The size and form of DNA molecules from microsporocytes of Lilium longiflorum at different stages of meiosis

DNA molecules from lysates of microsporocytes at different stages of meiosis of Lilium longiflorum were examined in the electron microscope. In all of the preparations, only linear (non-circular) molecules were found. They had a mean length of between 59 and 75 , and a range of lengths which varied from 1 to 115 , to 1 to 210 . From the data obtained, there was no indication of a change in molecular form or size of DNA molecules associated with the process of meiosis.     

In vitro control of blastema cell proliferation by extracts from epidermal cap and mesenchyme of regenerating limbs of axolotls

Summary The presence of a mitogenic activity in limb blastemas of axolotls was detected in crude extracts of blastemas at the mid-bud stage. The mitogenicity of the extracts was estimated from the mitotic index of blastema cells grown for 6 days in the presence of limb blastema extracts, with colchicine present for the last 2 days. All the extracts tested (whole blastema, blastemal mesenchyme, epidermal cap) significantly enhanced proliferation of blastema cells. The highest stimulation factors we observed were 7 × with 7 g protein/ml whole blastema extracts, 5.2 × with 14 g/ml blastemal mesenchyme extracts, and 11 x with 3.5 g/ml epidermal cap extracts. Hence the epidermal cap extracts appeared to be the most mitogenic. Extracts from the blastemal mesenchyme, although less mitogenic than the epidermal cap extracts, were more potent than nerve extracts [Albert P, Boilly B (1986) Biol Cell 58:251–262]. These results are discussed with regard to the production of growth factors during limb regeneration.     

Effect of a swim training on homocysteine and cysteine levels in rats

Summary. The purpose of this study was to investigate the effects of a 8-week of swim training on total plasma homocysteine and cysteine levels in 16 male Sprague-Dawley rats aged 17 weeks. We also evaluated the activity of hepatic cystathionine -synthase (CBS), an enzyme involved in the metabolism of Hcy, the concentration of plasma glutathione, taurine, and a fraction of vitamin B6: the pyridoxal 5-phosphate (PLP). After one week of acclimatization, rats were randomly divided into two groups: 8 non-trained (NTR) and 8 trained rats (TR). Following the training period, body weight gain was lower in TR than in NTR. Plasma homocysteine did not differ among groups while significantly lower plasma cysteine and taurine levels were found in TR (157.83±8.6mol/L; 133.01±9.32mol/L; P<0.05) compared with data of NTR (176.19±4.9mol/L; 162.57±8.16mol/L; P<0.05). No significant changes in hepatic CBS activity were observed in TR compared with NTR. Moreover, values for plasma glutathione and PLP concentrations were not affected by training.These results indicate that training reduces plasma cysteine and taurine levels whereas it does not modify other studied parameters. Thus, physical training may regulate cysteine metabolism.     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Changes with age in the absorption spectrum of chlorophyll α in a diatom

Summary Absorption spectra of a young and an old culture of the diatom Pheodactylum tricornutum were measured in thin layers between two opal glass sheets. The spectra at 24° and at -196°C were replotted to give equal areas from 730–625 m to allow direct comparison. At 24°C the spectrum for the difference between the two cultures had a negative component of 18 m half width centered at 675 m and a positive region of W_0.5=26 m near 700 m.The spectra at -196°C may be somewhat distorted by clumping of the cells during freezing but nevertheless the 16 day culture clearly showed a smaller proportion of Ca 670 to Ca 680. This older culture has a shoulder due to a 707 m component. The difference curve at -196°C shows the decrease of an unsymmetrical band peaking at 669 m and an increase at 695 m in addition to the 707 m component. Due to the possibility of distortion, the presence of an actual component at 695 is doubtful in these particular cultures.The room temperature spectrum in the chloropyhll a region for the 5 day culture can be closely fitted by a single probability curve at 675 m having a half-width of 31 m. The sum of two components, with widths more reasonable for chlorophylls, also matched the data well enough. These two probability curves, of 22 m half width, centered on 669 and 683.2 m and had a height ratio, h₆₆₉/h₆₈₃ of 1.18. In the 16 day culture the ratio for these bands changed to 1.11 and there was extra absorption around 700 m.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

A novel technique for the study of bacterial cell mechanical properties

A micromanipulation method is described for measuring the bursting forces of bacteria and relating them to cell size. At a compression speed of 6.2 m s^–1, bursting forces of three samples of rapidly growing Staphylococcus epidermis from a batch culture varied from 3 to 34 N with an average value of 13.8 N (standard error 0.8 N). Escherichia coli grown in continuous culture at a specific growth rate of 0.5 h^–1 had bursting forces varying from 1 to 9 N with an average value of 3.6 N (standard error 0.4 N). In squeeze-hold experiments, force relaxation was observed, which was attributed to water loss from the cells, or viscoelasticity, or both. At high compression speed, such as 6.2 m s^–1, this relaxation could be neglected. Micromanipulation strength measurements might be used in studies of cell mechanical disruption and of the dependence of cell strength on cell physiology.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

In vitro micropropagation of Piper longum – an important medicinal plant

Efficient and rapid tissue culture systems were developed for Piper longum, an important medicinal plant, through shoot tip multiplication and direct regeneration. Multiple shoots were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44–22.19 M benzyladenine (BA) and 4.64–13.9 M kinetin (K). Maximum number of shoots were induced with 8.9 M BA and 4.64 M K. Adventitious shoot regeneration from leaf segments was achieved on MS containing 3.6–22.19 M BA along with 3.31–12.4 M picloram (P). Shoot differentiation occurred directly from the leaf bases without intermediale callus formation. Maximum shoot buds were obtained on MS medium with 17.76 M BA and 8.28 M P. Elongated shoots were separated and rooted in MS supplemented with 2.46 M indole butyric acid (IBA). Plantlets, thus developed were established in soil.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater,Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth,Choloepus didactylus (Linn.)

Summary Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Pará State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 m: the wall is 1.5 to 2.0 m thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 m, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are 20.0 × 15.7 m some produce 12 to 20 merozoites which are 8.7 × 2.0 m, and others 10 to 26 merozoites which are 11.4 × 2.0 to 15.0 × 3.0 m. Mature microgamonts which are 27.5 × 24.1 m, produce from 150 to 170 microgametes of 7.1 × 1.0 m: microgametes have two flagella of unequal length. Mature macrogamonts are 28.4 × 24.5 m Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 m, have a wall 2.0 to 2.5 m thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 m, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either 13.0 × 2.0 m or 13.0 × 3.0 m. Microgamonts produce 50 to 80 microgametes of 8.0 × 1.0 m. Mature macrogamonts are 18.3 × 17.9 m. ac]19820212     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

In vitro somatic embryogenesis and plant regeneration in Acacia arabica

In vitro somatic embryogenesis and subsequent plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Acacia arabica on semi-solid Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 MBA, 6.78 M2,4-D and 30 g l^–1 (w/v) sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to MS medium supplemented with 6.66 M BA, 6.78 M 2,4-D. The maximum number of somatic embryos per callus was 72.6 after 8 weeks of culture on medium containing 6.66 M BA and 6.78 M 2,4-D. The isolated somatic embryos germinated on half-strength basal MS salts and vitamins supplemented with 0.04 M BA, 0.94 M ABA and 2% (w/v) sucrose. The embryo-derived plantlets were acclimatized in the greenhouse and subsequently showed normal growth.