Change in light quality due to a blue-green pigment, marennine, released in oyster-ponds: effect on growth and photosynthesis in two diatoms, Haslea ostrearia and Skeletonema costatum

Two prominent diatoms encountered in oyster-ponds,Haslea ostrearia and Skeletonema costatum,were grown in batch and in a semi-continuous modeunder light of different spectral quality, white, blueor blue-green. The last corresponded to white lightmodified by a water-soluble pigment, marennine,produced by H. ostrearia. After acclimation tothe different light treatments, the growth rates ofboth species showed little variation with respect tolight quality. The parameters for photosynthesisvs irradiance curves were very similar in H. ostrearia grown under the three light conditions,whereas S. costatum the maximum photosyntheticcapacity (on a chlorophyll a basis) wassignificantly reduced under blue-green light. Fluorescence analyses confirmed the data forphotosynthesis, with the operational fluorescenceyield decreasing faster with increasing irradiance inS. costatum grown under blue-green light. InH. ostrearia, fluorescence yields undersaturating irradiance were closely similar in thethree light conditions. The results are discussed inrelation with the prominent development of H.ostrearia that can outcompete other diatoms inoyster-ponds.     

Method for the quantification of the blue-green pigment “marennine” synthesized by the marine diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis> (Gaillon/Bory) Simonsen using HPLC gel-filtration and photodiode-array detection

This paper describes a new approach for quantifying marennine, a blue-green pigment synthesized by the marine diatom Haslea ostrearia, which is known to be responsible for the greening of cultured oysters in French coastal areas. The method uses gel-filtration HPLC interfaced with a photodiode-array detector (PDA). Under the chromatographic conditions applied, the peak of marennine is identified on the dextran pattern at 674 nm by its elution time (ET = 21–22 min) and its UV-Visible absorption spectrum. After calibration with pure marennine sample as external standard, consisting in plotting peak area as a function of marennine concentration, one can back-calculate concentrations of unknown samples. Results for quantitative determination of marennine using this procedure are validated and discussed in relation to those obtained by the spectrophotometric methods, the most often employed until now.     

Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

Light effects on in vitro rooting of pear cultivars of different rhizogenic ability

The effect of varying light regimes on in vitro rooting of microcuttings of two pear (Pyrus communis L.) cultivars was investigated. Cultures of the easy to-root Conference and the difficult-to-root Doyenne d'Hiver were incubated for 21 days with or without indole-3-butyric acid (IBA) in the medium in darkness or under continuous far-red (8 µmol m^–2 s^–1), blue, white or red (15 or 36 µmol m^–2 s^–1) light. Conference rooted without IBA when exposed to red, blue or white light while no rooting was observed under far-red light and in darkness. The high rooting efficiency under red and, by contrast, the inhibition under far-red light and darkness suggest the involvement of the phytochrome system in rhizogenesis. The addition of IBA to the culture medium enhanced root production under all light regimes in both cultivars. Red light, especially at the lower photon fluence rate, had a positive effect by increasing root extension (number × length of roots) and stimulating secondary root formation.Abbreviations IBA Indole-3-butyric acid - R red light - B blue light - FR far-red light - W white light - D darkness - P_fr active (far-red light absorbing) form of phytochrome - P_tot total phytochrome - BA benzyl-adenine     

Plantlet regeneration of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> var. adik-adik by tissue culture

Three color morphotypes of Kappaphycus alvarezii var. adik-adik (brown, green and red) collected from a farming area in Tictauan Is., Zamboanga City, Philippines were used as explants in the study in order to micropropagate ‘new’ plants. Individual sections of sterile Kappaphycus alvarezii var. adik-adik, initially cultured in a 48-well culture plate containing ESS/2 + E3 + PGR, released callus cells after 4–5 days of incubation at 23–25°C, 13:11H LD cycle and 10–15 μmol photons m⁻² s⁻¹ light intensity. True calli were formed after 29–35 days following dense formation of filaments or undifferentiated round cells at the medullary and inner cortical layers of the section. Plantlets (2–3 mm long) of Kappaphycus alvarezii var. adik-adik were able to regenerate after 98, 150 and 177 days in-vitro among the reds, greens, and browns, respectively. This study established successful methods for the production and regeneration of tissue explants of Kappaphycus alvarezii var. adik-adik which can possibly be used to mass produce ‘new’ cultivars for land- and sea-based nurseries as sources for commercial farming. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

A new blue-pigmented hasleoid diatom,Haslea provincialis,from the Mediterranean Sea

Haslea provincialis Gastineau, Hansen & Mouget, sp. nov., is a new, morphologically semicryptic blue diatom discovered on the French shores of the Mediterranean Sea. Like H. ostrearia and H. karadagensis, H. provincialis shares the capacity to synthesize a marennine-like blue pigment. Sexual reproduction between clones of H. provincialis has been repeatedly observed and resulted in viable initial cells. There were no sexual interactions with sexually competent clones of H. ostrearia or H. karadagensis, as would be expected for a separate biological species. There are strong similarities between the H. provincialis pigment and the marennine produced by H. ostrearia, evidenced by UV-visible spectrophotometry and Raman spectrometry. However, unlike the marennine from H. ostrearia, no differences were found between the extracellular and the intracellular forms of the pigment in H. provincialis. This indicates that the synthesis pathways and excretion mechanisms among the three ‘blue’ Haslea may be species-specific. Molecular taxonomy and phylogeny (based on rbcL, cox1 and SSU V4 DNA sequences) confirmed the distinct position of this species among the blue Haslea species. Haslea provincialis occurs in environments from which H. ostrearia has already been reported (mostly based on the presence of the blue cell vacuoles). Possible species misidentifications and the impact of the complex geological history of the Mediterranean Sea on blue diatom diversification are also discussed.     

Haslea karadagensis (Bacillariophyta): a second blue diatom,recorded from the Black Sea and producing a novel blue pigment

A new species of raphid pennate diatom producing a blue pigment, Haslea karadagensis Davidovich, Gastineau & Mouget, sp. nov., was recently isolated from the Crimean coast of the Black Sea (Ukraine). This organism is very similar to the well-known Haslea ostrearia, the first described ‘blue’ diatom, which produces marennine, the pigment involved in the greening of oysters. The Ukrainian diatom, H. karadagensis, differs slightly from H. ostrearia in the structure of its frustule, and the two organisms are unable to interbreed. Two molecular markers, rbcL and the ITS1–5.8S–ITS2 sequences, showed 2% and >50% differences, respectively, between the two species. UV-visible spectrophotometry and in vivo confocal micro-Raman spectroscopy were used to compare the pigment of H. karadagensis with marennine. Both pigments showed absorption bands in the UV and red regions, but the positions of the maxima differ between the pigments. Significant differences were observed by micro-Raman spectroscopy in the 1000–1700?cm^?1 wavenumber range, revealing that the pigments are different molecules. Haslea karadagensis is the first example of a new ‘blue’ diatom and produces a novel blue pigment.     

Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.     

<Emphasis Type="Italic">Sesbania exaltata</Emphasis> biocontrol with <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> microsclerotia formulated in ‘Pesta’ granules

The weed Sesbania exaltata (Raf.) Rydb. ex A.W. Hill (hemp sesbania) was effectively controlled in soybean [Glycine max (L.) Merr.] field test plots over a 2-year testing period (1995–1996) with microsclerotia of the bioherbicidal fungus, Colletotrichum truncatum, formulated in wheat gluten-kaolin granules called ‘Pesta’. Weed control averaged 84% and 88%, respectively, in plots treated pre-plant incorporated (PPI) at ‘Pesta’ rates of 168 or 336 kg ha⁻¹, and 71% and 78%, respectively, in plots treated pre-emergence (PE) at ‘Pesta’ rates of 168 or 336 kg ha⁻¹ over the testing period. Post-emergence (POE) control averaged 30% and 50%, respectively, for the 168 and 336 kg ha⁻¹ treatments, and was significantly less effective than either PE or PPI treatments. Although pathogenesis and mortality occurred in hemp sesbania tissues, satisfactory weed control was not achieved in plots treated at rates of 17 or 84 kg ha⁻¹ with any of the application methods. Soybean yields were significantly greater in test plots treated PPI or PE, as compared to yields from test plots treated either POE, with inert ‘Pesta’ granules, or from untreated controls. Microsclerotia formulated in ‘Pesta’ granules exhibited excellent shelf-life, retaining high viability after storage for 10 years at 4 °C. These results suggest that microsclerotia of C. truncatum formulated in ‘Pesta’ granules offers an effective method for controlling this important weed and preserving the activity of this bioherbicide.     

Carbon fixation and carbonic anhydrase activity in Haslea ostrearia (Bacillariophyceae) in relation to growth irradiance

The metabolic pathway of primary carbon fixation was studied in a peculiar pennate marine diatom, Haslea ostrearia (Bory) Simonsen, which synthesizes and accumulates a blue pigment known as “marennine”. Cells were cultured in a semi-continuous mode under saturating [350 μmol(photon) m⁻² s⁻¹] or non-saturating [25 μmol(photon) m⁻² s⁻¹] irradiance producing “blue” (BC) and “green” (GC) cells, characterized by high and low marennine accumulation, respectively. Growth, pigment contents (chlorophyll a and marennine), ¹⁴C accumulation in the metabolites, and the carbonic anhydrase (CA) activity of the cells were determined during the exponential growth phase. Growth rate and marennine content were closely linked to irradiance during growth: higher irradiance increased both growth rate and marennine content. On the other hand, the Chl a concentration was lower under saturating irradiance. The distribution between the Calvin-Benson (C₃) and β-carboxylation (C₄) pathways was very different depending on the irradiance during growth. Metabolites of the C₃ cycle contained about 70 % of the total fixed radioactivity after 60 s of incorporation into cells cultured under the non-saturating irradiance (GC), but only 47 % under saturating irradiance (BC). At the same time, carbon fixation by β-carboxylation was 24 % in GC versus about 41 % in BC, becoming equal to that in the C₃ fixation pathway in the latter. Internal CA activity remained constant, but the periplasmic CA activity was higher under low than high irradiance.     

Effects of daylength,irradiance and settlement density on the growth and reproduction of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> gametophytes

The effects of daylength, irradiance and spore settlement density on the growth, maturation and sporophyte production of Undaria pinnatifida (Harvey) Suringar gametophytes were examined using a factorial experimental design in culture. The growth of Undaria gametophytes increased with increasing daylength (8, 12 and 16 h), but the maximum fertility occurred at a daylength of 12 h followed by 8 and 16 h. Gametophytes grew better at the 16 h daylength under the same mean daily irradiance (MDI) of 20 μmol photons m⁻² s⁻¹. However, the fertility was higher at the short daylength (8 h), indicating that the maturation of U. pinnatifida gametophytes is influenced by daylength rather than by the MDI. Vegetative growth and sporophyte production of gametophytes were better at 60 μmol photons m⁻² s⁻¹ than at 30 μmol photons m⁻² s⁻¹ under a 8:16 h LD (Light: Dark) cycle, and their growth and maturation were density-dependant in 16 and 12 h daylength, respectively. These results suggest that the U. pinnatifida gametophytes require a certain amount of light for the growth and reproduction, and intraspecific competition occurred under the optimal growth and maturation conditions. However, the sporophyte recruits per unit has been enhanced with increasing spore settlement density at 8 and 12 h daylengths indicating that high settlement density gives a benefit for maintaining population, even though the sporophyte production of each female plant is inhibited. In conclusion, the vegetative growth, reproduction and sporophyte production of U. pinnatifida gametophytes are retarded at a low irradiance above growth saturation and a high settlement density, and are determined by daylength.     

Growth Promotion of Vegetative Gametophytes of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> by Blue Light

Through an acclimation period of 10 days, compared to white light, the maximal net photosynthetic rates were significantly higher for gametophytes of Undaria pinnatifida cultivated under blue light (400–500 nm), and were lower under red light (600–700 nm). Chlorophyll c and the carotenoid content of gametophytes were similar under blue light and red light but were much lower under white light. The growth rate of female gametophytes under blue light was higher than that under other lights, and the growth rate of male gametophytes showed little variation with respect to blue and white light. Male and female gametophytes were mixed together to form sporophytes under white, blue and red light. After approximately 5 days, 50% gametophytes became fertile under blue and white light, but remained vegetative under red light after 10 days.     

A microcomputer method for continuous measurement,recording and control of ion activity during nitrate uptake byLolium perenne

A microprocessor controlled apparatus is described which can measure, control and record nitrate uptake byLolium perenne in nutrient solution, comparing seven selection lines in duplicate. Nutrient solution flowed at 1 min⁻¹, and linear response was found from 10⁻¹ to 10⁻⁴ M NO ₃ ⁻ . Uptake rates for Lolium were between 10⁻⁵ and 10⁻⁴ M NO ₃ ⁻ , plant⁻¹, h⁻¹, which agreed with previous, manually determined, rates, ‘Overshoot’ in nitrate dosing, which was a problem with manual systems, was eliminated. Nitrate concentration was controlled (±3%) in modified Hoagland’s solution.     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

Response of antioxidant activity to excess copper in two cultivars of <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino

Changes in ascorbic acid content and antioxidant enzyme activities were investigated in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) leaves of ‘Wutacai’ and ‘Erqing’ exposed to excess copper (Cu). Cu treatment reduced the fresh weight of shoot and root by 57% and 46% in ‘Wutacai’, and 60 and 54% in ‘Erqing’, respectively. The accumulation of copper in leaves was higher in ‘Wutacai’ than that in ‘Erqing’. Compared to the control, ascorbic acid (AsA) contents were significantly decreased after copper treatment in both cultivars, while they were higher in ‘Wutacai’ than in ‘Erqing’, which may explain the higher copper-tolerance of ‘Wutacai’ with higher copper accumulation. The higher AsA contents of ‘Wutacai’ resulted from their lower activities of degrading enzymes, such as ascorbate oxydase (AAO) and ascorbate peroxidase (APX), as well as the increasing activity of dehydroascorbate reductase (DHAR) after copper treatment compared with ‘Erqing’. Copper stimulated superoxide dismutase (SOD) activity in both cultivars, but for catalase (CAT), there was little difference between both cultivars. Peroxidases (POD) activity was decreased after copper treatment in ‘Erqing’, while in ‘Wutacai’, it was significantly increased at 14 days, and POD activity was higher in ‘Wutacai’ than that in ‘Erqing’ at 21 and 28 days. Therefore, the induced increasing activity of POD in ‘Wutacai’ also played an important role in its copper tolerance.     

Microsatellite diversity in tetraploid <Emphasis Type="Italic">Gossypium</Emphasis> germplasm: assembling a highly informative genotyping set of cotton SSRs

A series of 320 mapped simple sequence repeats (SSRs) have been used to screen the allelic diversity of tetraploid Gossypium species. Fourty-seven genotypes were analyzed representing (i) the wide spectrum of diversity of the cultivated pool and of the primitive landraces of species G. hirsutum (‘marie-galante’, ‘punctatum’, ‘richmondi’, ‘morrilli’, ‘palmeri’, and ‘latifolium’, and ‘yucatanense’), and (ii) species G. barbadense, G. darwinii and G. tomentosum. The polymorphism of 201 SSR loci revealed 1128 allelic variants ranging from 3 to 17 per locus. Neighbor-joining (NJ) method based on genetic dissimilarities produced groupings consistent with the assignments of accessions both at species and at race level. Our data confirmed the proximity of the Galapagos endemic species G. darwinii to species G. barbadense. Within species G. hirsutum, and as compared to the other 6 races, race yucatanense appeared as the most distant from cultivated genotypes. Race yucatanense also exhibited the highest number of unique alleles. The important informative heterogeneity of the 201 SSR loci was exploited to select the most polymorphic ones that were assembled into three series of genome-wide (i.e. each homoeologous AD chromosome pair being equally represented) and mutliplexable (× 3) SSRs. Using one of these ‘genotyping set’, consisting of 39 SSRs (one 3-plex for each of the 13 AD chromosomes pairs) or 45 loci, we were able to assess the relationships between accessions and the topology in the genetic diversity sampled. Such genotyping set of highly informative SSR markers assembled in PCR-multiplex, while increasing genotyping throughput, will be applicable for molecular genetic diversity studies of large germplasm collections. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.