Production of Bacillus subtilis-fermented red alga Porphyra dentata suspension with fibrinolytic and immune-enhancing activities

The fermented marine alga Porphyra dentata suspension was tested for its fibrinolytic and immune-enhancing activities. An isolated Bacillus subtilis N2 strain was selected for its fibrinolytic activity on fibrin plates. After investigating the effects of biomass amounts of P. dentata powder in water, various additives including sugars, nitrogen-containing substances, lipids and minerals, and cultural conditions of temperature and agitation in flask, the highest fibrinolytic activity in the cultural filtrate was obtained by cultivating N2 strain in 3% (w/v) P. dentata powder suspension containing 1% peanut oil at 37?°C, 150?rpm for 48?h. A fermentor system was further established using the same medium with controlled pH value of 7.0 at 37?°C, 150?rpm, 2.0 vvm for 48?h for the best fibrinolytic activity. The fermented product also showed its immune-enhancing activity by increasing cell proliferation and stimulating the secretion of IL-1β, IL-6, and TNF-α in J774.1 cells.     

Isolation and culture of soybean hypocotyl protoplasts

Summary Protoplasts were isolated seedling hypocotyls of soybean (Glycine max), and cultured in both liquid and agarose-solidified, modified K8P medium. Nuclear staining revealed that only 2% of protoplasts lacked a nucleus, 93% contained a single nucleus, and 5% contained more than one. Maximum protoplast yields and subsequent division frequencies, in liquid medium, were obtained from 5 days-old seedlings. Maximum division frequencies (54%) were obtained from hypocotyl protoplasts plated at a density of 5×10⁴ ml⁻¹. Using different osmolality reduction régimes for liquid cultures, hypocotyl protoplasts developed into green, nodular callus, similar to that which has previously given rise to shoot buds in perennialGlycine species. This tissue, however, did not produce shoot buds in soybean. N. H. was supported by a SERC CASE studentship and a postdoctoral fellowship from Shell Research Ltd., Sittingbourne, Kent, UK.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Protoplast isolation and regeneration in the marine red alga Porphyra nereocystis

We have developed a method for isolating viable protoplasts from the blade phase of the epiphytic marine red alga Porphyra nereocystis Anderson, using a two-step enzymatic digestion with commercially available enzymes. The first step uses papain, the second step uses abalone acetone powder. The method is rapid and gives a high yield of viable protoplasts. In liquid culture in enriched seawater medium, the protoplasts can undergo regeneration along three pathways: they directly form filaments resembling the conchocelis phase of Porphyra; they form calli with relatively thick-walled, pigmented cells; and they indirectly form blades from the edges of these calli. Porphyra nereocystis protoplasts also may serve as an alternative propagation method in aquaculture and be useful for studies of cell-wall formation, cell division, and thallus differentiation. They may also be used in somatic selection, somatic hybridization and gene-transfection experiments.Abbreviations AAP abalone acetone powder - PAP papain - FDA fluorescein diacetate This paper is dedicated to the memory of the late Dr. Munenao Kurogi (1921–1988), Professor Emeritus of Hokkaido UniversityThis research was supported by the Washington Sea Grant Program (National Oceanic and Atmospheric Administration). We thank Professor Y. Fujita (Nagasaki University, Japan), Professor S.-J. Wang (Shanghai University of Fisheries, P.R. China) and Dr. H. Kito (Seikai Regional Fisheries Research Laboratory, Nagasaki, Japan) for sharing their experience with Porphyra protoplast production with us. We thank J.S. Charleston for expert technical assistance in preparation of the electron-microscopy specimens. We also thank Dr. S.K. Herbert and John Carrier (Friday Harbor Laboratories) and Dr. John Merrill and D. Gillingham (American Sea Vegetable Co. and Applied Algal Research, Seattle) for collections of P. nereocystis.     

Feasibility Study of Phragmites karka and Christella dentata Grown in West Bengal as Arsenic Accumulator

A survey was undertaken, in arsenic (As) contaminated area of the Nadia district, West Bengal, India, to find native As accumulator plants. As was determined both in soil and plant parts. The results showed that the mean translocation factor of Pteris vittata L, Phragmites karka (Cav.) Trin. Ex. Steud and Christella dentata Forssk were higher than 1. It thus appeared that these plants can be efficient accumulators of As.

Phytoremediation ability of C. dentata and P. karka was evaluated and compared with known As-hyperaccumulators -P. vittata and Adiantum capillus veneris L. Plants were grown in the As spiked soil (25, 50, 75 and 100 mg kg^?1). As accumulation was found to be highest in P. vittata, 117.18 mg kg^?1 in leaf at 100 mg kg^?1 As treatment, followed by A. capillus veneris, P. karka and C. dentata being 74, 83.87 and 40.36 mg kg^?1, respectively. Lipid peroxidation increased after As exposure in all plants. However, the antioxidant enzyme activity and molecules concentration also increased which helped the plants to overcome As-induced oxidative stress. The study indicates that P. karka and C. dentata could be considered as As-accumulators and find application for As-phytoextraction in field conditions.     

Protoplasts of Gelidium robustum (Rhodophyta)

Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 10⁵ protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg^–1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg^–1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.Dedicated to the memory of Professor Michael Neushul.     

A SIMPLE METHOD FOR ISOLATING FILAMENTS AS “ALGAL SEED STOCK” FROM MONOSTROMA LATISSIMUM (CHLOROPHYTA) GERMLINGS,AND APPLICATION FOR MASS CULTIVATION1

Germlings were grown from Monostroma latissimum Wittr. reproductive cells on nylon ropes. Holdfast threads and some uniseriate filaments were observed to have penetrated the fibers of the dispersed ropes. The algal filaments were easily isolated and prepared for cultivation, in comparison to the methods of enzymatically isolated algal protoplasts. Under low light (60–100 μmol photons · m^?2 · s^?1), the algal filaments grew to form a filamentous mass. When cultivated under stronger light (300–600 μmol photons · m^?2 · s^?1), they grew to initially form tubular thalli and then, when cultivated under light intensities >700 μmol photons · m^?2 · s^?1, formed foliaceous thalli. Consequently, the filaments were homogenized into small sections and then sewed on the nylon rope for algal mass cultivation. Under high‐intensity natural light, they grew to form leafy thalli.     

Confirmation of Neoporphyra cf. dentata on Shikinejima,Izu Islands,southcentral Japan,and comparison with co-occurring Neoporphyra haitanensis

The Izu Islands of southcentral Japan are thought to fall within the distribution range of Neoporphyra dentata. However, the gametophytic blades of Bangiales collected from Shikinejima and Hachijojima, Izu Islands, were identified as Neoporphyra haitanensis in our previous study. Thus, it became uncertain whether N. dentata is distributed in the Izu Islands, including Shikinejima. To clarify whether N. dentata grows on Shikinejima, we conducted a further distribution survey of N. dentata on the island. The morphological features of the blade samples collected from an additional sampling site on Shikinejima were more similar to those of N. dentata than to those of N. haitanensis: the blade thickness and the division formula of spermatangia resembled those of the former species rather than the latter species. However, the division formula of zygotosporangia was different from those of either species. The phylogenetic analyses of the rbcL gene indicated that the samples were resolved in a clade including N. dentata collected from Shirahama, Chiba Prefecture, and Enoshima, Kanagawa Prefecture, Honshu, Japan. The p-distances of the chloroplast rbcL gene and nuclear 18S rRNA also supported identification of the samples as N. dentata. The results demonstrated that N. dentata is also distributed on Shikinejima with co-occurring N. haitanensis, and that the island materials of the two species are genetically different from other materials of the two species, respectively.     

An experimental study of 226Ra and 45Ca accumulation from the aquatic medium by freshwater turtles (fam. Chelidae) under varying Ca and Mg water concentrations

Snapping turtles Elseya dentata (Gray) from Magela Creek, Northern Territory, were exposed under laboratory conditions for up to 30 days to waters resembling the inorganic composition of Magela Creek water during the Wet season, with background and elevated Ca and Mg concentrations, that were labelled with ²²⁶Ra and ⁴⁵Ca. The resulting concentrations of ⁴⁵Ca in muscle, skin, gut, liver, shell bone and leg bone of E. dentata equilibrated or approached equilibrium by 12–18 days. Among the experiments, the concentrations of ⁴⁵Ca in all six tissues were inversely related to turtle mass. An increase in the Ca water concentration by a factor of 15 increased the ⁴⁵Ca concentration in all six tissues. The arithmetic factors of increase in the concentration in each tissue were proportional or more than proportional to the factor of increase in Ca water concentration; this factor was highest for muscle tissue (26.6). An increase in the Mg water concentration by a factor of 48 reduced the ⁴⁵Ca concentration in all tissues except skin where it increased. The concentration of ²²⁶Ra in each tissue (except the gut) was positively related to its ⁴⁵Ca concentration and inversely related to turtle mass in muscle, skin and liver. With the exception of the skin, the increased Ca water concentration did not reduce the ²²⁶Ra in the tissues but increased the ²²⁶Ra concentration in bone and muscle. The increased Mg water concentration had an inverse effect on the ²²⁶Ra concentrations in all tissues except shell. With the exception of the skin the effects of increased Ca and Mg water concentrations and turtle size on ²²⁶Ra concentrations in the tissues of E. dentata were similar to their effects on ⁴⁵Ca tissue concentrations, indicating the similar metabolic behaviour of ²²⁶Ra and ⁴⁵Ca in E. dentata.Exposures of the species Elseya latisternum (Gray), Emydura signata (Ahl) and Chelodina longicollis (Shaw), which are the same or closely related to species reported to occur in Magela Creek, to ⁴⁵Ca-labelled Sydney tap water for 7 days demonstrated their ability to also accumulate ⁴⁵Ca from their aquatic medium. The patterns of ⁴⁵Ca concentrations in the tissues of these species indicated that they were inversely related to turtle mass, as demonstrated in E. dentata. The concentrations of ⁴⁵Ca accumulated in the tissues were also comparable to those found in single specimens of E. dentata and E. victoriae (Gray) that were exposed for 7 days to simulated Magela Creek water. The data also indicated the larger long-necked C. longicollis accumulated less ⁴⁵Ca per gram of tissue than similar-sized, short-necked species E. signata and E. latisternum, suggesting that long-necked turtles from Magela Creek would accumulate less ²²⁶Ra from their aquatic medium than similar-sized short-necked species.The capacity of E. dentata to accumulate ²²⁶Ra from the aquatic medium is about two orders of magnitude less than that of the tissue of the freshwater mussel Velesunio angasi (Sowerby) exposed under similar experimental conditions.     

Transformation of <Emphasis Type="Italic">Platymonas</Emphasis> (<Emphasis Type="Italic">Tetraselmis</Emphasis>) <Emphasis Type="Italic">subcordiformis</Emphasis> (Prasinophyceae,Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25°C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10⁷ cell ml⁻¹. The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10⁻⁵, and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.     

A comparison of photolyase activity in three Australian tree frogs

Ultraviolet-B (UV-B) irradiation of DNA generates mutagenic photoproducts such as cyclobutane pyrimidine dimers (CPDs) which can affect the growth and development of amphibian embryos. Differential ability to repair UV-B-induced DNA damage may be␣responsible for differences in population stability between␣some amphibian species. Photoreactivation via the enzyme photolyase is a major mechanism used to remove CPDs from DNA. The aim of this study was to determine if photolyase activity differed in three sympatric Australian amphibian species, one of which has suffered marked population declines (Litoria aurea) and two whose populations do not appear to be in decline (L. dentata and L. peronii). The specific activity of photolyase was measured in each species and compared to the hatching success of their eggs under unfiltered summer sunlight. The mean specific activities of photolyase were 1.10 ± 0.18 × 10¹¹, 5.76 ± 1.01 × 10¹¹, and 2.66 ± 0.15 × 10¹¹ CPDs repaired per hour per microgram of egg protein extract, for L. aurea, L. dentata and L. peronii, respectively. When intrinsic differences in hatching success between species were controlled for, the relative percentage hatching success under unfiltered sunlight of L. aurea (77%) was lower than that of L.␣peronii (91%) and L. dentata (98%); however, these values did not differ significantly. L. aurea had the lowest photolyase activity of the three species and showed a non-significant trend of reduced hatching success under UV-B exposure. Received: 15 December 1997 / Accepted: 9 March 1998     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

Protoplast Preparation from <Emphasis Type="Italic">Laminaria japonica</Emphasis> with Recombinant Alginate Lyase and Cellulase

Functional recombinant abalone alginate lyase (rHdAly) and β-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30°C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35°C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (<1 × 10³ protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 × 10⁶ protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 μm, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.     

Optimization of protoplast yields from the red algae <Emphasis Type="Italic">Gracilaria dura</Emphasis> (C. Agardh) J. Agardh and G. <Emphasis Type="Italic">verrucosa</Emphasis> (Huds.) Papenfuss

This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10, 0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields of 3.7 ± 0.7 × 10⁶ cells g⁻¹ fresh wt for G. dura and 1.2 ± 0.78 × 10⁶ cells g⁻¹ fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region, were also observed in G. verrucosa.     

Winged-bean protoplasts: Isolation and culture to callus

A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean,Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cullulysin, 1% Macerase, 1% desalted Rhozyme, 0.4M sorbitol, and 0.1M CaCl₂ at pH 5.5. Average yields of protoplasts were 6.5 × 10⁶ per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4 g/l glucose, 250 mg/l xylose, 0.1 mg/l 2,4-D, 0.5 mg/l BAP, 250 mg/l N-Z amine type AS, and 20 ml/l coconut water. After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating density was 1–2 × 10³ protoplasts/ml. The division frequency ranged between 40%–60% for most experiments with a high of 72% in one experiment. After three weeks, cell colonies could be transferred to solid MS medium containing N-Z amine and coconut water where callus developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation involving legumes.     

Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10³ (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 10³ (number/ml min)] and Wasabia japonica [kv = 14.2 × 10³ (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.     

A preliminary comparison of the mariculture potential of Porphyra purpurea and Porphyra umbilicalis

Due to their rapid growth and nutrient assimilation,Porphyra spp. are good candidates for bioremediation and polyculture. The production potential of two strains of P. purpurea and P. umbilicalis from north-east USA was evaluated by measuring rates of photosynthesis (as O₂evolution) of material grown at 20 ^°C. Photosynthetic rates of P. umbilicalis were 80%higher than P. purpurea over the temperature range 5–20 ^°C, at both sub-saturating andsaturating irradiances (37 and 289 μmol photonm^-2 s^-1). Porphyra umbilicalis was more efficient at low irradiances (higher α) and had a higher P_max (23.0 vs 15.6 μmolO₂ g^-1 DW min^-1) than P.purpurea, suggesting that P. umbilicalis is a better choice for mass culture, where self-shading maybe severe. This revised version was published online in August 2006 with corrections to the Cover Date.     

Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales,Chlorophyta)

Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 10⁵ cells ml⁻¹ before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.