Introduction

The genus Daphne L. (Thymelaeaceae Juss.) contains deciduous and evergreen shrubs and trees that grow mainly in Europe and Asia (Herber 2003). Desirable ornamental characteristics such as fragrant flowers, diverse plant habit (i.e., branching, compact, or procumbent), and coloration of flowers and leaves have promoted the horticultural use of Daphne species. Several species are propagated commercially, and garland flower (Daphne cneorum) has become one of the most popular shrubs among floriculturists (Halda 2001). The introduction of wild Daphne species into breeding programs is currently being pursued because of their natural resistance to fungal root pathogens. For example, infections caused by Thielaviopsis basicola (Berk. & Br.) Ferraris significantly limit the production of vegetative cuttings, although selection of resistant genotypes among Daphne species can be accelerated using in vitro assays (Noshad et al. 2007; Hanus-Fajerska et al. 2008). Root formation from conventional cuttings of Daphne is difficult (Marks and Simpson 2000) and these rooting difficulties could be overcome by means of in vitro culture (Leifert et al. 1992; Marks and Simpson 2000).

The successful initiation of in vitro cultures depends on the composition and physiochemical characteristics of the nutrient medium. The medium should provide an optimal environment for cell differentiation and organ development. The nutrient medium serves as a source of assimilable carbon, nitrogen, and other minerals as well as regulatory compounds (Neumann et al. 2009). Commercial formulations of defined plant tissue culture media are available, making culture establishment techniques repeatable and convenient. However, to reduce the overall expense of commercial micropropagation, there is a need for the development of inexpensive options for so called ‘low-cost tissue culture technology’.

Low-cost tissue culture technology utilizes practices and specific equipment to reduce the unit cost of micropropagule and plant production; savings are achieved by improving process efficiency and utilization of resources without compromising the quality of the plants (Savangikar 2004). One promising method for low-cost tissue culture is enrichment of nutrient medium with alternative organic supplements such as miscellaneous carbon sources (Kinnersley and Henderson 1988; Tyagi et al. 2007; Alkhateeb 2008; Agrawal et al. 2010), gelling agents (Thorpe et al. 2008; Agrawal et al. 2010), and plant extracts (Kitsaki et al. 2004; Peixe et al. 2007; Agampodi and Jayawardena 2009; Agrawal et al. 2010). Some natural compounds derived from fungal and plant cell walls can act as regulating molecules that control plant growth, development, organ differentiation, and defense against pathogens (Bois 1992). Commercially available cell wall compounds such as arabinogalactans and chitosan can also positively influence plant micropropagation. Addition of arabinogalactan to media enhanced shoot differentiation in cotyledonary explants of Vigna radiata (Das and Pal 2004), and chitosan addition accelerated plantlet growth in Vitis vinifera culture (Barka et al. 2004). Such supplementation made the medium improved differentiation and growth, since it supplied the cultured explants with signal compounds.

Nutrient medium can be also enriched by addition of conditioned medium, which is prepared from rapidly growing cells in culture (Matsubayashi and Sakagami 1996). Cells produce and release many different growth factors into the culture medium. Certain algae are a suitable source of conditioned medium, and exploitation of algae filtrates or exudates as media additives has been utilized for improving the tissue culture response for some higher plants (Wake et al. 1992; Hanus-Fajerska et al. 2009; Shrivastava and Banerjee 2009). Algae naturally occur as unicells, and algal cell suspension cultures can be inexpensively established. Algal growth rate is usually very high and algae have low nutrient requirements (no organic carbon in the medium, only mineral salts), making it easy to rapidly prepare inexpensive conditioned medium with high biological activity (Grabski and Tukaj 2008).

We report the improved micropropagation of three Daphne species (Daphne caucasica Pall., Daphne jasminea Sibth. & Sm., and Daphne tangutica Maxim) in media supplemented with organic additives, including plant extracts, cell exudates, and commercially available cell wall components. The objective of the study was to evaluate the biological activity of several supplements and their influence on vitality of shoot cultures, propagation rate, and differentiation processes, in order to induce rhizogenesis and produce plantlets. The results from this study will facilitate the development of low-cost micropropagation protocols for Daphne species.

Materials and Methods

Plant material and culture

In vitro shoots of D. caucasica, D. jasminea, and D. tangutica were kindly provided by Dr A. Riseman from the University of British Columbia Botanical Garden and Centre for Plant Research, Vancouver, Canada. Organogenesis was induced using 15 mm (D. caucasica and D. tangutica) or 5 mm (D. jasminea) microcuttings cultured on the media described below. Shoot cultures were initially maintained on medium D1 for 4 wk, consisting of woody plant medium (WPM) salts (Lloyd and McCown 1981), MS vitamins (Murashige and Skoog 1962), 12.3 μM N6-[2-isopentyl]adenine (2iP), 5.37 μM 1-naphthaleneacetic acid (NAA), 0.5 g L−1 polyvinylpyrrolidone (PVP), 0.5 g L−1 2-N-morpholino-ethanesulfonic acid (MES), 0.6 g L−1 activated charcoal, 0.65 g L−1 calcium gluconate, and 20.0 g L−1 sucrose, and solidified with 0.8% Difco agar. The following organic additives were added separately: 20.0 mg L−1 arabinogalactan (Sigma, St. Louis, MO), 15.0 mg L−1 chitosan (Sigma), 10.0 ml L−1 coconut water (Sigma), and 10.0 ml L−1 pineapple pulp. The pH of the medium was adjusted to 5.6.

The pineapple pulp was produced according to Kitsaki et al. (2004). Five ripe pineapples were homogenized using a blender and the pulp (~660 ml) was filtered through cheesecloth, deproteinized by boiling for 10 min, and stored in small batches in 1.5-ml microcentrifuge tubes at–20 °C.

The unicellular green alga Desmodesmus subspicatus Chodat (formerly named Scenedesmus subspicatus) (Chlorophyceae) strain 2594 was obtained from the UTEX (University of Texas, TX) collection (Starr and Zeikus 1993). Cells were grown in liquid Bold’s Basal Medium (Bischoff and Bold 1963) for 7 d and were removed from the medium by centrifugation at 1,000×g for 15 min. The supernatant was filtered through a series of Synpor membrane filters (0.80, 0.45, and 0.2 μm; VCHZ Syntezia, Pardubice, Czech Republic) and the filtrate, designated as CM, was stored at 4 °C. CM was added to D1 media at 20% (v/v) (CM1) or 50% (v/v) (CM2) for evaluation of Daphne proliferation.

All media were prepared directly before culture establishment/subculture and autoclaved at 121 °C, 0.1 MPa for 15 min. The organic additives, conditioned medium, and gelling agent were added prior to autoclaving.

All cultures were maintained in a growth chamber under 16-h photoperiod (irradiance 80 μmol m−2 s−1) at 24 ± 2 °C for 12 wk with subculture to fresh medium after 4 and 8 wk. Cool-white fluorescent lamps were used as the light source.

Acclimatization stage

Microplantlets with developed root systems were transferred into pots containing a mixture of perlite, potting compost, and sand in 1:1:1 ratio. For 3 wk, plants were grown under translucent covers, and watered every 2 d. Plants were periodically ventilated for 3 wk for acclimatization, and then transferred to greenhouse conditions (24 °C, 51% relative humidity, 750 μmol m−2 s−1).

Characterization of pineapple pulp properties

Thin-layer chromatography (TLC) was used to investigate whether pineapple pulp contains auxin-like substances, according to the procedure of Tien et al. (1979). TLC chromatograms were run on 0.5-mm-thick preparative silica gel plates, coated with SiO2 and F254 fluorescent indicator. The solvent system was chloroform/ethyl acetate/formic acid [50:40:10 (v/v)] to separate the indole compounds from the pineapple pulp extract. Apart from the sample, the following indole compounds were run as standards on the chromatogram: indole-3-acetic acid (IAA), tryptophan, 4-(3-indolyl)butyric acid (IBA), 3-indoleacetonitrile, indole-3-aldehyde, and indole-3-acetic acid ethyl ester. The positions of the indole compounds were then visualized on TLC plates in UV light (λ = 254).

Pineapple pulp was fractionated by dialysis through 500-Da membrane and the two fractions, dialyzate and concentrate, were tested as medium supplements at concentrations of 10 mg L−1. The dialyzate contained compounds of low molecular weight (<500 Da) while the concentrate contained those of higher molecular weight (>500 Da). Shoot cultures of Daphne species were then established on D1 medium supplemented with fractionated pineapple pulp and rooting percentage data were collected after 12 wk of culture.

Experimental design

The main experiment was set up as a randomized complete block design. The results were subjected to STATISTICA 9.0 (StatSoft inc., Tulsa, OK) ANOVA analysis and a post-hoc Tukey’s test was used to study differences between treatments at P < 0.05.

The experiment was performed independently three times (three replicates). At least 30 explants per treatment were used in a single replicate and all were subject to analysis. The number of explants in one 200-ml Erlenmeyer flask depended on the examined species: for D. tangutica and D. caucasica, five microcuttings per flask; for D. jasminea, 15 microcuttings per flask. Every replicate lasted 12 wk, with subculture to fresh medium every 4 wk. At the end of 12-wk period (replicate), data were collected on the number, length, and morphology of Daphne microcuttings. The micropropagation coefficient (MC) was calculated after 12 wk of culture. For D. jasminea and D. tangutica, MC = (number of induced adventitious shoots/total number of explants); for D. caucasica, MC = (number of developed axillary shoots/total number of explants).

The percentage of rooted shoots was recorded after 12 wk of culture. Daphne plants with an in-vitro-developed root system were successively subjected to acclimatization as described previously. For the survival percentage of acclimatized plants, data were recorded 3 wk after transplanting into pots.

Results

Non-supplemented media

In non-supplemented D1 medium, micropropagation rate, morphology, and plant habit varied among species. D. caucasica exhibited a propagation rate (MC) of 3.0 (Table 1) and an elongated and branchless habit. Shoots were pale green with convoluted upper leaves. Since no adventitious bud formation occurred, D. caucasica was propagated through 15-mm-long microcuttings that included leaf nodes and axillary buds. No roots were formed during culture. Daphne jasminea and D. tangutica proliferated more vigorously than D. caucasica, due to the formation and development of adventitious buds. D. tangutica was propagated in 15-mm long microcuttings of both axillary and adventitious shoots. Obtained shoots had a branched habit with shortened internodes. The leaves of D. tangutica became slightly chlorotic. Interestingly, D. jasminea shoots spontaneously formed roots and flowers. The flowering shoots became lignified and were unsuitable for further propagation.

Table 1. Effects of medium enrichment with organic supplements on the micropropagation of three Daphne species after 12 wk of culture
Full size table

Medium enrichment stage

The greatest improvement in cultures of D. caucasica was achieved through medium enrichment. The highest frequency of adventitious rhizogenesis (57.1% of shoots) occurred when tissues were cultured on the medium containing pineapple pulp. Rhizogenesis also occurred to a lesser extent on media with chitosan and coconut water (11.6% and 37.5%, respectively; Table 1). Both pineapple pulp and coconut water stimulated adventitious bud formation and ameliorated shoot morphology. Shoots were highly branched with dense leaves (Fig. 1a ). Chlorosis was not apparent. The lowest propagation rate (MC = 2.0) was observed when tissues were placed on medium supplemented with arabinogalactan, and no rhizogenesis occurred in this medium.

Figure 1.
figure 1

Micropropagation of Daphne sp. in media supplemented with organic supplements. (a) Morphology of typical D. caucasica shoots in control medium (C) and media supplemented with arabinogalactan (Ag), chitosan (Ch), coconut water (CW), and pineapple pulp (P). (b) D. tangutica shoots in non-supplemented and supplemented media. (c) Rhizogenesis in D. jasminea on medium without plant growth regulators and supplemented with pineapple pulp. (d) Rhizogenesis in D. caucasica on medium without plant growth regulators and supplemented with pineapple pulp. (e) Cultures of tested Daphne species in optimal media. (f) Acclimatized plants of D. caucasica. (g) Acclimatized plants of D. jasminea.

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Proliferation of D. tangutica shoots was significantly improved by medium supplementation with coconut water (MC = 16.6), pineapple pulp (MC = 13.4), and arabinogalactan (MC = 13.2), compared with MC = 10.4 in control medium. Shoots from the coconut water and pineapple pulp treatments were significantly longer than in the control treatment, with dense, green leaves (Table 1, Fig. 1b ). Although efficient micropropagation of D. tangutica was achieved, no rhizogenesis occurred. In this species, chitosan had a negative effect on shoot cultures, leading to chlorosis and poor proliferation (Table 1).

Shoot cultures of Daphne jasminea, established on supplemented media, exhibited slight deterioration of the tested characteristics in comparison with the non-supplemented medium. Limited root formation occurred only on media containing coconut water (nearly 16%) and pineapple pulp (19%; Table 1). In media supplemented with arabinogalactan or pineapple pulp, senescence of small adventitious buds occurred. Pineapple pulp and arabinogalactan also reduced a frequency of in vitro flowering.

Cultures of Daphne species were also established on media without synthetic plant growth regulators (medium D0), but supplemented with either pineapple pulp or coconut water (data not shown). The newly developed shoots were shorter than those from the corresponding media with plant growth regulators, and 12-wk-old cultures exhibited no aging symptoms (lignification, leaf chlorosis, and senescence). All three Daphne genotypes formed adventitious buds, and rhizogenesis was initiated in the D. caucasica and D. jasminea cultures (Fig. 1c,d ). In D. jasminea the addition of pineapple pulp rather than coconut water stimulated rhizogenesis. The mean number of roots/microcutting developed on medium with pineapple pulp reached 3.9 ± 0.9 and the rooting percentage was 14.0 ± 2.6%, while in medium supplemented with coconut water the roots per plant and rooting percentage were 1.0 ± 0.6 and 8.9 ± 2.2%, respectively.

Shoots of D. caucasica and D. tangutica were cultured on D1 and D0 media supplemented with conditioned medium obtained from green algae culture. In treatment CM1 (D1 medium supplemented with 20% CM) MC increased significantly over the respective control values, to 3.5 in D. caucasica and to 12.3 in D. tangutica (Table 2). We observed significant (P < 0.05) increases in adventitious organogenesis in D. caucasica: in the CM1 treatment, the frequencies of adventitious shoots and roots reached 25% and 80%, respectively (Table 2, Fig. 2). In this species, rhizogenesis was also stimulated by CM2 treatment (D1 medium supplemented with 50% CM), although to a lesser extent (48%; Table 2), but adventitious bud formation was inhibited. The least effective combination tested was D0 medium supplemented with 50% CM (D0-CM2). In D0-CM2, both species produced shortened shoots (not exceeding 15 mm in length) and micropropagation coefficients decreased, even though D. caucasica produced a high percentage of adventitious buds (Table 2). The buds did not develop shoots long enough to be further propagated. Surprisingly, in D0-CM treatment cultures appeared dormant and juvenile; growth and development of shoots were inhibited, but they had shiny, pale-green leaves, and showed no signs of vitrification. Cultures could be maintained for up to 3 mo without subculture.

Table 2. Effect of medium enrichment with conditioned medium obtained from Desmodesmus subspicatus culture on the micropropagation of Daphne caucasica and D. tangutica after 12 wk of culture
Full size table
Figure 2.
figure 2

Rhizogenesis and shoot culture of D. caucasica in medium supplemented with conditioned medium obtained from the green alga D. subspicatus. (a) Roots obtained on medium containing 20% CM. (b) Shoots obtained on medium containing 20% CM. (c) Roots obtained on medium containing 50% CM. (d) Shoots obtained on medium containing 50% CM.

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We determined optimal culture environment for effective shoot multiplication of tested Daphne species: non-supplemented D1 medium for D. jasminea, D1 medium supplemented with 10.0 ml L−1 pineapple pulp for D. caucasica and D1 medium supplemented with 10.0 ml L−1 coconut water for D. tangutica (Fig. 1e ).

We obtained rooted plantlets of D. caucasica and D. jasminea that were transferred ex vitro (Fig. 1f,g ). A significantly higher survival rate was noted for D. caucasica; in D. jasminea, some plantlets rotted at the shoot base and consequently died. The overall percentage of acclimatized plants was 83% and 65% for D. caucasica and D. jasminea, respectively. The treatments tested here were ineffective for rhizogenesis induction in D. tangutica. Thus, although we obtained viable shoot cultures of this species, no plants were produced that could be acclimatized to greenhouse conditions.

Determination of active compounds in pineapple pulp

Additional experiments were aimed at determining whether the biological activity of pineapple pulp was due to the presence of auxins. In TLC, UV visualization of bands revealed that the sample of pineapple pulp extract had not migrated through the stationary phase (silica gel), in contrast with the indole compounds standards (tryptophan, IAA, and IAA derivatives). Therefore, we conclude that the analyzed pineapple pulp did not contain auxin-like substances.

In another experiment, pineapple pulp was fractionated by dialysis into two fractions, dialyzate (<500 Da) and concentrate (>500 Da), and tested in a bioassay. We observed significant increases in the frequency of root formation in the medium containing 10 mg L−1 dialyzate. The frequency of rooted shoots was approximately 59% in D. jasminea, 20% in D. caucasica, and 8% in D. tangutica (data not shown). This was the first experiment in which rhizogenesis occurred in our D. tangutica culture. In medium supplemented with concentrate, only 4% of the D. jasminea shoots developed roots, compared with 35% in the non-supplemented medium. Shoots of D. caucasica and D. tangutica did not form roots in either concentrate-enriched medium or the non-supplemented medium.

Discussion

For Daphne spp., the previously used tissue culture media are far from ideal for organogenesis or micropropagation. Noshad et al. (2009) noted that repeated trials were necessary to establish a reproducible protocol for complete plantlet regeneration. Low frequency of in vitro rooting was problematic. The presence of exogenous auxin was crucial for rhizogenesis induction in Daphne species (Marks and Simpson 2000). For in vitro rooting, IBA and NAA (alone or in combination) were evaluated (Leifert et al. 1992; Marks and Simpson 2000; Mala and Bylinsky 2004; Noshad et al. 2009). In the present study, the NAA-containing non-supplemented medium induced rhizogenesis in almost half of the D. jasminea shoots. Since the medium containing high concentrations of cytokinin was designed for shoot multiplication the induction of rooting was unexpected and fortuitous. Refinement of culture conditions could further improve in vitro rooting. However, our observations are in an agreement with those of Noshad et al. (2009), who reported that the response to culture conditions remains species-specific, since D. caucasica and D. tangutica did not root on the non-supplemented medium.

In order to overcome barriers to micropropagation of Daphne ssp., we have studied the effects of medium enrichment. To our knowledge, alternative organic supplements have not been tested before in in vitro cultures of Daphne. Differences in response of the tested genotypes became evident on the supplemented medium. Especially D. caucasica proliferated rather poorly on the non-supplemented medium. In the course of experiments on each of tested Daphne species, we determined the most suitable culture environment for effective shoot multiplication (Fig. 1e ). By using medium enrichment, we were also successful in obtaining viable rooted and acclimatized plants of D. caucasica.

Overall, significant improvements in Daphne cultures were achieved by supplementing the medium with the following plant extracts: coconut water, pineapple pulp, and green algae exudates. Plant extracts may provide in vitro cultures with nutrients and minerals as well as growth regulators. The stimulatory role of coconut endosperm has been highlighted. For example, the addition of 5% coconut milk enhanced micropropagation in cultures in Echinacea purpurea, in which up to 10 shoots per explant were obtained (Mechanda et al. 2003). In cultures of olive (Olea europaea), the addition of coconut water enabled the reduction of the synthetic zeatin level in the medium; in this way, a high micropropagation rate was achieved on medium composed of relatively inexpensive ingredients (Peixe et al. 2007). In numerous studies, plant growth regulators have been detected in coconut liquid endosperm (Thorpe et al. 2008). Interestingly, Dix and van Staden (1982) observed that the use of isolated coconut water components individually or in various combinations had not produced as favorable results as those obtained with complete coconut water/milk. Moreover, in rooting experiments on Dracena purplecompacta, Agampodi and Jayawardena (2009) confirmed that an auxin extract from coconut water was superior to a commercial product containing IAA. Although the use of this supplement has decreased because of the availability of defined growth regulators, its use is still supported for in vitro cultures of species poorly responding to standard medium formulations. In practice, the benefits from the application of coconut water (either commercially available or directly prepared from fresh coconuts) are greater than its biological benefits alone, because its use may allow the cost of medium preparation to be reduced significantly (Peixe et al. 2007).

In Daphne micropropagation, pineapple pulp was successfully used as a medium supplement. Similar positive influences of this additive have been described for cultures of several Ophrys orchids (Kitsaki et al. 2004), although the origin of the growth promoting activity was not discussed there. Pineapple pulp is not as often exploited in in vitro cultures as coconut liquid endosperm, so little is known about its composition and stimulatory properties. Mercier et al. (2003) detected an auxin (IAA) and five different cytokinins in the basal leaf region of pineapple plantlets grown in vitro. In the present study, IAA and other auxins were not found in pineapple pulp. However, enhanced rooting of Daphne cultures could be obtained on medium containing pineapple compounds of low molecular weight (<500 Da). Effective formation of adventitious roots may result from the presence of rooting cofactors. Some plant extracts may contain rooting cofactors and polyamines (Wilson and van Staden 1990). When added exogenously, these low-molecular-weight compounds can stimulate or inhibit in vitro rooting processes, depending on the concentration and the rooting phase (Arena et al. 2005). Several polyamines, including serotonin and putrescine, have been recently detected in pineapple fruits (Santiago-Silva et al. 2011). Melatonin, a serotonin derivative, affects root development in higher plants and is an important metabolic regulator (Murch and Saxena 2002). The presence of melatonin in pineapple has been also reported (Hattori et al. 1995).

In addition to auxins and cytokinins, other naturally occurring growth promoters are present in plant extracts. Gibberellin-like substances and salicylic acid were detected in coconut milk (Dix and van Staden 1982; Wu and Hu 2009). The activity of such compounds may be responsible for the increased proliferation of Daphne shoot cultures.

Despite the generally beneficial activity of the tested additives, we observed a decrease in rooting efficiency of D. jasminea shoots in media enriched with pineapple pulp and coconut water in comparison with non-supplemented medium (Table 1). This may be the result of a species-specific reaction to the compounds in the applied plant extracts. It is also possible that an interaction between exogenous auxin and rooting cofactors had a negative effect on rhizogenesis. Similar results were found for micropropagated Acacia (Scholten 1998). Further understanding of the growth regulatory effect of pineapple pulp could contribute to the development of novel media formulations to make the micropropagation process more effective.

Utilization of natural plant extracts as medium supplements does have several disadvantages. Organic medium prepared from plants from different habitats or growth conditions may not contain uniform concentrations of growth regulatory compounds. For example, coconut water samples from different coconut fruits contain different amounts of abscisic acid (usually deleterious to growth), which may be an indication of environmental stress encountered by the parent plant (Ma et al. 2008). Such fluctuations may cause unwanted variability in commercial micropropagation.

In the present study, the best results were obtained when culture medium was supplemented with conditioned medium containing algal exudates. A stimulatory effect of D. subspicatus spent medium has been already reported in systems simpler than shoot culture, e.g., for cell biomass production in suspension culture (Grabski and Tukaj 2008; Hanus-Fajerska et al. 2009). Here we confirm that exudates of D. subspicatus released into the medium affect in vitro growth of higher plants, and the proportion of conditioned medium influences both propagation rate and rooting efficiency. Previous analyses revealed that one conditioned medium component with growth factor-like properties is a low-molecular-weight peptide (around 500 Da) with prolonged activity (Grabski et al. 2010). Shrivastava and Banerjee (2009) proposed utilization of algal filtrate as a cost-efficient medium supplement for Jatropha curcas micropropagation. In the present study, the optimal combination contained 20% conditioned medium.

Arabinogalactan and chitosan were ineffective for micropropagation improvement in the three Daphne species. Deterioration of growth in medium supplemented with chitosan could be a result of a hypersensitive response to the applied concentration, as suggested by Lesney (1989) for Pinus elliottii cell suspensions. Arabinogalactan did not influence Daphne cultures as negatively as chitosan, but given the superior results obtained for other supplements, its use as a medium supplement for Daphne species is not supported.

Conclusions

The supplementation of media with plant extracts and cell exudates is beneficial in Daphne spp. micropropagation. In addition to vigorous appearance of shoots, enhancement in rooting efficiency can be achieved. Moreover, some difficult-to-root genotypes of this genus can be more easily rooted on media enriched with pineapple pulp or its low-molecular weight fraction, or with spent algal medium.

Complex, plant-derived natural medium supplements promoted proliferation of Daphne shoots and production of viable microplantlets. However, microshoot response to the applied treatments remained genotype-specific. Cell wall derivatives such as arabinogalactan and chitosan were not found to be as effective as undefined products such as pineapple pulp or spent algal medium. This suggests that the observed improvement may be due to supplying the cultured organs with nutrients rather than to oligosaccharide-type signal compounds.

Moreover, medium supplemented with conditioned medium but without synthetic growth regulators can be proposed for maintaining Daphne stock cultures. Such medium allows a reduction in labor-intensive subculturing. Production of conditioned medium is inexpensive because algal cells are cultured in pure mineral solution, without organic compounds. In laboratory practice, algal cells may be exploited for miscellaneous studies, and the spent medium reused for higher-plant culture. This approach may contribute to economical use of resources such as water during medium preparation. Therefore, the use of culture media based on natural organic supplements may facilitate and simplify horticultural production. The protocols developed in our study should now be tested on other Daphne species, including commercial varieties and wild endangered species.