Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Isolation and culture of suspension-derived protoplasts ofBeta vulgaris L.

Sugar beet protoplasts (Beta vulgaris L.) were isolated from hypocotyl-derived suspension cells and cultured on modified Murashige and Skoog medium supplemented with 5 μM naphthaleneacetic acid (NAA) and 2 μM 6-benzyl-aminopurine (BAP). Protoplasts were plated at a density 1.0–1.5×10⁵ cm⁻³ and incubated in either liquid medium or in medium solidified by 1.2% agarose, at 25°C in the dark. Comparison of two methods of culture unequivocally showed the second to be superior. Immobilizing the protoplast in agarose proved to be essential for obtaining sustained protoplast division and reproducible colony formation. The plating efficiency after two weeks of culture, expressed as the percentage of protoplasts which developed to form colonies, reached 40%. Subsequent subcultures of protoplast-derived callus to regeneration media with different concentrations of BAP (5 μM, 10 μM, 20 μM, 30 μM) resulted in very good callus proliferation at the three lowest concentrations, although organogenesis was not achieved.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 10⁶ g^-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.     

Protoplasts to plants of Gentianaceae. Regeneration of lisianthus (Eustoma grandiflorum) is affected by calcium ion preconditioning,osmolality and pH of the culture media

A reliable method has been developed for regeneration of whole plants from isolated protoplasts of five cultivars of lisianthus,Eustoma grandiflorum (Griseb.) Schinners (Gentianaceae). Protoplasts were isolated from either cotyledons or leaves and cultured in agarose beads surrounded by liquid V-KM media containing 5.37 µM 1-naphthyleneacetic acid (NAA) and 2.28 µM zeatin. When microcalli were approximately 1 mm in diameter, the agarose beads were transferred to shoot regeneration media containing 0.1 µM indolebutyric acid (IBA) and 4.44 µM 6-benzylaminopurine (BAP). Shoots were produced from the calli during several sub-culture periods. Protoplast viability and the subsequent regeneration of plants were dependent on calcium levels and growth regulator presence in thein vitro seed germination media, on the osmolality of the protoplast purification solution, and osmolality increase and pH of the culture media. Shoots were rooted in Murashige & Skoog (1962) media containing 5.71 µM indole-3-acetic acid (IAA). Plantlets derived from protoplasts of five lisianthus cultivars (Fresh White, Hakusen, Miss Lilac, Fresh Purple and Doremi Wine Red) have been successfully transferred to the glasshouse.     

Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

High Frequency Divisions in Leaf Base Protoplasts of Wheat (Triticum aestivum L.)

Protoplasts were isolated from the basal meristematic region of leaves from 6-day-old seedlings of wheat (Triticum aestivum). Protoplasts divided when cultured on MS medium (as agarose beads) in presence of nurse tissue. Donor seedlings when grown on BAP-supplemented MS medium were found to be considerably superior for protoplast isolation and culture than when grown on MS basal medium, in terms of protoplast viability, cell wall formation and cell division frequency. In addition, reduction of ammonium content of the culture medium, together with a dark Incubation, led to a high protoplast division frequency of about 70%. Microcolonies of 10-to 12-celled stages were obtained in Triticum aestivum, varieties HD 2329, HD 2285, Kalyan Sona, Arjun and CPAN 1676.     

Studies of protoplast culture types and plant regeneration from callusderived protoplasts of Asparagus officinalis L. cv. Lucullus 234

Plating efficiency and colony formation of callus-derived protoplasts of Asparagus officinalis L. cv. Lucullus 234 differed significantly with different protoplast culture media and types of culture. Osmotic conditions and hormone concentrations of liquid media produced the greatest influence on plating efficiency and colony formation in bead culture. Protoplasts grew best in bead culture with a solid modified Kao & Michayluk protoplast culture medium (KM) supplemented with 0.5 mg l^–1 -naphthaleneacetic acid (NAA), 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l^–1 kinetin, and 0.6% agarose (KM6) and a liquid modified KM medium differing from KM6 medium in sugar content, having 0.18 M sucrose and 0.18 M mannitol (A8). An average plating efficiency of 19.1% and colony formation of 15.5% was obtained one week after isolation in bead culture with the KM6 and A8 media. The highest average shoot regeneration of 92.3% was obtained with a Murashige & Skoog medium (MS) containing 0.125 mg l^–1 NAA, 0.125 mg l^–1 2,4-D, 0.25 mg l^–1 6-benzylaminopurine (6-BAP) and 3% sucrose. Plants have been regenerated and transferred to the greenhouse.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Plant regeneration fromBrassicanigra (L.) Koch stem protoplasts

Summary Protoplasts ofBrassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 mg/liter glutathione, 50 mg/literl-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH, 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 mg/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/liter 6-(γ,γ-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. THis is the first report of plant regeneration fromB. nigra protoplasts.     

Cytoplasmic-nuclear interaction and medium effect for protoplast culture in sunflower

Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with a density of 2×10⁴ protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from 0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions. Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from protoplast cultures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan. and P. cincinnata Mast.

Summary Protoplasts isolated from seedling cotyledons of yellow passionfruit (Passiflora edulis var. flavicarpa Deg.) and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., divided in culture and produced calli. Shoot regeneration was obtained in MS medium (Murashige and Skoog 1962) containing 2.0 mg/l 6-benzylaminopurine (BAP). Regenerated plants produced roots in half-strength hormone-free MS medium and could be transferred to soil after being acclimatized.     

Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum

Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 10³ per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H² 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 10⁴ cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Plant regeneration from protoplasts of wild barley (Hordeum murinum L.)

Summary We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - L1, L2 medium according to Lazzeri et al. 1991 - L3 medium medium according to Jähne et al. 1991a     

A Simple Protocol for Regenerating Mesophyll Protoplasts of Vegetable Brassicas

We report here a simple protocol for regenerating plants from leaf protoplasts of vegetable Brassicas, viz., cabbage, cauliflower and broccoli. Protoplasts from in vitro grown leaf material were cultured in Kao’s medium with a supplementation of 2,4-D, NAA, BAP and glucose, initially in dark for 3d and subsequently in light. Dilution of protoplast cultures was effected on the 7th, 10th and 13th day of culture initiation with Kao’s medium supplemented with sucrose, and reduced 2,4-13 content; NAA was omitted. Micro-colonies were plated on a K3 medium having 2,4-D, BAP and sucrose gelled with agarose. Transfer of calli to another K3 medium with zeatin regenerated shoots from cauliflower protoplast derived calli, whereas a medium with kinetin and zeatin supported shoot regeneration in cabbage and broccoli. Shoot regeneration occurred within 6-6 weeks of culture initiation. Shoots were easily rooted on MS medium without growth regulators.     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae)

Summary Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization.