Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N⁶-benzyladenine and N₁-(2-chloro-4-pyridyl)-N₂-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4–6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10³ RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.Abbreviations BA N⁶-benzyladenine - [PU]-30 N₁-(2-chloro-4-pyridyl)-N₂-phenylurea - UMP undine 5-monophosphate - UTP udine 5-triphosphate     

Growth and Endogenous Cytokinins in Tobacco Callus as Affected by N-(2-chloro-4-pyridyl)-N′-phenylurea

The effect of N-(2-chloro-4-pyridyl)-N-phenylurea (4PU-30) on the growth and content of endogenous cytokinins of adenine type in tobacco (Nicotiana tabacum L.) callus was investigated. Biomass accumulation in calli grown on Murashige and Skoog (MS) medium with 4PU-30 was higher than that on MS medium with kinetin. The obvious presence of isopentenyladenine type cytokinins and traces of zeatin type cytokinins supposes modification in the endogenous cytokinin metabolism of the tobacco callus grown on 4PU-30.     

Embryogenic cell lines from somatic embryos of grape (Vitis vinifera L.)

Somatic embryo formation occurred on leaf callus of grape (Vitis vinifera cv. Koshusanjaku). An embryogenic callus was induced from somatic embryo clusters cultured on vitamin-, inositol- and glycine-free Nitsch and Nitsch (1969) medium supplemented with 1.0M 2,4-D. This callus has retained a high embryogenic activity after repeated subculture on the same medium for over two years, and has produced numerous embryos after transfer to a hormone-free medium. The effect of cytokinin treatment on somatic embryogenesis from leaf callus was also examined. N-(2-chloro-4-pyridyl)-N-phenylurea (KT-30) and N-(1,2,3-thiadiazol-5-yl)-N-phenylurea (TAG), both synthetic cytokinins, were found to be effective for the induction of somatic embryogenesis. When leaf callus was induced by these cytokinins combined with 2,4-D at either 5.0 or 10.0M, somatic embryos were produced.Abbreviations B₅ Basal medium, Gamborg et al. (1968) - BA 6-benzylaminopurine - 2,4-D 2,4- dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP (2-isopentenyl)adenine - KIN kinetin - KT-30 N-(2-chloro-4-pyridyl)-N'-phenylurea, also called 4PU-30, Kyowa Hakko Kogyo Co., Japan - NAA 1-naphthaleneacetic acid - NN Basal medium, Nitsch and Nitsch (1969) - MS Basal medium, Murashige and Skoog (1962) - TAG N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea, also called thidiazuron or TDZ, Tomono Noyaku Co., Japan - ZEA zeatin     

Endogenous levels of abscisic acid and gibberellins in leaf protoplasts competent for plant regeneration in Betula platyphylla and Populus alba

Differences in endogenous levels of abscisic acid and gibberellinsbetween Betula platyphylla and Populusalba leaf protoplasts were determined using micro-scale extractionand purification steps, including thin layer chromatography ormicro-high-performance-liquid-chromatography and quantification by enzymelinkedimmunosorbent assay or micro-bioassay. The content of abscisic acid was tentimes higher in B. platyphylla than in P.alba on the basis of both cell number and dry weight; in contrast,levels of gibberellins were lower in the former. Leaf protoplasts of bothspecies are competent for plant regeneration through the exogenous supply ofauxins and cytokinins. The function of abscisic acid in these protoplastcultures is discussed in relation to the need for a strong cytokinin,N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU) for colony proliferation inB. platyphylla, in contrast to a weak cytokininrequirementin P. alba.     

Expansion during early apple fruit development induced by auxin and N-(2-chloro-4-pyridyl)-N′-phenylurea: Effect on cell wall hemicellulose

Expansion in apple fruit (Malus domestica Borkh. cv. Braeburn) during early development was induced by injecting 2,4-dichlorophenoxyacetic acid (2,4-D) through the calyx of the fruit and by dipping the apples in N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU). Cell wall composition was analysed, focusing on the hemicellulose fraction containing xyloglucan, a polysaccharide believed to play an important role in cell expansion. Changes were observed in the yields of the cell wall fractions of the fruit treated with either 2,4-D or CPPU, although the monosaccharide composition of the fractions exhibited few differences. There was no decrease in the molecular weight of the xyloglucan from treated fruit. These results are discussed in terms of current cell wall expansion mechanisms.     

Somatic embryogenesis and plant regeneration in Sorghum bicolor (L.) Moench

Summary Callus induction, somatic embryogenesis and plant regeneration were obtained in two cultivars of Sorghum bicolor (L.) Moench. Transverse thin cell layers from roots/epicotyls of 15-day-old seedlings or of regenerated plantlets were used. Callus response depended on the genotype, the size of transverse thin cell layers, the level at which transverse thin cell layers were excised on the epicotyl, the composition of growth substances and the number of in vitro regeneration cycles undergone by the donor plant. Somatic embryos were differentiated under a defined dark/light sequence, from epidermised compact calluses (i.e having already differentiated an epidermis), obtained directly with dicamba or from friable callus initiated with kinetin and 2,4 dichlorophenoxyacetic acid. The importance of kinetin and dicamba on the induction of embryogenic potential is reported.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - 2iP N6-(2-isopentyl)adenine - BAP 6-benzylaminopurine - CaMV cauliflower mosaïc virus - CPPU N-(2-chloro 4-pyridyl)-N-phenylurea - dicamba 3,6-dichloro-o-anisic acid - IAA indole-3-acetic acid - K kinetin - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PEPC phosphoenolpyruvate carboxylase - SD standard deviation - tTCL transverse thin cell layer     

Agrobacterium-mediated transformation and plant regeneration from hypocotyl segments of Japanese persimmon (Diospyros kaki Thunb)

Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea, zeatin or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4–5 months. Received: 18 August 1997 / Revision received: 8 October 1997 / Accepted: 11 November 1997     

The response of a cytokinin resistant mutant is highly specific and permits a new cytokinin bioassay

We have previously isolated six independent cytokinin-resistant mutants of Nicotiana plumbaginifolia which define three complementation groups, zeal, zea2 and zea3. We report here the characterization of the phenotypic response to cytokinin treatment of the mutant 1–64, belonging to the zeal group, and the result of the study of the specificity of this response. The phenotype of this mutant grown in the presence of cytokinin concentrations higher than 0.1 M is characterized by a hypertrophy of the cotyledons and hypocotyl which results in an increase of plantlet fresh weight. This hypertrophy is correlated to cytokinin concentration in a range between 0.01 to 10 M. The specificity of this response has been verified by using adenine and urea type cytokinins, as well as enantiomers of methylzeatin and methylbenzyladenine which differ widely in their cytokinin activities. We show that the high specificity of the hypertrophic response to cytokinins can be used as a convenient bioassay to screen the cytokinin activity of adenine or urea type molecules.Abbreviations zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine] - iP isopentenyladenine [6-(3-methylbut-2-enylamino)purine] - BA benzyladenine [6-(benzylamino)purine] - (R)-(+)-MeZea [(R)--methylzeatin] - (S)-(–)-MeZea [(S)--methylzeatin] - (R)-(+)-MeBA [(R)--methylbenzyladenine] - (S)-(–)-MeBA [(S)--methylbenzyladenine] - CPPU N-(2-chloro-4-pyridyl)-N-phenylurea - thidiazuron N-(1,2,3-thiadiazol-5-pyridyl)-N-phenylurea The authors dedicate this paper to the memory of Jean-Pierre Bourgin, Director of the Laboratoire de Biologie Cellulaire, who died suddenly on October 29, 1994.     

Effects of the growth regulator 4PU-30 on growth,K+ content,and alkaloid production in tobacco callus cultures

Growth, K⁺ content, and alkaloid production were compared in nonorganogenetic callus cultures ofNicotiana tabacum cv. Burley 21 grown at 25°C in the dark on two different media: a basal medium with 1 M -naphthaleneacetic acid and 1 M kinetin, and one with 1 M -naphthaleneacetic acid and 1 M 4PU-30 (N-(2-chloro-4-pyridyl)-N-phenylurea). These callus tissues behaved differently not only in growth and K⁺ content but also in alkaloid production. In comparison to cultures grown with kinetin, those grown with 4PU-30 showed a significantly higher fresh weight and dry weight and K⁺ content during the growth period studied. The data clearly indicate a positive correlation between K⁺ uptake rate stimulated by 4PU-30 and cell enlargement rate. However, the alkaloid biosynthesis in the callus tissues was activated by the supply of kinetin and diminished by that of 4PU-30. It thus appears that cellular enlargement of meristematic tissue stimulated by 4PU-30 limited alkaloid production.     

Effect of 2,4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus

Callus induction, somatic embryogenesis and plant regeneration were obtained in lemon [Citrus limon (L.) Burm. cv `Femminello'] and sweet orange [C. sinensis (L.) Osb. cv `Washington Navel GS'] from cultures of stigma and style transverse thin cell layer explants [(t)TCLs]. Explants were cultured on 16 different media, based on the nutrients and vitamins of Murashige and Tucker medium (MT) supplemented with different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU). Sucrose (146 mM) was used as the sole carbon source. Somatic embryos arose from callus at the surface of stigma and style (t)TCLs 3–5 months after culture initiation of both sweet orange and lemon. The percentages of embryo formation from style (t)TCLs ranged from 0% (the media containing 2,4-D) to 16.0% (the medium supplemented with 4 M 4-CPPU) for C. limon. Better results were obtained when stigma (t)TCLs from C. limon were used; in fact, percentages ranged from 0% on the media containing 2,4-D, with the only exception for the medium supplemented with 0.4 M 2,4-D, to 24.8% on medium with 4 M 4-CPPU. The embryogenic response of lemon (t)TCLs was usually higher than for sweet orange (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies ranging from 53% to 75% for sweet orange and lemon style derived embryos, respectively.     

An improved protocol for the culture of cassava leaf protoplasts

Viable protoplasts (yield > 1.9 × 10⁷ g^–1 fresh weight; mean viability 85±2%, n=5) were isolated from leaves of axenic shoot cultures of Manihot esculenta Crantz. cv. M. Thai 8. Protoplasts were cultured for up to 50 days in liquid, ammonium-free MS medium, overlaying agarose-solidified B5 medium with short glass rods embedded perpendicularly within, and protruding from, the agarose layer. Control protoplasts were cultured identically, but without glass rods. Sustained protoplast division was observed only in the presence of glass rods, where the initial plating efficiency was almost 6-fold greater than control (p < 0.05). The mean final plating efficiency of treated cultures was 1.0±0.2% while, in contrast, significant colony formation was not observed in controls.Abbreviations BA 6-benzyladenine - CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea - MES 2[N-morpholino]ethane sulphonic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - IPE initial plating efficiency - FPE final plating efficiency     

Propagation of banana through encapsulated shoot tips

Plants were regenerated from encapsulated shoot tips of banana. Shoot tips (ca 4 mm) isolated from multiple shoot cultures of banana cv. Basrai were encapsulated in 3% sodium alginate containing different gel matrices. The encapsulated shoot tips regenerated in vitro on different substrates. Use of White's medium resulted in 100% conversion of encapsulated shoot tips into plantlets. The plantlets were successfully established in soil.Abbreviations BA Benzylaminopurine - NAA Naphthalene acetic acid - DMSO Dimethyl sulphoxide     

Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski)

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.Abbreviations BAP 6 Benzylaminopurine - NAA -Naphthalene acetic acid - MS Murashige and Skoog's medium - IAA Indole acetic acid - KN Kinetin     

Plant regeneration from stem-derived protoplasts of Brassica alboglabra bailey

Protoplasts isolated from stem tissue of Brassica alboglabra Bailey devided rapidly and formed microcalli on medium supplemented with 0.2 mg/l dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/l 6-benzyladenine (BA). On shoot regeneration medium, the presence of 0.25-1 mg/l BA was most effective in inducing shoot and plant regeneration from protoplast-derived callus at a frequency of 18 and 2–4%, respectively. While low cytokinin concentrations enhanced rhizogenesis, callus growth was promoted in the presence of 2,4-D or naphthalene acetic acid (NAA). All 20 regenerated plants were successfully acclimatized of which 19 were diploid and one was aneuploid.     

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tsatsai</Emphasis>)

Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.     

Response of NADH oxidation and growth in K-562 cells to the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984)

Summary Growth of K-562 cells in culture is inhibited by the antitumor sulfonylureaLY181984 (N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea) with an ED₅₀ of about 30 M. LY181984 was shown previously to inhibit NADH oxidation by plasma membranes from HeLa cells and other sources and to influence mitochondrial oxidative phosphorylation. With K-562 cells, NADH oxidation by plasma membranes was transiently stimulated and then inhibited by LY181984. NADH oxidation by whole cells was transiently stimulated and then inhibited by 0.1 to 100 M LY181984 as well. Both the stimulations and inhibitions of activity were time-dependent. NADH oxidation by lower phase membranes depleted of plasma membranes by aqueous two-phase partition also was inhibited by micromolar and submicromolar concentrations of LY181984. Inhibition did not correlate with mitochondrial presence but rather with membranes that appeared to be fragments of the Golgi apparatus. The oxidation of NADH by whole cells and of plasma membranes that was inhibited by LY181984 was distinguished from mitochondrial NADH oxidation by resistance to inhibition by cyanide and by proceeding under oxygen-depleted conditions or an argon atmosphere. In contrast to the active antitumor agent LY181984, the inactive but chemically-related analog, LY181985 (N-(4-methylphenyl-sulfonyl)-N-(4-phenylurea), inhibited neither growth nor NADH oxidation with K-562 cells or cell fractions.     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

Micropropagation of Raphanus sativus L. var. longipinnatus (Japanese radish) cv. Gungjung

Shoot cultures were established from seedling shoot tips of Raphanus sativus var. longipinnatus Bailey cv. Gungjung, (Japanese radish) cultured on a Murashige-Skoog medium supplemented with ca. 4.5–135 M kinetin or N⁶-benzyladenine. The latter cytokinin supported overall better growth, and 22.2 M was adopted for maintenance of established cultures. The nitrate: ammonium levels in the medium proved optimal for growth and shoot proliferation and both these parameters were significantly increased by addition of adenine sulfate or sodium phosphate. Rooting of excised shoots was achieved on auxin containing medium. Indole-3-butyric acid (ca. 5 or 10 M) also enhanced shoot growth. Plants were easily established in soil, appeared morphologically normal, and flowered.     

Genetic instability in calamondin (<Emphasis Type="Italic">Citrus madurensis </Emphasis>Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives

Somatic embryos were regenerated in vitro from calamondin style–stigma explants cultured in the presence of N ⁶-benzylaminopurine (BAP) cytokinin and three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), N-phenyl-N′-benzothiazol-6-ylurea (PBU) and N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU). The phenylurea derivative compounds tested at micromolar level (12 μM) were able to induce a percentage of responsive explants significantly higher from that obtained with BAP and hormone-free (HF) conditions. In order to verify the genetic stability of the regenerants, 27 plants coming from different embryogenic events were randomly selected from each different culture condition and evaluated for somaclonal variations using inter-simple sequence repeat and random amplified polymorphic DNA analyses. We observed that 2,3-MDPU and PBU gave 3.7% of somaclonal mutants, whereas 4-CPPU gave 7.4% of mutants. No somaclonal variability was observed when plantlets were regenerated in BAP or HF medium. Although diphenylurea derivatives show a higher embryogenic potential as compared to BAP, they induce higher levels of somaclonal variability. This finding should be taken in consideration when new protocols for clonal propagation are being developed.