Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Optimization of culture conditions for the direct regeneration of Kappaphycus alvarezii (Rhodophyta,Solieriaceae)

Cultivation of seaweeds on a commercial scale requires a large number of propagules with desirable phenotypic traits which include high growth rates and resistance to diseases. Seaweed micropropagation can be considered as one of the best methods to provide a large amount of seedlings for commercial cultivation. This study was carried out to optimize the parameters known to affect the growth of Kappaphycus alvarezii in vitro and subsequently improve the production of seedlings through micropropagation. Suitability of media, concentration of phytoregulators, types and concentration of fertilizers, culture density, light intensity, interval of aeration activity, salinity, and pH were found to be critical factors for the growth of K. alvarezii. The optimum condition for direct regeneration of K. alvarezii in a culture vessel was found to be cultivation of explants in Provasoli's enriched seawater (PES) media supplemented with 2.5 mg L^?1 6-benzylaminopurine (BAP), 1.0 mg L^?1 indole-3-acetic acid (IAA), and 3.0 mg L^?1 natural seaweed extract (NSE) with culture density of 0.4 %?w/v, under light intensity of 75 μmol photons m^?2 s^?1, continuous aeration of 30.0 L h^?1, salinity of 30.0 ppt, and pH 7.5. An airlift photobioreactor was constructed for the mass propagation of K. alvarezii explants with optimum culture conditions obtained from the study. The optimum growth rates of the K. alvarezii explants in culture vessels (5.5 % day^?1) and photobioreactor (6.5 % day^?1) were found to be higher than the growth rate observed in field trials in the open sea (3.5 % day^?1). The information compiled during the course of this study will be of utility to commercial seaweed cultivators.     

PROTEOMICS OF THE RED ALGA,GRACILARIA CHANGII (GRACILARIALES,RHODOPHYTA)1

The application of proteomics in alga research is still quite limited. The present report describes the establishment of the proteome of a red alga of economic importance, Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Initially, four protein extraction methods including direct precipitation by trichloroacetic acid/acetone, direct lysis using urea buffer, Tris buffer, and phenol/chloroform extraction were compared for their suitability to generate G. changii proteins for two‐dimensional gel electrophoresis (2‐DE). The phenol/chloroform protein extraction method gave the best 2‐DE resolution of the proteins. Using these 2‐DE gels and mass spectrometry, several proteins including pigment proteins, metabolic enzymes, and ion transporters were identified. These findings highlight the potential of using proteomic approaches for the investigation of G. changii protein function.     

In vitro culture of Aloe barbadensis Mill.: Morphogenetic ability and nuclear DNA content

In vitro culture of Aloe barbadensis is very difficult for both callus Induction and plant regeneration. By contrast, micropropagation from shoot meristems is easily achieved. A DNA microdensitometric study was performed on different organs of A. barbadensis and during in vitro culture of different explants. It was shown that morphogenetic ability is correlated to nuclear DNA content in the explant and/or in the callus. In particular, plant regeneration (or micropropagation) is obtained only from calli (or from explants) in which the amount of DNA per nucleus falls strictly within the diploid limits (2C and 4C) of the species.     

Micropropagation of Gagnepainia godefroyi K Schum and Gagnepainia thoreliana (Baill) K Schum — Rare Medicinal Plants of Thailand

A micropropagation protocol for Gagnepainia godefroyi K Schum and G thoreliana (Baill) K Schum, two rare medicinal plants of Thailand, has been developed using terminal buds. After a total of twenty weeks of culture (4 weeks on MS added with 36.33 μM TDZ and 16 weeks on PGR-free MS), 28.77 and 13.50 shoots/explant were obtained, respectively. Rooting was spontaneous and regenerated plants were successfully transplanted to soil.     

Branch,micropropagule and tissue culture of the red algaeEucheuma denticulatum andKappaphycus alvarezii farmed in the Philippines

Three forms of the iota-producing carrageenophyte,Eucheuma denticulatum, and four forms of the kappa-producing carrageenophyte,Kappaphycus alvarezii, obtained from seaweed farms in the Philippines have been grown in the laboratory under unialgal and axenic conditions. Comparison of media indicates that seed stocks of both species can be cultured using enriched seawater media ranging from ESS and SWMD-1 to inexpensive soil extract (Erdshreiber's) or holding in sterile seawater for up to three weeks. Micropropagation has been successful with at least two forms of each species resulting in clonal propagation from axenic explants within 4 to 8 weeks. Callus development and branch regeneration has also been induced in two forms of each species. The results indicate that culture facilities in the farming areas of the Philippines could maintain high-yielding and rapidly growing seed stock for the seaweed farmers.     

In vitro shoot proliferation of 5-leaf aralia explants from field grown plants and forced dormant stems

A. Sieboldianus (5-leaf aralia) is recalcitrant for micropropagation, but has very good landscaping potential. This research was conducted with the following objectives: (1) to study effects of BA, TDZ, CPPU, 2iP, kinetin and zeatin in woody plant medium on the performance of softwood shoot nodal explants produced by field grown 5-leaf aralia plants; (2) to investigate influences of BA or TDZ in the forcing solution on subsequentin vitro shoot initiation of nodal explants taken from forced softwood growth. Shoot initiation of softwood nodal explants from field-grown plants was promoted by adding BA, TDZ or CPPU to the culture medium. Kinetin, zeatin and 2iP were ineffective for micropropagation ofA. Sieboldianus. The forced softwood growth for use as explants was “primed” by forcing dormant stems in solution containing 200 mg 8-HQC per liter plus 2% sucrose, 44.4, 222, or 444 μM BA, or 45.4, 227, or 454 μM TDZ. BA and TDZ in the forcing solution enhanced subsequentin vitro axillary shoot initiation of nodal explants taken from forced stems by doubling the number of shoots produced per explant to 3.3 from 1.65 shoots per explant taken from field grown plants. This forcing solution technique also reduced the time needed from culture initiation to potted plants to half of the time needed for the conventional micropropagation method (12 to 14 vs. 25 to 27 weeks), thus expediting the micropropagation ofA. Sieboldianus.     

Optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte <Emphasis Type="Italic">Kappaphycus</Emphasis>

To improve the production of Kappaphycus plantlets in tissue culture, optimum media concentrations of an Ascophyllum nodosum extract (Acadian Marine Plant Extract Powder, AMPEP), plant growth regulators (PGR), pH–temperature combinations, and explant density were determined. Kappaphycus alvarezii var. tambalang purple (PUR), kapilaran brown (KAP), vanguard brown (VAN), adik-adik (AA), tungawan green (TGR), and K. striatum var. sacol green (GS) were used as explants. Based on the shortest period for shoot emergence and the economical use of AMPEP, the optimum enriched media was 3.0 mg L⁻¹ AMPEP and 0.1 mg L⁻¹ AMPEP + PGR 1 mg L⁻¹ each phenylacetic acid (PAA) and zeatin for PUR, 1.0 mg L⁻¹ AMPEP + PGR for KAP and GS, 0.1 mg L⁻¹ AMPEP + PGR for VAN, and 3.0 mg L⁻¹ AMPEP and 0.001 mg L⁻¹ AMPEP + PGR for AA and TGR. Results showed that the addition of PGR to low concentrations of AMPEP hastened shoot formation. pH–temperature combinations for the most rapid shoot formation were determined for the brown (KAP) and purple (PUR) color morphotypes of K. alvarezii var. tambalang and the green morphotype of K. striatum var. sacol (GS) cultured in 1.0 mg L⁻¹ AMPEP + PGR. The brown morphotype produced the most number of shoots at pH 7.7 at 20°C after as little as 20 days. Purple K. alvarezii showed an increased shoot formation at pH 6.7 at 25°C and the green K. striatum morphotype at pH 8.7 at 25°C. The optimum number of explants added to the culture media was also determined for tungawan green (TGR), brown (KAP), and tambalang purple (PUR) varieties of K. alvarezii in 1.0 mg L⁻¹ AMPEP + PGR. The number of explants and the volume of the culture media combination were also tested. The highest average number of shoots formed occurred in two explants:1 mL culture media (2:1) for KAP and PUR (35.00% and 16.67%, respectively) and 1 explant: 2 mL culture media for the TGR (100.00%) with a range of 0.5–3.0 mm shoot length after 40 days in culture. The earliest shoot formation was observed after 21 days for the brown and 9 days for both the green and purple color morphotypes of Kappaphycus, in all densities investigated. This indicated that within the range tested, the density of explants did not have a significant effect on the rate of shoot formation but did influence the average number generated from the culture. The rate of production of new and improved Kappaphycus explants for a commercial nursery stock was improved through the use of AMPEP with optimized culture media pH, temperature, and density conditions.     

In vitro antimicrobial activity against Pseudomonas aeruginosa and acute oral toxicity of marine algae Gracilaria changii

Methanol extract of the Gracilaria changii has been screened for antimicrobial activity against Pseudomonas aeruginosa. Antimicrobial activities were carried out using disc diffusion assay and broth dilution method against P. aeruginosa. The methanol extract of G. changii showed a good antimicrobial activity against P. aeruginosa with MIC (Minimum Inhibitory Concentration) value of 6.25 mg/ml. Exposure of P. aeruginosa cells to 6.25 mg/ml of methanol extract of G. changii resulted in complete inhibition of the bacterial cells. The main abnormalities noted via SEM and TEM studies were the alterations in morphology and cytology of the bacterial cells. The main reason for this deterioration was discussed. The effect of the methanol extract on the growth profile for the bacteria was also done and confirmed the bactericidal effect of the G. changii methanol extract on P. aeruginosa by changing the normal growth profile of P. aeruginosa. In an acute toxicity study using mice, the median lethal dose (LD₅₀) of the extract was greater than 2000 mg/kg, and we found no pathological changes in macroscopic examination by necropsy of mice treated with extract. We conclude that G. changii might be safely used as an antimicrobial agent.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (“Edison de Paula”, EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch’s (VS 50) and Guillard & Ryther’s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L⁻¹) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.     

Callus ontogeny of the Kappaphycus alvarezii (Rhodophyta, Gigartinales) brown tetrasporophyte strain

This study analyzes the callus ontogeny of the Kappaphycus alvarezii brown strain tetrasporophyte using micropropagation and tissue culture. Calli were induced from axenic explants and grown for 2 months. Ontogenic analysis was made in explants collected from days?7 to 60 of culture and observed under light and transmission electron microscopy (TEM). At the end of the experimental period, 60-day samples were also analyzed by confocal microscopy. Formation of calli was initiated in the first week from medullary and cortical cells of the explant region in air contact. Alterations in the apical region of the cells were observed in TEM as cell wall thickening, proliferation of convoluted membranes, increased number of mitochondria and chloroplast alterations, indicating the dedifferentiation of these cells. Throughout the experiment, the thick cell wall of the cells which compose callus filaments showed the presence of acidic polysaccharides, suggesting a large amount of carrageenan and neutral polysaccharides. The cytoplasm showed the following main characteristics: large amount of starch grains, presence of chloroplasts with altered plastoglobules, as well as disorganized thylakoids, presence of several convoluted membranes, and vacuoles. These filaments maintained their uniseriate organization and irregular branching with many intercellular connections between them. Despite the initial stress caused by explant isolation and the beginning of callus formation, results showed that filaments were capable of reorganization and development, retaining their dedifferentiated cells, which, in turn, strongly suggested the potential of callus-based micropropagation.     

Investigation of the application of Acadian Marine Plant Extract Powder (AMPEP) to enhance the growth, phenolic content, free radical scavenging, and iron chelating activities of Kappaphycus Doty (Solieriaceae, Gigartinales, Rhodophyta)

An optimization study on concentration (viz. 0.01, 0.1, and 1.0 g L^?1) and dipping time (i.e., 30 and 60 min) was conducted on three different color morphotypes (i.e., reddish brown, yellowish brown and purple) of the commercial carrageenophyte Kappaphycus alvarezii (Doty) Doty. The study tested the efficacy of Acadian Marine Plant Extract Powder (AMPEP) on the growth rate and occurrence of macro-epiphytes from August to November, representing the wet season of the Philippines. The optimum concentration and dipping time were obtained at 0.1 g L^?1 and 30 min, respectively. These optimum parameters were then further verified in a commercial nursery using the yellowish brown morphotype. In another experiment, K. alvarezii (tambalang purple morphotype) and Kappaphycus striatum (Schmitz) Doty (sacol green morphotype) with, and without, AMPEP dippings were tested for their total phenolic content, free radical scavenging and iron chelating activities. Seaweed dipped in AMPEP demonstrated higher growth rates than the control. Lower concentrations (i.e., 0.01-0.1 g L^?1) and shorter dipping time (e.g., 30 min) produced higher growth rates than the highest concentration (1.0 g L^?1) and longer (60 min) dipping time. The presence of macro-epiphytes such as filamentous Ulva did not adversely affect the robust growth of the three color morphotypes of K. alvarezii. The lowest and highest growth rates obtained in a commercial seaweed nursery using the optimum concentration and dipping time of AMPEP were observed in July and January with 0.8% and 6.7% day^?1, respectively. The antioxidant content of K. alvarezii (tambalang purple) and K. striatum (sacol green) responded differently to AMPEP dipping. The changes in total antioxidant activity followed almost the same trend as in phenolic content, in both K. alvarezii (tambalang purple) and K. striatum (sacol green), whereas, the iron chelating ability of both seaweeds with and without AMPEP dipping varied monthly. The results obtained for the use of AMPEP dips for commercial Kappaphycus cultivation demonstrated an effective management tool for improved farming protocols.     

Clonal propagation of Lilium nepalense D.Don, a threatened medicinal plant of Nepal

A protocol for the micropropagation of Lilium nepalense D.Don, a highly prized medicinal plant of Nepal, has been developed. Axillary shoots were regenerated from twin-scale explants prepared from mature bulbs. Multiplication was carried out on MS medium supplemented with 20 µM zeatin using longitudinally split shoot halves. On average, more than seven shoots were obtained from one explant in a 4-week culture period. After rooting the cloned plantlets were successfully hardened to ex vitro conditions. Preliminary trials in Nepal showed that the method can be successfully applied for the production of plants suitable for field cultivation. This may be an alternative to the over-exploitation of the natural resources of the species.     

Micropropagation,callus and root culture of Phyllanthus urinaria (Euphorbiaceae)

Efficient micropropagation, callus culture and root culture protocols were developed for the medicinal plant Phyllanthus urinaria(Euphorbiaceae) using single node explants. Maximum multiplication (16–20 shoots per explant) was achieved on Murashige and Skoog media supplemented with 5.0 M kinetin. Murashige and Skoog and Anderson Rhododendron media promoted significant shoot culture growth in terms of numbers of shoots and nodes produced per explant. Rooting was achieved with 93–100% of the microshoots on Murashige and Skoog medium without growth regulators, although 1.25–5.0 M -naphthaleneacetic acid significantly increased the number of roots per explant. Regenerated plants were successfully acclimatized and 91% of plantlets survived under ex vitro conditions. Flowering was observed on micropropagated plants after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when single node explants were inoculated in the horizontal position on Murashige and Skoog medium supplemented with 5.0 M indole-3-butyric acid. Other auxins such as 2,4-dichlorophenoxyacetic acid and -naphthaleneacetic acid promoted moderate callus fresh weight increase, when used separately. Root cultures were successfully established on Murashige and Skoog medium containing 1.1 M -naphthaleneacetic acid. The optimized micropropagation, callus culture and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies.     

Temperature and salinity responses of drifting specimens of Kappaphycus alvarezii (Gigartinales,Rhodophyta) farmed on the Brazilian tropical coast

Bioinvasion events causing serious environmental damage have been a concern with the mariculture of Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, suggesting the importance of studying the biological aspects of drifting specimens of K. alvarezii for monitoring programs. The present study aims to evaluate the tolerance and growth of drifting color variants of K. alvarezii under different temperatures and salinities to determine their physiological capacity for growing outside cultivation rafts. Drifting color variants were collected in Paraíba State, Brazil, in November 2011(dry month) and August 2012 (rainy month), and cultivated in the laboratory under different temperatures (20, 24, 28, and 32 °C) and salinities (15, 25, 35, 45, and 55 psu). Growth rates as well as pigment and protein contents were determined. Results showed that drifting specimens collected in the dry month showed higher tolerance to variation in temperature (20 to 28 °C) and salinity (25 to 35 psu) than drifting specimens collected in the rainy month. Higher growth rates occurred in samples cultured at 20 and 24 °C (2.8–3 % day^?1) and 25 to 35 psu (3.4–3.5 % day^?1), suggesting temperature and salinity optima. Higher phycobiliprotein levels were observed in the red and brown variants under hypersaline conditions (45 and 55 psu). Higher chlorophyll a contents were associated with samples cultivated at 20–24 °C and 24–35 psu. Based on the results of the present study, drifting specimens collected in dry month are more tolerant to temperature and salinity variations, suggesting that the drifting K. alvarezii should be monitored especially during this period to prevent its establishment outside the cultivation rafts and dispersion along the northeastern coast of Brazil.     

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells

Background and Aims

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.

Methods

Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression.

Key Results

Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature.

Conclusions

The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.Key words: Callus, dedifferentiation, leaf veins, Medicago truncatula, pluripotency, procambium, protoplasts, reactive oxygen species, SERK, somatic embryogenesis, stem cells, totipotency     

Hyperosmotic stress-induced somatic embryogenesis and its continuous culture in Japanese honewort (Cryptotaenia japonica)

Japanese honewort (Cryptotaenia japonica) is consumed as a traditional vegetable and has medicinal applications. In Japan, C. japonica is mainly produced using hydroponic culture systems; however, damping-off is often caused by the adherence of pathogens to its seeds. Therefore, the use of sterile artificial seeds in hydroponic culture is likely to be effective for preventing disease. In this study, we established methods for stress-induced somatic embryogenesis and artificial seed production in Japanese honewort. Shoot apex explants from seedlings were treated with 0.7 M sucrose as a hyperosmotic stress for 3 or 6 weeks, and then transferred to stress-free conditions. Somatic embryos were formed after culture in stress-free conditions for 7 weeks. Stress-treated shoot apex explants that formed somatic embryos were cultured in Murashige and Skoog liquid medium with shaking. After 2 weeks of culture, approximately 800 somatic embryos were formed from each explant. Somatic embryos were formed continuously during 37 weeks under the same culture conditions. Thus, somatic embryogenesis was effectively induced in Japanese honewort via hyperosmotic stress, and embryogenic competence was maintained under stress- and phytohormone-free conditions. The somatic embryos produced by liquid culture were used to produce artificial seeds by enveloping the embryos in whipped alginate gel to avoid hypoxic conditions. The artificial seeds had a high germination rate (72%). This system is suitable for the sterile, highly productive hydroponic culture of Japanese honewort.