Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l^-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l^-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.     

Improvement of maize (Zea mays L.) anther culture responses by algae-derived natural substances

This is the first report on the beneficial effect of microalgal and cyanobacterial biomass on anther cultures of maize (Zea mays L.). Investigations were made on the cytokinin- and auxin-like activity and content of terrestrial and fresh-water living microalgal and cyanobacterial strains. The influence of media supplemented with biomass from four selected strains on the anther induction, the frequency of microspore-derived embryo-like structures, and regeneration capacity in anther cultures of maize was also studied. The addition of cyanobacterial and microalgal biomass to the induction and regeneration media in concentrations of 1 or 2 g/L improved the androgenic response, and was able to reduce the quantity of the synthetic auxin 2,4-dichlorophenoxy-acetic acid (2,4-D) required, or replace it completely.     

Isolation of protoplasts from different Eucalyptus species and preliminary studies on regeneration

Protoplasts were isolated from different Eucalyptus clones and hybrids using mesophyll tissue, calli and cell suspension cultures. The protoplast yields differed greatly according to the starting material and adaptations of the basic procedure had to be designed in specific cases. Eucalyptus protoplasts are representative of recalcitrant woody plant systems since their proliferation is limited in culture. The best results were obtained with protoplasts from cell suspension cultures. A screening of factors increasing proliferation was performed. When some of these factors were combined the cell division frequency was enhanced and microcalli were obtained.Abbreviations B.A.P. Benzylaminopurine - 2-4D 2-4 dichlorophenoxy-acetic acid - F.D.A. Fluorescein diacetate - F.W. Fresh weight - M.E.S. Morpholino ethane sulfonic acid - M.S. Murashige & Skoog (1962) - N.A.A. Naphtalene acetic acid - TRIS Tri (hydroxymethyl amino methane) - V.KM. Medium-Kao and Michayluk medium modified by Vasil (Vasil & Vasil 1980)     

Actual,potential and speculative applications of seaweed cellular biotechnology: some specific comments on Gelidium

Cellular biotechnology is a promising application in the propagation and selection of superior strains of seaweeds. Although axenic cultures, organogenetic tissue cultures, vegetative micro-propagation, callus induction and high yields of agar from calli have been described for several species of Gelidium, a number of basic problems remain to be solved. These include standardized methods for obtaining axenic cultures, identification of requirements for organic nutrients, PGR's, cellular disorganization and reorganization, somaclonal variation and somatic incompatibilities. Future progress in seaweed biotechnology will depend on the resolution of many of these problems.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

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.     

Osmotic microenvironments within tissue culture well plates are an important cause of variability in plant protoplast cultures

Summary To determine if the medium osmolality of plant protoplast cultures in 24-well tissue culture plates changes sufficiently during the culture period to affect development of the protoplasts the osmolality and the division of the protoplasts was monitored within the plates over a three week period. Large increases in osmolality were measured in cell-free experiments indicating that overall evaporation from the plates was substantial. The amount of evaporation from a given culture well depended on the position of the well within the plate; three microenvironments corresponding to the corner, edge, and middle positions of the plate consistently developed. Water placed in the recessed area between each culture well moderated the desiccation of the medium but did not eliminate the formation of microenvironments. The osmolality of the medium in protoplast cultures was higher than in the cell-free experiments but similar trends in terms of plate position were recorded. After 3 weeks of incubation of plates with water added between the wells, the osmolality of the medium in the protoplast cultures had increased 209 mOsm in the corner wells, had increased 77 mOsm in the edge wells, and had decreased 39 mOsm in the middle wells. As a result, there was a three-fold higher incidence of division of the protoplasts in the middle wells than in the corner wells. The non-uniformity of medium osmolality in protoplast cultures within tissue cultures well plates is important in experimental design, in the reproducibility of procedures between different laboratories, and in the preparation of replenishment medium for protoplast cultures to minimize osmotic shock. This work was supported by the College of Agriculture and Life Sciences and the Graduate School, University of Wisconsin-Madison; by McIntire-Stennis project WIS 3082; and by the USDA-Forest Service, North Central Forest Experiment Station (B. Haissig, Project Leader).     

The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway Spruce)

Embryos of Picea abies at various developmental stages were cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (10^?5 M) and N⁶-benzyladenine (BA) (5×10^?6 M). The immature embryos gave rise to a highly friable and embryogenic callus which could be maintained by subculture and contained polarized and organized structures (somatic embryos) consisting of long highly vacuolated cells at one end (suspensor) and a group of small meristematic cells at the other (embryonal end). These structures closely resembled the early stages of normal zygotic embryogeny. Upon further culture these structures formed a bipolar shoot-root axis with an independent and closed vascular system. In many instances either the shoot or the root meristems failed to differentiate. Embryogenic tissues obtained on agar media could be transferred to liquid media and maintained by subculture for at least 6 months. The development of somatic embryos was observed in the liquid cultures also.     

Degradation of 2,4-dichlorophenoxyacetic acid by mixed cultures of bacteria

Summary We explored the feasibility of using mixed cultures for herbicide degradation, with the ultimate aim of application for effluent treatment. The present study reports on mixed cultures which were developed to grow aerobically with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon substrate. Degradation of 2,4-D was verified by HPLC and UV-spectroscopic analysis of the residual 2,4-D concentration in the test cultures. Cultures that were initially developed with 2,4-D also grew readily with glucose, but the degradation of 2,4-D was effectively prevented under mixed substrate conditions. Mamor intermediates or metabolites resulting from 2,4-D degradation were not detected with the HPLC methodology except 2,4-dichlorophenol which appeared to accumulate transiently in the growth medium.     

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml^-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×10⁶ viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×10⁵ protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [¹⁴C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN₃, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.     

Factors promoting sustained divisions of mesophyll protoplasts isolated from dihaploid clones of potato (Solanum tuberosum L.) and a cytological analysis of regenerated plants

A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina).Abbreviations BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid     

Callus formation from protoplasts of cell suspension cultures of Rosa 'Paul's Scarlet'

Sustained divisions of protoplast-derived cells obtained from cell suspension cultures of Rosa‘Paul's Scarlet’ leading to colony and callus formation was achieved. The rather low plating efficiencies observed for agar-plated protoplasts were partly due to early arrests in further development of dividing protoplast-derived cells and cell clusters. Successful cultivation of dividing protoplast-derived cells was particularly dependent upon frequent subculturing of the precultures and on an efficient procedure for viable protoplast isolation. Colony formation was largely independent of medium composition.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Culture of Isolated Single Cells from <Emphasis Type="Italic">Taxus</Emphasis> Suspensions for the Propagation of Superior Cell Populations

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4×10⁵ cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mm), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days⁻¹ was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.     

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid