (Italy).
References
AGENC, 1994. Restauration de Dunes a faible dynamique edificatrice en Corse. Agence pour la Gestion des Espaces Naturels de Corse Bastia.
Alpert, P., Holzapfel, C. J., M. Benson, J.M., 2002. Hormonal modification of resource sharing in the clonal plant Fragaria chiloensis. Functional Ecology 16, 191-197.
Balestri, E., Bertini, S., 2003. Growth and development of Posidonia oceanica seedlings treated with plant growth regulators: possible implications for meadow restoration. Aquatic Botany 76, 291-297.
Balestri, E., Lardicci, C., 2006. Stimulation of root formation in Posidonia oceanica cuttings by application of auxins (NAA and IBA). Marine Biology 149, 393-400.
Balestri, E., Luccarini, G., Cinelli, F., 2001. Isolation of leaf protoplasts from Pancratium maritimum L. and two other dune plants: possible applications. Journal of Coastal Research 17(1), 188-194.
Barbour, M.G., de Joung, T.M., Pavlik, B.M., 1985. Marine beach and dune plant communities. In: Chabot, B.F., Mooney, H.A., (Eds.), Physiological ecology of north American plant communities, Chapman & Hall, New York. pp. 298-322.
Benavent Olmos, J.M., Collado Rosique, P., Martì Crespo, R.M., Muñoz Caballer, A., Quintana Trenor, A., Sánchez Codoñer, A., Vizcaino Matarredona, A., 2004. La restauración de las dunas litorales de la Devesa de l`Albufera de Valencia. Ajuntament de Valencia. 65 p. Bird, E.C.F., 1996. Beach Management, Wiley, Chichester, UK, 292 pp.
Blakesley, D. 1994. Auxin metabolism and adventitious root initiation. In: Davis, T.D., Haissig, B.E. (Eds.), Biology of Adventitious Root Formation., Plenum Press, New York, pp. 143– 154.
Bollmark, M., Eliasson, L., 1986. Effects of exogenous cytokinins on root formation in pea cuttings. Physiologia Plantarum 68, 662-666.
Bovina, G., Callori di Vignale, C., Amodio M., 2003. L’approccio dell’ingegneria naturalistica nella conservazione degli ambienti dunali. In: Manuale di Ingegneria Naturalistica vol. 2, Regione Lazio, Ass. Ambiente.
Brown, A.C., McLachlan, A., 2002. Sandy shore ecosystems and the threats facing them: some predictions for the year 2025. Environmental Conservation 29(1), 62-77.
Butola, J.S., Badola, H.K., 2007. Vegetative propagation of Angelica glauca and Heracleum candicans. Journal Tropical Medicinal Plants 8(1), 85-91.
Caniglia, G., Casetta, D., Nascimbeni, P., Pizzicato, C., 1998. Aspetti del dinamismo della vegetazione nell’edificazione di un sistema dunoso artificiale (Venezia – Cavallino). Atti conv. International Association for Environmental Design, La progettazione ambientale nei sistemi costieri, quad. 12.
Cho, D.Y, Soh, W.Y., 1989. Effect of endogenous IAA transport on adventitious root formation in Phaseolus vulgaris hypocotyl cuttings. Korean Journal of Botany 32, 323-330.
Cho, D.Y., 1985. Distribution and quantitative determination on IAA by high performance liquid chromatography on adventitious root formation in Azukia epicotyl cuttings. Korean Journal Plant Tissue Culture 12, 79-87.
Clements, A.M., Simmonds, A., Hazelton, P., Inwood, C., Woolcock, C., Markovina, A.L., O’Sullivan, P., 2010. Construction of an environmentally sustainable development on a modified coastal sand mined and landfill site - Part 2. Re-Establishing the natural ecosystems on the reconstructed beach dunes. Sustainability 2, 717-741.
Converio, F., Fanelli, G., Villani, M.G., 2007. La protezione dell’ecosistema dunale a Focene (Litorale romano). Fitosociologia 44 (1), 111-116.
