Conditions for a high plating efficiency of free cell suspensions of Haplopappus gracilis (Nutt) gray

Summary Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 m. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency.     

Haplopappus gracilis cell strains resistant to pyrimidine analogues

Summary Strains of Haplopappus gracilis (Nutt.) Gray cells resistant to 6-azauracil have been isolated from cultures of diploid cells. These strains are also resistant to 8-azaguanine, as is their parent. The variants are 100- to 125-fold more resistant to 6-azauracil than their parent, and they exhibit different spectra of cross resistance to other pyrimidine analogues. The phenotype of each variant is stable in the absence of selection. The majority of cells in cultures of the variants are diploid; all others examined were tetraploid. Initial rates of uptake of uracil are not reduced in the variants. Fluorouracil, to which two variants are resistant, is taken up by one of them as well as by the parent. Responses of the other two to fluorouracil are not correlated with decreased ability to accumulate this analogue.     

Efficient isolation of non-chimeric tetraploids artificially induced in a stable culture of Haplopappus gracilis

A method for reducing cytochimerism and inducing homogeneous tetraploids in Haplopappus gracilis (2n = 4) was developed in which masses of shoot primordia treated with 0.5 mg/ml of colcemid for 3 days were cut into small meristematic domes. All of the shoot primordia sampled just after the colcemid treatment were cytochimeras that were mixoploids of 2x, 4x and 8x cells. However, when they were allowed to recover in a colcemid-free medium, the frequency of 4x cells spontaneously increased in most of the shoot primordia. Thirty days after the recovery, chimeric masses containing shoot primordia, each of which consisted uniformly of 4x or 2x cells, were observed. In order to obtain a completely homogeneous tetraploid mass, we then cut these primordia into small pieces, each of which had approximately one meristematic dome. Subsequent to this homogeneous tetraploid masses were easily obtained. Tetraploid shoot primordia could propagate with chromosomal stability over a year, and plants regenerated from these tetraploid shoot primordia were also completely tetraploid. These results show that non-chrimeric masses can be easily isolated from artificially induced cytochimeras using masses of shoot primordia as material.     

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum

Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - TLC thin layer chromatography     

Cytogenetic analysis of plants regenerated from colchicine-treated callus cultures of an interspecificHordeum hybrid

Summary Hybrids of Hordeum vulgare (HV) x H. jubatum (HJ) were synthesized for purposes of introgressive breeding, but were sterile and the recovery of pure diploid tillers by colchicine applications in vivo was difficult. Plant regeneration from colchicine-treated callus cultures of the hybrid (HV x HJ) was investigated as a means to produce high numbers of pure diploid, fertile intermediates. 10 of 50 plants regenerated in this manner exhibited variable chromosome numbers with means of approximately 37 (expected diploid number = 42). Cytological examinations of microsporogenesis in all such plants revealed a high incidence of bivalent formation at metaphase I (as compared to nearly complete asynapsis in the F₁), but spindle and chromosome abnormalities in later meiotic stages led to complete sterility. Approximately 40% of HJ plants regenerated from colchicine-treated calli appeared to be pure tetraploids of high fertility. These techniques are hence useful for high frequency production of diploid or polyploid plants.     

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation decreased with an increasing concentration of 2,4-D up to 10 mg l⁻¹, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses using half-strength SH medium supplemented with 0.1 mg l⁻¹ of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to mass propagation and conservation of this species.     

Optimization of growth and jaceosidin production in callus and cell suspension cultures of Saussurea medusa

Calli cultures derived from the leaves of Saussurea medusa were selected on the basis of colour into three callus, A, B and C, which suggested different levels of metabolite accumulation. An improved reversed phase high performance liquid chromatographic method provided selective determination of the jaceosidin content of these samples. The jaceosidin concentration in callus B was higher than that of the callus A and C. By using 12-day old culture and 9-day old inoculum, jaceosidin yield of 72.91 mg l^–1was obtained from cell line B in cell suspension cultures. The influence of some factors affecting jaceosidin formation, i.e. temperature, light, inoculum size, type of media, phytohormones, nitrogen and carbon source etc. were also examined. Light irradiation and combination of 3% (w/v) sucrose with 1% glucose brought about a marked increase of jaceosidin production. The effect of blue light on jaceosidin was markedly superior to other kinds of monochromatic light (red and far-red) or white light. Analysis of growth and jaceosidin content of callus cultures and cell suspension cultures demonstrated that the production of jaceosidin was growth-dependent in both cell solid culture and cell suspension culture.     

Rearrangement of enzyme patterns in maize callus and suspension cultures

The development of enzyme patterns was followed in the course of: (a) the irreversible cell differentiation via division and expansion to maturity in the root tip and coleoptile of the intact seedlings, (b) the irreversible cell dedifferentation associated with induction and establishment of callus from the growing internodes, and (c) the growth cycle (proliferationstationary phase) in callus and cell-suspension cultures of maize (Zea mays L.). By measuring the activities of glycolytic, mitochondrial, microbody and hydrolytic enzymes cells proliferating in vivo and in vitro could be compared and changes related to cessation or resumption of cell division could be studied.Proliferating cells of callus and suspension cultures maintained by serial culture did not differ from those of the root meristem and coleoptile in the specific activities of hexokinase, phosphoglycerate kinase and phosphopyruvate hydratase. Proliferation in vitro resulted in an enormous increase in the ratio g glutamate-dehydrogenase/cytochrome-oxidase activity and in the level of acid-phosphatase activity, with concomitant drop in galactosidase and xylosidase activity. A 3-5-fold increase of alcohol-dehydrogenase, lactate-dehydrogenase and catalase activities was characteristic of dividing callus cells, while a ca. 100-fold increase in the fructofuranosidase-to-glucosidase activity ratio marked cell proliferation in suspension-cultured cells.Changing enzyme activities after cessation of proliferation were quite similar in root tips and coleoptiles, except those of alcohol dehydrogenase and catalase. The enzyme rearrangement during callus establishment and in the growth cycle of callus cultures was in most cases comparable to that in the intact tissues, while the changes from the dividing to the non-dividing cells in suspension cultures, in contrast, differed widely from those in the intact tissues and callus. Galactosidase and xylosidase were the only activities that showed a similar trend of changes in all the investigated, intact and in-vitro-grown cells.Thus, judged by the pattern of enzyme development, the cell suspension appears to be a unique system, virtually unrelated to the growing cells of the intact tissues. It is also very difficult to draw a definite distinction between the metabolic consequences of cell growth and enzyme modulations in cell suspensions as the cells adapt their metabolism to the environmental changes in liquid medium.     

Programmed cell death in Datura innoxia Mill. callus cultures cultivated in light and in dark

It has been established that dimedone in solid culture medium influencedthe growth of Datura innoxia Mill. callus tissues and theapoptotic processes of cells. This formaldehyde (HCHO) capture reagent appearsto modify the metabolism of plant cells, resulting in quantitative changes inthe apoptotic index (A_i). Apoptotic cells were detected insix-week-old callus tissues by the TUNEL reaction. The amount of TUNEL positivenuclei showed a characteristic spatial distribution. Enhanced DNA fragmentationwas observed in the cell layers close to the surface of the cultures. ElevatedA_i was determined in cultures grown in the dark compared to thetissues grown in the light. High doses of dimedone considerably decreasedapoptosis in tissue cultures under both light and dark conditions.     

Improved plant regeneration from barley callus cultures by increased copper levels

Incorporation of cupric sulfate into callus induction, maintenance, and regeneration media significantly enhanced plant regeneration from callus cultures of barley (Hordeum vulgare L.) immature embryos. Embryos from the cultivars Hector and Excel were cultured on MS medium containing 0, 0.1 (MS level), 0.5, 1.0, 5.0, 10.0, 50.0, or 100.0 M cupric sulfate. Plants were regenerated beginning at 8 weeks and continuing through 36 weeks. For Hector, medium containing 50 M copper regenerated significantly more plants than any other medium, with an average of 17 plants per embryo. In comparison, medium with MS copper levels (0.1 M) regenerated only 5 plants per embryo. For Excel, medium containing 5.0 M copper was the best, regenerating 1.4 plants per embryo. No Excel regenerants were obtained on medium with MS copper levels. Increased copper levels also increased the percentage of embryos that regenerated at least one plant, in both cultivars. The results indicate that MS copper levels are not optimized for barley callus cultures, and that improved plant regeneration can be obtained at higher copper concentrations.Abbreviations MS Murashige & Skoog (1962) - 2,4-d 2,4-dichlorophenoxyacetic acid The US Government's right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged     

First evidence of trans-resveratrol production in cell suspension cultures of cotton (Gossypium hirsutum L.)

For the first time, trans-resveratrol, a stilbene, has been identified in cotton cell suspensions. Cell suspensions of Coker 312, a cultivar which produces embryogenic structures, acccumulate trans-resveratrol contrary to those of cultivar R405-2000, which do not. This stilbene may be a good phenolic marker for induction of somatic embryogenesis in cotton.     

Spectral composition of irradiation regulates the cell growth and flavonoids biosynthesis in callus cultures of Saussurea medusa Maxim

Cell growth, flavonoids biosynthesis and L-phenylalanine ammonia-lyase (PAL) activity were studied in callus cultures of Saussurea medusa Maxim. under different types of spectral radiance. After 21 days, red light significantly improved the callus growth, but inhibited the biosynthesis of flavonoids in callus cultures. However, blue light was found to enhance the biosynthesis of flavonoids, although callus growth under this spectrum was comparable with that under white and other coloured spectra, such as green and yellow. The accumulation of flavonoids in callus cultures was related to the PAL activity, which was found to be stimulated by the spectral composition of irradiation.     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were Corbit (first subculture); GAF/Park and 88Ab3073 (second subculture); and GAF/Park and 87Ab5932 (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. GAF/Park, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes GAF/Park, 88Ab3073, and Tibor. The smaller clusters only regenerated plants for GAF/Park and 88Ab3073. From one gram of callus used to initiate suspensions of GAF/Park and 88Ab3073, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of GAF/Park, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase     

Ploidy dependence of chromosomal variation in callus cultures ofHyoscyamus muticus L.

Summary Remarkable variation for chromosome number was observed in both diploid (2x) and autotetraploid (4x) callus cultures continuously examined for 12 months at monthly subsculture intervals. Initially, the subcultures exhibited predominantly the genomic level of the starting material which subsequently and gradually developed into heterogenous populations of euploid and aneuploid cells imparting the subcultures a aneusomatic status. The comparison of chromosomal instability recorded in 2x vs. 4x callus cultures revealed that with time both types of calli stabilized at a chromosome number around the 4x level. However, the chromosomal examination of the adventitious roots emerging from the disorganized calli revealed the euploid (both 2x and 4x) chromosome levels suggesting the occurrence of amorphogenetic sieve.This paper is dedicated to Professor A. K.Sharma of Calcutta University on his 65th birthday by his former student U. C. L. and grandstudent S. S. (CIMAP Publication no. 739).     

Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures

Summary A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37–80 kb subpopulation, which was predominant in whole plants, to 18–34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5–10 kb significantly increased in suspension culture cells. In addition, a new peak (10–12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4–2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine