In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 μmol m^–2 s^–1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed. Received: 14 June 1996 / Revision received: 14 December 1996 / Accepted: 25 January 1997     

Chain elongation of pectic β-(1→4)-galactan by a partially purified galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) hypocotyls

Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified a β-(1→4)-galactosyltransferase (GalT) involved in the synthesis of the β-(1→4)-galactan side chains of pectin. The enzyme protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized β-(1→4)-galactoheptaose (Gal₇-AB), leading to the formation of Gal_8–11-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for 2-aminopyridine-derivatized β-(1→4)-galactooligomer acceptors (Konishi et al. in Planta 218:833–842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains to about Gal₃₅-AB, thus almost reaching the length (43–47 Gal units) of native β-(1→4)-galactan side chains found in pectic polysaccharides from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301–1313, 2002). Enzyme activity increased with increasing chain length of β-(1→4)-galactooligomers and reached maximal activity at heptaose, whereas galactooligomers higher than heptaose showed lower acceptor efficiency. Sugars described in this paper belong to the d-series unless otherwise noted.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

In vitro cold storage of cork oak shoot cultures

Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM 4-amino 3,5,6-trichloropicolinic acid, 400 mg l^-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions. This revised version was published online in June 2006 with corrections to the Cover Date.     

ENZYME ACTIVITIES ASSOCIATED WITH RIBOSOMES FROM SOYBEAN SEEDLINGS

Ribosomes from cotyledons of soybean seeds soaked overnightin water (resting-cotyledon ribosomes) and respective ribosomesfrom cotyledons (working-cotyledon ribosomes) and hypocotyls(hypocotyl ribosomes) of 5 day-old seedlings were prepared.These ribosomes mainly consisted of 80 S particles. However,hypocotyls contained 115 S particles in a small amount and working-cotyledonscontained 115 and 150 S particles which may correspond to thepolymers of ribosomes or polysomes. The abundant 80 S ribosomeswere fractionated by the sucrose density gradient centrifugation,and enzyme activities in the fractionated ribosomes were estimated.RNase, PDase, acid phosphatase, 5'-nucleotidase, XTPase, peroxidaseand ß-glucosidase were found in the ribosomes. Theenzyme activities were lower in the resting-cotyledon ribosomesand higher in the hypocotyl ribosomes. Working-cotyledon ribosomesshowed the middle of them. All these enzymes were also foundin the cytoplasmic solution (supernatant) of cotyledon and hypocotylcells. However, RNase, PDase, 5'-nucleotidase and peroxidasewere concentrated in ribosomes, and the specific activitiesof 5'-nucleotidase and ß-glucosidase were increasedby washing the ribosomes. The status of the enzymes found inthe ribosomes was discussed. (Received May 12, 1966; )     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Characterization of novel thermostable dextranase from Thermotoga lettingae TMO

Biochemical properties of a putative thermostable dextranase gene from Thermotoga lettingae TMO were determined in a recombinant protein (TLDex) expressed in Escherichia coli and purified to sevenfold apparent homogeneity. The 64-kDa protein displayed maximum activity at pH 4.3, and enzyme activity was stable from pH 4.3–10. The optimal temperature was 55–60°C during 15 min incubation, and the half-life of the enzyme was 1.5 h at 65°C. The enzyme showed higher activity against α-(1 → 6) glucan and released isomaltose and isomaltotriose as main products from dextran T2000. An unusual kinetic feature of TLDex was the negative cooperative behavior on the reaction of dextran T2000 cleavage. Enzyme activity was not significantly affected by the presence of metal ions, except for the strong inhibited by 1 mM Fe²⁺ and Ag²⁺. TLDex may prove useful as an enzyme for high temperature sugar milling processes.     

Growth and protopectinase production of Geotrichum klebahnii in batch and continuous cultures with synthetic media

