Protoplast fusion and the cell cycle

Protoplasts isolated from rapidly dividing cell suspension cultures of either Nicotiana sylvestris or tumor derived cultures of Crepis capillaris were fused by PEG or liposome treatments to form homokaryons. Analysis of binucleates by Feulgen microspectrophotometry, and autoradiography, has revealed that whereas fusion products of all cell cycle combinations occur, protoplasts of certain cycle phases participate in fusions more frequently than expected, and there is a slight predominance of like-with-like cycle combinations. It is argued that this tendency towards specificity of fusion may be explained by cycle related variation in surface charge on protoplasts, and the mechanisms of action of the fusogens used.Abbreviations PEG polyethylene glycol - CAPT tumorous cell culture of Crepis capillaris - NS-1 cell culture of Nicotiana sylvestris     

Cytological changes associated with alkaloid production in cultured cells of Coptis japonica and Thalictrum minus

Cell structures were compared between alkaloid-producing and non-producing cell cultures of Coptis japonica and Thalictrum minus by electron microscopic observation. In alkaloid-producing cells of C. japonica, prior to the onset of alkaloid synthesis, the vacuoles showed a greater volume than in non-producing cells. These were characterized by a number of large starch grains in the cytoplasm. Furthermore, alkaloid-producing cells contained stacks of rough endoplasmic reticulum. Comparison of different cell lines suggested that there might be a negative correlation between accumulation of alkaloids and starch. Similar cytological differences were observed with T. minus cell cultures that release berberine into the culture medium. Alkaloid producing cells were found to contain an abundance of cytoplasmic vesicles (0.5 – 1 m in diameter).     

A new bioassay system for screening high berberine-producing cell colonies of Thalictrum minus

A new method of estimating the amount of berberine released from minute cell colonies of Thalictrum minus has been devised to facilitate the selection of high berberine-producing cell lines. In this system, cell aggregates obtained from a cell suspension culture are grown on small pieces of an agar culture medium and the concentration of berberine which has been released from the cells into the agar piece is assayed by the antibacterial activity against Bacillus cereus MT2026. Screening of 1000 cell colonies by the agar piece method has resulted in the isolation of four, high berberine-producing cell lines, although they have been found to be more or less unstable with respect to the biosynthetic capability during successive subcultures.     

Effects of “skeleton” photoperiods and high frequency light-dark cycles on the rhythm of cell division in synchronized cultures of Euglena

Summary Two further lines of evidence support the contention (Edmunds, 1966) that the cell cycle in autotrophically grown Euglena can be coupled to an endogenous, circadian biological clock under certain conditions. So-called skeleton photoperiods (LD: 3,6,3:12 and LD: 4,4,4:12) following a complete photoperiod regime entrain the cell division rhythm in the population to a precise 24 hr period, although the step-sizes of the successive fission bursts are always less than 2.00, indicating that not all cells divide in any one 24 hr interval. These findings imply that the continuous action of light is not required for synchronization and suggest that the putative oscillation underlying the rhythm can be phased by discrete light (or dark) pulses or signals.The effects of high frequency LD cycles whose periods were integral submultiples of 24 hr were also investigated. In most regimes (LD:1/4,1/2; LD:1/2,1; LD: 1,2; LD: 1,3; LD: 2,4; LD: 2,6; LD: 4,4) synchronous cell division iccurred in the culture with an average period of 26–27 hr, although only a fraction of the cells divided during any one burst. Similar results were obtained if (i) a synchronized culture was exposed to certain high frequency cycles whose periods were not integral submultiples of 24 hr (e.g., LD: 5,5 or LD: 8,8); (ii) an asynchronous culture (grown in LL) was subsequently exposed to a high frequency cycle; or (iii) a synchronized culture was subjected to a random LD cycle. The synchrony does not break down as long as the given LD regime is imposed and shows some indications of persistence in certain ensuing conditions of continuous illumination.A general formula was derived which predicts the time of division, t _D , for an individual cell: t _D =k+n, where k is the initial phase delay, n is an integer, and is the free-running period of the rhythm observed in the population. These results are interpreted as indicating that the high frequency cycles employed were unable to entrain the circadian oscillation(s) hypothesized to underly and gate cell division, with the result that the rhythm reverted to its free-running period. Exposure to such cycles, however, apparently either initiates a rhythm or synchronizes the phases of the individual oscillations in the populations of cells. The possible direct interaction between energy supply and the observed somewhat variable period lengths is discussed; also, the relevance of stochastic models for the decay of division synchrony in the absence of a recurrent synchronizing procedure is considered.Some of these results were initially reported at the 5th International Congress on Photobiology, Hanover, N.H., U.S.A., August 26–31, 1968.This work was supported by NSF research grants #GB-4140 and #GB-6892 to L. Edmunds.     

Transient expression of the β-glucuronidase gene in embryogenic callus of Picea mariana following microprojection

A microprojection protocol using the DuPont Biolistic particle delivery system and the -glucuronidase (GUS) reporter gene fused with the 35S promoter of Cauliflower mosaic virus (CaMV) was developed for Picea mariana callus. Comparison of four tungsten microprojectile sizes showed the highest transient gene expression with 1.11m diameter particles. Adsorption of DNA on the microcarriers using calcium chloride led to higher GUS gene activity than using polyethylene glycol. GUS gene activity in P. mariana was the highest when cells were treated 5 and 6 days after subculturing to fresh media. The wheat ABA-inducible Em gene promoter yielded 4.5 times higher GUS gene activity than the 35S CaMV promoter. Comparison of transient GUS gene expression among 10 P. mariana embryogenic cell lines from six different open-pollinated families showed comparable gene activity, with the exception of one family showing no GUS gene activity.     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Production of virus resistant and insect tolerant transgenic tobacco plants

The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis -endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r₀ and R₁ tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.Abbreviations CMV cucumber mosaic virus - Bt toxin Bacillus thuringiensis -endotoxin - CaMV cauliflower mosaic virus - NOS nopaline synthase - Kan Kanamycin - Spe spectinomycin - Carb Carbenicillin     

A study of the influence of the growth media on the fatty acid composition inCandida lipolytica diddens and lodder

Candida lipolytica YB 423-12 is able to incorporate fatty acid from the culture medium when lipids are used as carbon substrate. The composition of cell lipids is largely dependent on that of the culture medium. An important 9 desaturase activity acts on incorporated palmitic and stearic acids; and 11-eicosenoic and erucic acids are shortened to oleic acid.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Cellulase and beta-glucosidase production by mexied culture of trichoderma reesei rut C30 and aspergillus phoenicis

Summary When lignocellulosic materials are hydrolysed using cellulase fromTrichoderma reesei, cellobiose accumulates due to a deficiency in -glucosidase activity of the enzyme complex. Cellobiose decreases the rate and extent of hydrolysis through feedback inhibition of exo -1,4-glucanase. Cellulase produced through mixed culture ofTrichoderma andAspergillus showed increased -glucosidase activity and greatly improved hydrolytic potential.     

Isolation of Robillarda sp. producing high β-1,4-glucan glucanohydrolase

Summary A fungus, Robillarda, sp. Y-20, which produces mainly, -1,4-glucan glucanohydrolase (Cx-enzyme) in the culture filtrate, was isolated from soil. The specific activity of the Cx-enzyme was up to 4.9 times higher than that of Trichoderma reesei QM 9414.     

Accumulation of silver by Chromatium vinosum from solutions containing silver thiosulfate

The photosynthetic sulfur bacterium, Chromatium vinosum, was cultured in inorganic photographic processing solutions containing silver thiosulfate complex salt (AgNa₃(S₂O₃)₂) under light. It was found that Chromatium was resistant to Ag and accumulated granular silver in the membrane during growth. The amount of Ag accumulated in the cells depended on the initial concentrations of the Ag salt in the culture solution. When the concentration of Ag was 300 mg/l, the bacteria accumulated Ag as high as 30% of the dry cell weight. The size of the granules was 0.1 to 0.3 m. Results from X-ray microanalysis indicated that these granules consisted mostly of Ag^o with small fractions of Ag₂S and AgCl.     

Diacetyl production by co-immobilized citrate-positiveLactococcus lactis subsp.lactis 3022 and homogenized bovin liver in alginate fibers with double gel layers

Summary Diacetyl production by (Citr⁺)Lactococcus lactis subsp.lactis 3022 cells immobilized in Ca-alginate fine fibers with single layer in the presence of catalase was three times higher than that in the absence of catalase. A co-immobilized culture system of the lactic acid bacterial cells (outer) and the homogenized bovine liver (inner layer) in Ca-alginate fibers with double gel layers was developed. The culture system gave high diacetyl productivity (30 mg/l) for ten repeated batch cultures.     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata Crandon, NC-5339 and P. nigra Betulifolia × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula Erecta) and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - NC-XXXX North Central Forest Experiment Station accession number assigned to hybrid poplar clones.     

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes

Hairy root cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of in vitro plant tissue culture for the production of valuable plant secondary products is identified and confirmed.     

Improved efficiency of plant regeneration from protoplasts of eggplant Solanum melongena L.

Eggplant (Solanum melongena L.) mesophyll protoplasts were obtained from in vitro growing plants of line 410 and cv. Classic. Relatively high (15%) plating efficiency was achieved using petri dishes with alternate quadrants containing reservoir medium (R medium + 1% activated charcoal) and culture medium. Shoot regeneration occurred within 6 weeks following initiation of protoplast culture.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan Israel, No. 1164-E, 1984 Series.