A comparison of genome modifications leading to genetic and epigenetic tumorous transformation in Nicotiana spp. tissue cultures

A single system is presented, where both genetic and epigenetic control of tumor induction can be studied at the same time. This system is offered by the amphidiploid tumorous hybrid Nicotiana glauca × N. langsdorffii, a nontumorous mutant of it and the nontumorous parent species N. glauca and N. langsdorffii. The aim of the present paper is to compare long-term in vitro cultures of tumorous (genetic and habituated), and nontumorous strains, through the characterization of their genomes according to several physico-chemical parameters. The data reported show that both qualitative and quantitative differences in DNA complexity are correlated with the tumorous transformation. Particularly, a high degree of mismatching between the DNAs of the tumorous and nontumorous hybrids and the lack, in the second genotype (nontumorous), of three DNA peaks in Ag⁺?Cs²SO⁴ analytical ultracentrifugation profile seem to support the hypothesis, suggested in a previous paper, of the presence, in the nontumorous mutant, of a gross chromosomal rearrangement, probably a deletion. Amplification and underreplication of specific sequences also seemed to be correlated with changes from the normal to the tumorous state, highly repetitive sequences being present in higher amounts in the normal strains and in the habituated N. glauca than in the case of the tumorous hybrid. Finally, DNA bound ion contents were found to be strikingly higher in tumorous than in nontumorous tissues. The results are discussed in the frame of the general hypothesis of high somatic genomic plasticity in plants.     

L'Indole et le Tryptophane "régulateurs" de croissance, en culture "in-vitro", pour les tissus de parenchyme médullaire de Tabac, normaux et tumoraux

Indoleacetic acid, tryptophan and indole promote growth of pith tissues of tumorous Nicotiana glauca×N. langsdorffii amphidiploïd hybrid, but only indoleacetic acid gives the potentiality of continuous development to the tumorous tissues after some days of contact on a medium without growth substances. In combination with 6-benzylaminopurine, tryptophan and especially indole are promoting substances like auxin for the non-tumorous pith tissues of tobacco, and allow a considerable organogenesis.     

Interaction of Kinetin and Abscisic Acid in the Avena Straight-Growth Test

Different amounts of abscisic acid, 2.7 × 10^?9? 5 × 10^?8 moles, were chromatographed in isopropanol: ammonia: water (100:14:6), firstly alone and secondly together with 5 × 10^?8 moles kinetin. The same amount of kinetin was also chromatographed alone. The chromatograms were tested biologically with the Avena straight-growth test. Whereas a large part of the chromatograms of kinetin gives growth stimulation, the Rf region 0.4–0.6 of abscisic acid chromatograms is strongly growth-inhibiting. The inhibition within this Rf region does not become less if abscisic acid and kinetin are chromatographed together.     

Dominant lethal assay in female mice after oral dosing with dichlorvos or exposure to atmospheres containing dichlorovos.

The effect of a synthetic auxin-like substance (2,4-D) and a synthetic cell division factor (kinetin) on the induction of chromosome aberrations was studied on tissue cultures of Nicotiana glauca and the tumorous amphidiploid hybrid Nicotiana glauca × Nicotiana langsdorffii.The aberration frequencies in normal Nicotiana glauca tissue were proportional to the length of time of culture in the presence of 2,4-D. Moreover, both 2,4-D and kinetin increased chromosome breakage in the habiatouated Nicotiana glauca tissue but not in the amphidiploid hybrid tissue.The data are discussed in terms of genotype-hormone equilibria in long-term development of plant tissue culture.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of H₂O-soluble and NaOH-hydrolyzable bound forms of indole-3-acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous ¹⁴C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in ¹⁴C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana.     

The Initiation and Maintenance of Vicia faba Tissue Cultures

Explants obtained by removing the radicle tip and the plumule from embryos of Vicia faba have been induced to form callus in culture. Of a range of agar-solidified culture media tested, only that of Schenk and Hildebrandt (1972) was consistently successful. Improved growth, measured as increasing fresh weight was obtained by increasing the nitrogen content of the medium, either as potassium nitrate or as ammonium nitrate. A kinetin concentration of 0.01 mg/1 (5 × 10⁻⁸M) and a 2,4-dichlorophenoxyacetic acid (2,4-D) concentration of 0.5 mg/1 (2.3 × 10⁻⁶M) allowed optimum initial callus growth. A 2,4-D concentration of 2.3 × 10⁻⁸M, while insufficient to induce callus formation was able to inhibit lateral root development which occurred from embryo explants cultured without added 2,4-D. Subcultured tissue grew well on media supplemented with casein hydrolysate or a mixture of the eight most common amino acids in casein hydrolysate. Growth in subcultures was inhibited by two other amino acid mixtures used by other workers for different species.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Tiba inhibition of <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in excised tobacco leaf explants

Summary Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.     

Stimulation of Growth and Nucleic Acid Biosynthesis at Low Concentration of Abscisic Acid in Tissue Culture of Spinacia oleracea

The effect of abscisic acid on growth, ultrastructure and nucleic acid biosynthesis was studied in tissue culture of spinach (Spinacia oleracea L.). Low concentration (0.01 mg l^?1) of abscisic acid increased fresh and dry weight of calluses, whereas 1.0 mg l^?1 was inhibitory. The stimulating effect was observed only in the presence of a relatively high concentration of kinetin (1 mg l^?1). The inhibitory effect was partly overcome by the same kinetin concentration. The low concentration of abscisic acid probably accelerated the induction of callus growth after subculture and stimulated cell division in the exponential phase of growth. Electron microscopy showed the presence of numerous polysomes and rough endoplasmic reticulum in callus cells grown at the stimulating abscisic acid concentration. Control cells and cells at the inhibitory concentration had slightly hyaline cytoplasm and were more vacuolated. Incubation of callus tissue with ³²P in the presence of stimulating concentration of abscisic acid showed a significant increase in the rate of biosynthesis of all nucleic acid classes after 8 h, whereas inhibitory concentration produced a decrease in ³²P incorporation. However, when the tissue was grown in the presence of abscisic acid for 20 days, both concentrations decreased the rate of nucleic acid biosynthesis, as compared to the controls.     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

Rapid Effects of Abscisic Acid on Ion Uptake in Sunflower Roots

Short-term effects of ABA, ABA + kinetin and kinetin on ion (⁸⁶Rb-potassium and phosphate) and water uptake in sunflower plants (Helianthus annuus var. californicus) were examined with a continuous-recording technique. Ion uptake in the roots and transport to the shoots were also investigated by conventional tracer uptake experiments and by sap bleeding experiments with excised roots. After addition of 5 × 10^?6-4 × 10^?5M ABA to the root medium there was an immediate decrease (30–70%) in the rate of ion uptake which lasted 30–70 min. The rate of water uptake was not significantly affected as measured with this method. Ion transport to the shoots and to the bleeding sap of excised roots was decreased by ABA. ABA-induced inhibition of ion uptake was abolished by the presence of kinetin, and uptake was slightly stimulated by 2 × 10^?5M kinetin alone. We suggest that concentration gradients of ABA or rapid changes in the ABA-kinetin balance in the roots affect ion uptake and transport.     

VARIATIONS OF MORPHOGENETIC BEHAVIOR IN PLANT TISSUE CULTURES I. CICHORIUM ENDIVIA

Several-years-old callus tissue derived from mature embryos of endive (Cichorium endivia Linn., Compositae) was grown on synthetic liquid and/or agar nutrient media. Incorporation of yeast extract or high concentrations of inositol, kinetin, casein hydrolysates (pancreatic and acid hydrolysates), etc., improved growth and organ formation. Rosettes of leaves, shoots and roots were differentiated on synthetic media. On agar media shoots arose first and were from marginal meristematic areas, while the roots arose later and were from pockets of meristematic tissue located in the deeper regions of the callus. In liquid media embryoids from single cells were formed which first developed roots and then shoots.     

Effect of synthetic auxins on callus induction from tea stem tissue

A study was initiated to establish an in vitro culture protocol for tea (Camellia sinensis). Explant sources, disinfestation methods and culture media were examined. Segments (divots) were dissected from greenwood stem (current year growth) internodes of field grown plants. Disinfestation was achieved by separate treatments of 3.75% sodium hypochlorite and 7.5% CaCl₂. MS medium with sucrose (30 g/L), inositol (100 mg/L) and thiamine-HCl (1.3 mg/L) and kinetin was used with combinations of the auxins: (2,4-dichlorophenoxy) acetic acid (2,4-D), (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T), (naphthalene) acetic acid (NAA) and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram). Picloram (10^-7M) induced the most callus proliferation without kinetin. At a constant level of kinetin (10^-5M), the concentrations inducing the most callus growth were 10^-7M for 2,4-D, 10^-6M for 2,4,5-T, 10^-7M for Picloram and 10^-8M for NAA. A factorial test of 2,4,5-T and kinetin concentrations showed the optimum for callus growth was 10^-7M and 10^-5M, respectively.Technical Contribution No. 2532 of the South Carolina Agricultural Experiment Station, Clemson University.Graduate Research Assistant and Professor, respectively.     

Changes in Nucleic Acid Metabolism of Excised Barley Leaves During Senescence and the Effect of Kinetin on these Changes

The changes in the amount, rale of synthesis and the nucleotide composition of different RNA fractions in excised barley leaves floated on water or kinetin (10 mg/l) in the dark were examined. In excised leaves floated on water all nucleic acid components declined and these declines were retarded by kinetin. Barley leaves floated on water showed a stimulation of ³²P incorporation into various RNA fractions within 48 hours followed by a decline after 96–144 hours. The leaves floated on kinetin, however, showed an even higher incorporation of ³²P into UNA by 48 hours which remained at a comparatively higher level throughout the experiment. In spite of the above changes in RNA synthesis significant differences in the ³²P sucrose gradient profiles or in the ³²P nucleotide composition of UNA from water and kinetin floated leaves were not noted. The results of this study show that important changes in nucleic acid metabolism occur during the early stages of leaf senescence and that alterations in nucleic acid metabolism during senescence and during kinetin treatment may involve quantitative and only subtle qualitative changes.     

Auxin-facilitated utilization of glutamic acid by tobacco crown-gall teratoma cells

Summary Teratoma tissues obtained by inoculating Nicotiana tabacum cv. Turkish with a moderately virulent strain of the crown-gall bacterium require the synthetic auxin, -naphthaleneacetic acid (NAA) when glutamic acid is used as a sole nitrogen source in the culture medium. In contrast, growth on culture media containing ammonium ion, nitrate ion or glutamine as an N source does not require NAA. Moreover, added NAA does not stimulate teratoma tissue grown on these N sources. Glutamic acid did not inhibit growth of teratoma tissue on media containing NO ₃ ^- . Growth on mixtures of glutamic acid and NO ₃ ^- was additive in the presence of NAA indicating that NAA promotes the utilization of glutamic acid in the culture medium. Increased concentration of potassium ion in the culture medium was required for growth on glutamic acid in the absence of added auxin. K⁺ did not stimulate growth on glutamine. When teratoma tissues were grown on media containing glutamic acid and varying concentrations of both K⁺ and NAA increasing concentrations of NAA reduced the stimulating effect of K⁺ and, conversely, increasing concentrations of K⁺ reduced the stimulating effect of NAA. It is concluded that K⁺ and auxin act either directly or indirectly at a common site to promote glutamic acid utilization.     

Growth of the Peritrich Opercularia coarctata in Axenic Culture*

A synthetic medium for Opercularia coarctata was developed that contains 20 amino acids, 10 vitamins, an 8-component balanced salt solution, Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O, Tween 80, stigmasterol, a 7-component nucleic acid mixture, phenol red as an indicator, and 2,500 U.S.P. units/ml penicillin to maintain sterility. This medium supported axenic survival for 96 hr. Multiple supplements of thioctic acid, niacin, niacinamide, inositol, PABA, oleic acid, and Fe(NO₃)₂·9H₂O instead of Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O coverted the survival medium into a growth medium, which permitted 36–45 days continuous cultivation of populations in excess of 4 × 10³ cells/3.0 ml final volume. Five generations were produced during the 48 hr logarithmic growth period. Serial transfers at 72 hr and during periods of greatest cell density produced a maximum of 8 generations 96 hr after initiation but the medium failed to sustain growth through more than 6 serial transfers. Extension of this investigation to formulating a minimal axenic medium is discussed.     

Purification and Properties of the Geminivirus Euphorbia Mosaic Virus

Euphorbia mosaic virus was purified from infected plants of Nicotiana benthamiana. Highest concentrations of virus particles were found in infected plant tissue between 10–12 days after inoculation. The enzyme driselase assisted in purification of the virus particles from the infected tissue yielding about 600 μg/kg of plant material. Purified preparations showed a maximum absorption at 260–263 nm and the ratio of absorption at 260 and 280 nm was 1.4. The viral nucleic acid was digestedby DNase I and S₁ Nuclease but not RNase A. A single coat protein with a MW of 32,000 d and two DNA bands with a MW 0.96 × 10⁶ d (2870 nucleotides) and 0.90 × 10⁶ d (2700 nucleotides) were associated with the purified virus particles. Virus specific DNA was isolated from infected tissue between 7 and 15 days after inoculations.     

Somatic embryogenesis in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Embryonal-suspensor masses from immature embryos from cones of Sitka spruce (Picea sitchensis (Bong.) Carr.) proliferated on a modified Murashige & Skoog medium with N⁶-benzyl-aminopurine, kinetin, 2,4-dichlorophenoxyacetic acid and an organic nitrogen source. The slimy white embryonal-suspensor masses with proembryos were maintained on a solid proliferation medium with reduced amounts of growth regulators. Transfer of embryonal-suspensor masses to a non-woven polyester carrier with liquid maturation media containing ±2-cis-4-trans-abscisic acid and a reduced amount of inositol and organic nitrogen resulted in synchronized embryo formation. Further development was achieved on a medium without ±2-cis-4-trans-abscisic acid and organic nitrogen. Somatic embryos were successfully transferred ex vitrum.Abbreviations ABA ±2-cis-4-trans-abscisic acid - BAP N⁶-benzyl-aminopurine - ESM embryonal-suspensor masses - KIN kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis of agriculturally important lupin species (Lupinus angustifolius,L. albus,L. mutabilis)

Somatic embryos were obtained from immature cotyledons of Lupinus angustifolius, L. albus and L. mutabilis but not from L. luteus. Different kinds of basal media and plant growth regulators in primary and secondary culture were tested. The best induction media were based on B5 and were supplemented with 5 mg I^-1 2,4-D alone or with 0.25 mg I^-1 kinetin. Mature stage somatic embryos were obtained on media containing ABA (0.1–0.5 mg I^-1) and a high NH₄/NO₃ ratio. Embryo germination and plantlet development occurred on MS media supplemented with glutamine or GA₃.