Nurse culture of low numbers of Medicago and Nicotiana protoplasts using calcium alginate beads

Protoplasts of two species, lucerne and tobacco, were cultured in semi-solid droplets of calcium alginate as a means of nurse culturing very low numbers of protoplasts. It was shown that increasing autoclave times decreased the gelling capacity of the alginic acid. A convenient measure of viscosity is described to allow appropriate adjustment of the alginate solution. Tobacco protoplasts are shown to be more sensitive to higher alginate concentrations than lucerne, however beads with a final alginate concentration of approximately 1.5% were suitable for both species. Agitation of the beads in liquid medium was needed for optimum division frequencies. The volume of liquid medium affected the culture response. Interestingly, the local cell density (bead cell density) was shown to be more influential than the total cell density. Nurse beads with higher densities of protoplasts of the same species were visually marked with activated charcoal. Experiments were performed to determine whether nursing was effective with calcium alginate encapsulation and to what extent the cell densities could be lowered. When there were no nurse beads, divisions effectively ceased at 10⁴ per ml with lucerne and 10³ per ml with tobacco. In the presence of nurse beads, protoplasts in the test beads grew at high frequency down to the lowest densities tested, namely 50 per ml for tobacco. With these methods transformed lucerne protoplasts from electroporation experiments and somatic hybrids have been recovered and plants regenerated with much greater efficiency that was hitherto possible.     

In vitro culture of tobacco protoplasts: Use of feeder techniques to support division of cells plated at low densities

Summary Tobacco protoplasts obtained from leaf mesophyll cells and suspended in agar nutrient medium divided and produced colonies only when plated at high densities, above 10⁴ cells per ml. At such densities coalescence of the expanding colonies occurred at high frequency. Nondividing X-irradiated protoplasts used as feeder cells supported division of viable protoplasts plated at densities as low as 10² cells per ml. The feeder cell technique should thus facilitate the application of screening procedures for the isolation of colonies originating from single mutated cells occurring in a suspended population.     

Plant regeneration from long term suspension culture-derived protoplasts of hexaploid wheat (Triticum aestivum L.)

Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×10⁶ protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - PCV packed cell volume - MES morpholinoethanesulfonic acid     

Preparatory studies for the use of plant protoplasts in space research

An experiment using plant protoplasts has been accepted for the IML-1 mission to be flown on a space shuttle in 1991. Preparatory experiments include studies of cell wall formation, cell division, the effect of simulated weightlessness using fast and slow rotating clinostats, and the development and testing of hardware for the IML-1 mission. After 24 h at 25°C, protoplasts isolated from hypocotyls or leaves of rapeseed seedlings, or from carrot suspension cells, show 60, 20 and 15% cell wall formation, respectively. The time course of formation of the cell wall and cell division could be delayed by treatment at low temperatures or immobilization in alginate or agarose. This aspect is of importance in connection with problems of late access to the space shuttle before launch. At 4°C only 18% of the rapeseed hypocotyl protoplasts had formed cell walls after 24 h. Protoplasts immobilised in agarose or alginate gradually regain their cell division capacity and after 72 h the frequencies are 51 and 26%, respectively, compared to non-immobilised control protoplasts. A significant decrease in cell division activity is observed after rotation for 6 h on the slow clinostat. A similar effect is not observed on the fast clinostat. Protoplasts, cultured in the specially designed plant chamber for up to 14 days established cell aggregates which have further developed into plants.     

Plant regeneration from mesophyll protoplasts of Matthiola incana (L.) R. Br.

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21–28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.Abbreviations 2,4-D 2,4-dichlorphenoxyacetic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-banzylaminopurine - IPA isopentenyladenine - IPAR isopentenyladenosine - MES (2-[N-morpholino]) ethanesulfonic acid     

A simple,versatile feeder layer system for Brassica oleracea protoplast culture

Protoplasts from cauliflower (Brassica oleracea ssp. botrytis) and broccoli (ssp. italica) leaves and hypocotyls were successfully cultured on membrane filters over a feeder layer of cells from a B. campestris suspension culture. Cells from rice, tomato and tobacco suspensions were not as effective as the B. campestris cells. Plants were recovered from protoplasts of previously recalcitrant Brassica genotypes. Protoplasts cultured in low numbers (10–100) on the feeder layer divided and formed colonies capable of plant regeneration, as did fused protoplasts.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PCV packed cell volume     

Fertile Indica rice plants regenerated from protoplasts isolated from microspore derived cell suspensions

Rice plants (Oryza sativa L., Chinsurah Boro II var. Indica) were regenerated from protoplasts isolated from microspore derived cell suspensions. A simple procedure for the establishment of such cell suspension cultures from embryogenic microcallus derived from cultured isolated microspores of Indica-type rice is described. Regenerating protoplasts could readily be isolated from 5–12 months old cell suspensions showing visible colony formation in the range of 180–1050 colonies/10⁶ protoplasts after about one month in culture. More than 100 independent green plantlets were regenerated via secondary embryogenesis from ca 20×10⁶ protoplasts. Out of 32 plants grown to maturity under greenhouse conditions 24 were fertile.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - NAA naphthaleneacetic acid     

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.     

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl₂ solution, and the alginate/cell suspension is gelled on contact with the CaCl₂ to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…     

Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet)

Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to <5% (<4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.     

Plant Regeneration from Protoplasts of Peucedanum Terebinthaceum (Fisch.) Fisch. ex Turcz

Calll with many embryogenic cell colonies were produced from segments of seedlling of Peucedanum terebinthaceum (Fisch.) Fisch. ex Turcz. which were cultured on the 1/2MS agar medium (with half quantity of macronutrients) containing 1 mg/l 2,4-D. Cell suspension culture with high percentage of embryogenic cell colonies was established from the calli shaking in liquid medium. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CeCl2, 1 mmol/l KH2PO4, 0.6 mol/l mannital at pH 5.8 and 25℃. Cultured in a modified MS liquid medium containing 1 mg/l 2,4-D+ 0.5 mg/l zeatin, the protoplasts emered division after four days, and formed cell colonies of 0.5–1mm after about forty days. When transfered to 1/2 MS liquid medium supplemented with zeatin (0.5 mg/l), the cell colonies differentiated in to embryoids, then developed into plantlets with many green leaves and roots on the 1/2 MS agar medium devoid of phytohormones.     

Hybridoma perfusion systems: a comparison study

The efficiency of lgM production by hybridoma cells (1) cultured in suspension; (2) entrapped in alginate beads; or (3) packed in hollowfiber cartridge bioreactors, were compared in long-term perfusion cultures. The results showed that steady-state cell concentration and antibody production, per liter of perfused medium per day, were similar when cells were either entrapped in alginate beads of maintained in suspension. These values were also similar whether cells were maintained at high density in a hollowfiber cartridge bioreactro, or at low density in suspension. This work points out that cell behavior and antibody yield are comparable overall in the various perfusion systems currently used. However, a significant reduction of antibody production appeared whenever a part of the viable cells was lost in the filtrate. The reduction was due both to a decrease of viable cell yield and a decline of lgM productivity on a percell basis. This result is well in agreement with the previously presented model of "grow or die" cell cycle system of hybridoma, which proposes that the ratio of arrested to proliferating cells in perfusion cultures, should be increased in proportion to cell retention in the bioreactor, with a concomitant increase of lgM productivity.     

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5–10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.Abbreviations B5 medium according to Gamborg et al. (1968) - SH-x medium according to Schenk and Hildebrandt (1972) supplemented with x M dicamba - dicamba 3,6-dichloro-o-anisic acid - KM-8p medium 8p of Kao and Michayluk (1975)     

Fertile Plants Regenerated from Protoplasts of Cold Tolerant Rice Line RCPL 1-2C

We report plant regeneration from suspension protoplasts of an advance breeding line suitable for high altitude areas of NorthEastern Hill of India. Protoplasts were cultured in liquid and low density agarose in presence as well as absence of nurse. Higher frequency of division and plant regeneration were obtained from the protoplasts cultured in presence of nurse. In contrast, only some colonies were recovered from the protoplasts cultured in absence of nurse. Frequency of plant regeneration from these calli was also very low. Use of nurse was therefore, critical in obtaining sustained division in the protoplasts. So far, 115 plants have been regenerated from ten independent experiments and twelve of them have tlowerd and set seeds.     

Regeneration from leaf protoplasts of Arabidopsis thaliana ecotype estland

Protoplasts (2 x 10(7)/g fresh wt) were isolated from leaves of A. thaliana ecotype estland, with a viability of more than 90%. Protoplasts cultured in calcium alginate beads or layers showed division while culture in liquid or agarose beads failed to elicit any division. Effect of culture density showed highest frequency of division occurring at 5 x 10(5) while no division was seen when cultured at a density of 5 x 10(4). Culture in MS medium resulted in higher division frequency and better sustenance of microcolonies as compared to B5 medium. Under optimized conditions, macrocolonies were formed at a frequency of 1.8%. Shoot regeneration was seen in 50% of microcalli transferred to shoot induction medium for regeneration. Shoots were rooted and plantlets transferred to pots. The plants produced flowers and were fertile.     

Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.     

Plants Regenerated from Mesophyll Protoplasts of Cottonwood New Clone

Poplar NL-80106 (Populus deltoides×P, simonii) mesophyll protoplasts were isolated from leaves of 30 days-old sterile shoot, with 4 × 107/g fr. wt of protoplast yield after purification. The protoplasts were cultured in KM8p and MS liquid media containing 2 mg/L 2, 4-D, 0. 5 mg/L NAA and 0.5 mg/L KT. Higher plating density and lower osmatic pressure (0.45 mol/L) were proved to be favourable to division of protoplast-derived cells. The first division initiated 5 days after culture, and the division frequency reached 4.5 % on the 10th day. A number'of cell colonies and microcalli was formed in 12 weeks. Using organic nitrates and glucose in protoplast culture medium was beneficial to increase division frequency and plating efficiency. The calli were allowed to grow to 4--6 mm in height with red colour and compact structure on the gelrite-sohdified NLZ1 proliferation medium in 3 weeks and were transferred onto NLF differentiation medium where the frequency of shoot formation could reach 100%. The 3 cm high shoots were then cut off from the callus and rooted on 1/2 MS medium.     

Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.