Craig, R.M., Smith, D.C., Roush, R.D., 1981. Propagation of two threatened coastal dune plants having beatification and erosion control features. Proceedings of the Florida State Horticultural Society 94, 202-204.
De Klerk, G.J., Keppel, M., Brugge, J.T., Meekes H., 1995. Timing of the phases in adventitious root formation in apple microcuttings. Journal Experimental of Botany 46, 965-972.
De Lillis, M., 1997. Gestione delle dune sabbiose in Europa: qualche esperienza dal Nord-Europa al Mediterraneo. Atti del 1° Congresso Conservazione e biodiversità nella progettazione ambientale, Perugia 28-30 nov. 1996, IAED International Association for Enviromental Design.
De Lillis, M., 1998. Restoration and management of European sand dunes. Journal of International Association for Environmental Design 2, 31-40.
De Lillis, M., Costanzo, L., Bianco, P.M., Tinelli, A. 2004. Sustainability of sand dune restoration along the coast of the Tyrrehnian sea. Journal of Coastal Conservation 10, 93-100.
Defeo, O., McLachlan, A., Schoeman, D.S., Schlacher T.A., Dugan, J., Jones, A., Lastra, M., Scapini., F., 2009. Threats to sandy beach ecosystems: A review. Estuarine, Coastal and Shelf Science 81, 1–12.
Doody, J. P., 2001. Coastal conservation and management: An ecological perspective. Kluwer, Academic Publishers, Boston, USA, 306 pp.
Drew, R.A., Simpson, B.W., Osborne, W.J, 1991. Degradation of exogenous indole3-butyric acid and riboflavin and their influence of rootings response of papaya in vitro. Plant Cell Tissue Organ Culture 26, 29-34.
EEC, 1992. Council Directive 92/43 of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora.
Escaray, F.J.., Rosique, F.J.C., Scamato, A.A., Bilenca, D., Carrasco, P., Matarredona, A.V., Ruizi, O.A., Menéndez, A.B., 2010. Evaluation of a technical revegetation action performed on foredunes at devesa de la Albufera, Valencia, Spain. Land Degradadation and Development 21, 239–247.
Fabijan, D., Taylor, J.S., Reid, D.M., 1981. Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings II. Action of gibberellins, cytokinins, auxins and ethylene. Physiologia Plantarum 53, 589-597.
Géhu, J.M., 1985. European dune and shoreline vegetation. Nature and environment no. 32. Council of Europe, Strasbourg, FR.
Gilbert, M., Pammenter, N., Ripley, B., 2008. The growth responses of coastal dune species are determined by nutrient limitation and sand burial. Oecologia 156, 169-178.
Gomes Neto, A., Cunha, S. R., Voigt, E. L., 2006. Vegetative propagation of the dune building plant for use in dune rehabilitation projects. Journal of Coastal Research 39, 1251 - 1254.
Gómez-Pina, G., 1999. Beach nourishment fundamentals. The Spanish experience. Proceedings of the Short Course on Coastal and Port Engineering, 5th International Conference on Coastal and Port Engineering in Developing Countries (COPEDEC), (Cape Town), pp. 6.1– 6.48.
Gómez-Pina, G., Muñoz-Perez, J.J., Ramirez, J.L. & Carlos, L. 2002. Sand dune management problems and techniques, Spain. Journal of Coastal Research 36, 325-332.
Greipsson S., 2002. Coastal dunes. In: Perrow, M.R., Davy, A.J. (Eds.), Handbook of ecological restoration. Vol. 2: Restoration in practice. Cambridge University Press, Cambridge UK. pp. 584.
Gutiérrez, J.L., Jones, C.G., Byers, J.E., Arkema, K.K., Berkenbusch, K., Commito, J.A., Duarte, C.M., Hacker, S.D., Hendriks, I.E., Hogarth, P.J., Lambrinos, J.G., Palomo, M. G., Wild, C., 2010. Physical ecosystem engineers and the functioning of estuaries and coasts. In: Heip, C.H.R., Philippart, C.J.M., Middelburg, J.J. (Eds.), Functioning of Estuaries and Coastal Ecosystems. Treatise on Estuarine and Coastal Science (Series eds., E. Wolanski, and D. McLusky), Elsevier, Amsterdam, The Netherlands, pp. 1-99.
Feagin, R.A., Sherman, D.J., Grant, W.E., 2005. Coastal erosion, global sea-level rise, and the loss of sand dune plant habitats. Frontiers in Ecology and the Environment 3, 359–364. Harris, G.P., Hart, E.M.H., 1964. Regeneration from leaf square of Peperomia sandersii A.,
D.C: a relationship between rooting and budding. Annals of Botany 28, 509-526.
Hertling, U.M., Lubke, R.A., 2000. Assessing the potential for biological invasion – the case of Ammophila arenaria in South Africa. South African Journal of Science 96, 520-527. Hesp, P.A., 1991. Ecological processes and plant adaptations on coastal dunes. Journal of Arid
Hobbs, R.J., Gimingham, C.H., Band, W.T., 1983. The effects of planting techniques on the growth of Ammophila arenaria (L.) Link and Lymus arenarius (L.) Hochst. Journal of Applied Ecology 20, 659-672.
Huiskes, A.H.L., 1979. Biological flora of the British Isles: Ammophila arenaria (L.) Link (Psamma arenaria (L.) Roem et Schult.: Calamagrostis arenaria (L.) Roth). Journal of Ecology 67, 363-382.
Huiskes, A.H.L., Harper, J.L., 1979. The demography of leaves and tillers of Ammophila littoralis in a dune sere. Oecologia Plantarum 14, 435-446.
Labouriau, L.G., Osborn, J.H., 1984. Temperature dependence of the germination of tomato seeds. Journal of Thermal Biology 9, 285–294.
Leopold, A.C., Noodén, L.D., 1984. Hormonal regulatory systems in plants. In: Scott, T.S. (Ed.), Hormonal regulation of development. II The functions of hormones from the level of the cell to the whole plant. Encyclopedia of Plant Physiology, New Series, vol. 10. Springer-Verlag, Berlin pp. 4-20.
Maun, M.A., 1997. Adaptations of plants to burial in coastal sand dunes. Canadian Journal of Botany 76, 713-738.
Maun, M.A., Fahselt, D., 2009. Dune systems in relation to rising seas. In: Maun, A.A. (Ed.), The biology of coastal sand dunes. Oxford University Press, Oxford New York pp. 197-214. McCown, B.H., 1988. Adventitious rooting of tissue cultured plants. In: Davis, T.D., Hassig,
B.E., Sankhla, N. (Eds.), Adventitious root formation in cuttings. Dioscorides Press, Portland, Oregon pp. 289-302.
Nadeem, M., Palni, L.M.S., Purohit, A.N., Pandey, H., Nandi, S.K., 2000. Propagation and conservation of Podophyllum hexandrum Royle: an important medicinal herb. Biological Conservation 99, 121-129.
O'Briain, M., 2005. EU biodiversity policy context for the conservation of estuaries and dunes. Proceedings ‘Dunes and Estuaries 2005’: International Conference on nature restoration practices in European coastal habitats, Koksijde, Belgium 19-23 September 2005. VLIZ Special Publication, 19: pp. 7-12.
Panayotova L.G., Ivanova T.A., Bogdanova Y.Y., Gussev C.V., Stanilova M.I., Bosseva Y.Z., Stoeva T.D., 2008. In vitro cultivation of plant species from sandy dunes along the Bulgarian Black Sea Coast. Phytologia Balcanica 14, 119–123.
Park, H.J., Min, B.M., Cha, H.C., 2004. Mass production of sand dune plant, Vitex rotundifolia, via micorpropagation. Journal of Plant Biotechnology 6(3), 165-169.
Pickart, A. J., 1990. Dune revegetation at Bhune Point, King Salmon, California. In: Berger, J.J. (Ed.), Environmental restoration. Science and strategies for restoring the earth. Inland Press, Washington, pp. 38-49.
Pierik, R.L.M., 1987. In vitro culture of higher plants. Dordrecht, Netherlands: Martinus Nijhoff Publishers, pp. 203-209.
Remeshree, A.B., Hariharan, M., Unnikrishnan, K., 1994. Micropropagation and callus induction of Aristolochia bracteolata Lam. - a medicinal plant. Phytomorphology 44, 247-252.
Roze´, F., Lemauviel, S., 2004. Sand dune restoration in north Brittany, France: a 10-year monitoring study. Restoration Ecology 12, 29–35.
Rozema, J., Bijwaard, P., Prast, G., Broekman, R., 1985. Ecophysiological adaptations of coastal halophytes from foredunes and salt marshes. Vegetatio 62: 499-521.
Salman, A.H.P.M., Strating K.M., 1992. European coastal dunes and their decline since 1900. European Union for Coastal Conservation (EUCC), Leiden (NL). pp. 439-449.
Sanjaume, E., Pardo, J., 1992. The dunes of the Valencian coast (Spain): past and present. Proceedings of the third European dune congress, Galway, Ireland, 17-21 June 1992. pp. 475-477.
Schlacher, T.A., Dugan, J., Schoeman, D.S., Lastra, M., Jones, A., Scapini, F., McLachlan, A., Defeo, O., 2007. Sandy beaches at the brink. Diversity and Distributions 13, 556–560. Sokal, R.R., Rohlf, F., 1995. Biometry: the principles and practice of statistics in biological
research. 3rd ed. W.H. Freeman, New York, 887 pp.
Terrados Muñoz, J., 1995. Effects of some plant growth regulators on the growth of the seagrass Cymodocea nodosa (Ucria) Ascherson. Aquatic Botany 51, 311-318.
Thetford, M., Miller, D., 2002. Propagation of 4 Florida coastal dunes species. Native Plants Journal 3(2), 112-120.
Tinelli, A., De Lillis, M., Costanzo, L., 1998. Riqualificazione ambientale del sistema dunale costiero del litorale romano. In: La progettazione ambientale nei sistemi costieri, IAED International Association for Environmental Design, Roma. pp. 59-68.
Valero-Aracama C., Wilson S.B., Kane, M.E., Philman, N.L., 2007. Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy- and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes. In Vitro Cellular & Development Biology—Plant 43, 237–246.
van der Laan, D., van Tongeren, O.F.R., van der Putten, W.H., Veenbaas, G., 1997. Vegetation development in coastal foredunes in relation to methods of establishing marram grass (Ammophila arenaria). Journal of Coastal Conservation 3, 179-190.
van der Meulen, F., Bakker, T.H.W., Houston, J.A., 2004. The costs of our coasts: examples of dynamic dune management from Western Europe. In: Martínez, M.L., Psuty, N.P. (Eds.), Coastal dunes: ecology and conservation. Springer-Verlag, Berlin, DE., pp. 259–278.
van der Meulen, F., Jungerius, P.D., Visser, J.H., 1989. Perspective in coastal dune management. SPB Academic Publishing, The Hague, NL. 333 pp.
van der Putten, W. H., 1989. Establishment, growth and degeneration of Ammophila arenaria in coastal sand dunes. Ph. D Thesis, Agricultural University, Wageningen, The Netherlands. van der Putten, W. H., 1990. Establishment of Ammophila arenaria (Marram Grass) from
culms, seeds and rhizomes. Journal of Applied Ecology 27, 188-199.
van der Putten, W. H., van Gulik, W.J.M., 1987. Stimulation of vegetation growth on raised coastal fore- dune ridges. Netherlands Journal of Agricultural Science 35, 198-201.
van der Salm, J., Unal, O., 2001. Towards a common Mediterranean Framework for potential beach nourishment projects. MEDCOAST 01: Proceedings of the Fifth International Conference on the Mediterranean Coastal Environment, Hammamet, Tunisia, October 23– 27, 2001, pp. 1333–1346.
Van Staden, J., Harty, A.R., 1988. Cytokinins and adventitious root formation. In: Davis, T.D., Haissig, B.E., Sankla, N. (Eds.), Adventitious Root Formation in Cuttings, Advances in Plant Sciences Series, Vol 2. Dioscorides Press, Portland, OR, pp. 185–201.
Vestergaard, P., Hansen, K., 1992. Changes in morphology and vegetation of a man-made beach-dune system by natural processes. In: Carter, R.W.G., Curtis, T.G.F., Sheehy-Skeffington, M.J. (Eds.), Coastal dunes: geomorphology, ecology and management for conservation. A.A. Balkema, Rotterdam, pp. 165-176.
Vincent, K., Mathew, K.M., Hariharan, M., 1992. Micropropagation of Kaempferia galanga L., a medicinal plant. Plant Cell Tissue Organ Culture 28, 229-230.
Willis, J.M., Hester, M.W., 2008. Evaluation of enhanced Panicum amarum establishment through fragment plantings and humic acid amendment. Journal of Coastal Research 24, 263-268.
Woodhouse, W.W., 1982. Coastal sand dunes of the U.S. In: Lewis, R.R. (Ed.), Creation and restoration of coastal plant communities. CRC, Boca Raton, FL, pp. 1–44.
Zhang, J., 1996. Interactive effects of soil nutrients, moisture and sand burial on the development, physiology, biomass and fitness of Cakile edentula. Annals of Botany 78, 591-598.
Legend of figures
Fig. 1. Ammophila arenaria. Effect of various auxins and cytokinins applied at different concentrations (50 and 100 mg l-1) on the cumulative number of new roots per rooted cutting over time. Untreated cuttings were used as control. Data are means (± SE). The asterisk indicates the means of treatments that differed significantly from control.
Fig. 2. Ammophila arenaria. Effect of various auxins and cytokinins applied at different concentrations (50 and 100 mg l-1) on the length of roots in rooted cuttings over time. Untreated cuttings were used as control. Data are means (± SE). The asterisk indicates the means of treatments that differed significantly from control.
Fig. 3. Ammophila arenaria. Effect of various auxins and cytokinins applied at different concentrations (50 and 100 mg l-1) on the cumulative number of culms (plus buds) produced in survived cuttings over time. Untreated cuttings were used as control. Data are means (± SE). The asterisk indicates the means of treatments that differed significantly from control.
Fig. 4. Sporobolus virginicus. Effect of various auxins and cytokinins applied at different concentrations (50 and 100 mg l-1) on the cumulative number of new roots per rooted cutting over time. Untreated cuttings were used as control. Data are means (± SE). The asterisk indicates the means of treatments that differed significantly from control.
Fig. 5. Sporobolus virginicus. Effect of various auxins and cytokinins applied at different concentrations (50 and 100 mg l-1) on the length of roots in rooted cuttings over time. Untreated cuttings were used as control. Data are means (± SE). The asterisk indicates the means of treatments that differed significantly from control.
Fig. 6. Sporobolus virginicus. Effect of various auxins and cytokinins applied at different concentrations (50 and 100 mg l-1) on the cumulative number of culms (plus buds) produced in survived cuttings over time. Untreated cuttings were used as control. Data are means ± SE. The asterisk indicates the means of treatments that differed significantly from control.
Fig. 7. Rooting and growth response of cuttings from A. arenaria (A-C) and S. virginicus (D-E) to some PGR treatments (NAA 100 mg l-1, Kinetin 50 and Kinetin 100 mg l-1). The photographs were taken 1 year after transplanting into pots. Untreated cuttings were used as control.