Geotrichum klebahnii ATCC 42397 produces a protopectinase (PPase-SE) with polygalacturonase (PGase) activity. The microorganism was aerobically cultivated in synthetic media. Glucose, fructose and xylose yielded the highest enzyme levels (10–11 PGase units ml⁻¹). Galacturonic acid repressed enzyme production and no growth was obtained with disaccharides and pectin. Specific enzyme activity obtained in an O₂-limited culture was similar to that found in nonlimited ones. A growth yield (Y _x/s) of 0.49 g of cell dry weight per gram of glucose consumed was obtained in a typical batch bioreactor culture. Enzyme production was growth associated, and no major products other than biomass and CO₂ were detected. The volumetric enzyme activity reached a maximum around D=0.3–0.4 h⁻¹ in glucose-limited continuous cultures. However, it varied strongly (together with microorganism morphology) even after retention times ≥8 at any D tested (0.035–0.44 h⁻¹) though the rest of the culture variables remained fairly constant. No correlation between morphology and enzyme activity could be obtained. Enzyme production was poor in urea- and vitamin-limited continuous cultures. In all cases, biomass and CO₂ accounted for ≅100% of carbon recovery though Y _x/s values were different. Journal of Industrial Microbiology & Biotechnology (2000) 25, 260–265. Received 20 April 2000/ Accepted in revised form 15 September 2000     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

High frequency plant regeneration from cotyledon and hypocotyl explants of ornamental kale

A high frequency shoot regeneration system for ornamental kale [Brassica oleracea L. var. acephala (D.C.) Alef.] was firstly established from seedling cotyledon and hypocotyl explants. The ability of cotyledon and hypocotyl to produce adventitious shoots varied depending upon genotype, seedling age and culture medium. The maximum shoot regeneration frequency was obtained when the explants from cv. Nagoya 4-d-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg dm⁻³ 6-benzylaminopurine (BA) and 0.1 mg dm⁻³ naphthaleneacetic acid (NAA). The frequency of shoot regeneration was 65.0 % for cotyledons, 76.1 % for hypocotyls; and the number of shoots per explant was 4.3 for cotyledons, 8.2 for hypocotyls. Hypocotyl explants were found to be more responsive for regeneration when compared with cotyledons. Among the 4 cultivars tested, Nagoya showed the best shoot regeneration response. The addition of 3.0 mg dm⁻³ AgNO₃ was beneficial to shoot regeneration. Roots were formed on the base of the shoots when cultured on half-strength MS medium.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Wing morph-specific differences in the metabolism and endocrine control of reserve mobilization in adult males of a flightless bug, <Emphasis Type="Italic">Pyrrhocoris apterus</Emphasis> (L.) (Heteroptera)

The differences in the metabolism and endocrine control of reserve mobilization in long-winged (macropterous) and short-winged (brachypterous) males of a flightless firebug (Pyrrhocoris apterus) were studied. We found that protein content in the gut was significantly lower in 5–10 day-old macropterous males due to their fasting and higher in 28 day-old ones than in the same aged brachypterous counterparts as the result of renewed food intake. Overall protease activity was significantly lower in 10–14 day-old macropters, while an abrupt increase in the activity starting on day 21 after adult ecdysis was also associated with renewal of the food intake. The levels of carbohydrates in haemolymph were only slightly lower in 1–10 day-old macropterous males than in the same aged brachypters. However, more than twofold higher lipid content in haemolymph of 7–10 day-old macropterous males than in the same aged brachypterous males was found. Higher mobilization of lipid reserves from the fat bodies in macropterous males was accompanied by more intensive adipokinetic response and higher levels of adipokinetic hormone in the body. It is the first report of endocrine regulation of wing morph-related differences in the lipid mobilization in males of wing-polymorphic insects.     

Activation and Inactivation of Alcohol Dehydrogenase in Germinating Pea Cotyledons

Alcohol dehydrogenase (E.C.1.1.1.1.) activity increases markedly in the germinating pea cotyledon in the first 2 days. The activity was not suppressed by the administration of actinomycin D, 6-methylpurine, DL-p-fluorophenylalanine, and D-chloramphenicol. The compounds rather depressed the decrease of alcohol dehydrogenase activity in cotyledons after 3 days of germination. The alcohol dehydrogenase activity in ungerminated pea seeds was activated by treatment with sodium lauryl sulphate, sodium dioctyl sulfosuccinate, dithiothreitol, 2-mercaptoethanol and NADH. The inhibitory effect caused by the extract from 7 day-old cotyledons was diminished markedly in the presence of dithiothreitol and 2-mercaptoethanol, as well as by addition of bovine serum albumin. If dithiothreitol was added to the extraction medium, the enzyme activity from older cotyledons was greatly enhanced.     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Kinetic characterization of enhanced lipase activity on oil bodies

Enzyme access, kinetic behavior, and protein–protein interactions are critical for explaining reaction of the metabolites contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the interface resulted in an enzyme–membrane complex that was catalytically enhanced 3–15-fold over the emulsion system for substrate concentrations in the measured range of approximately 1–5.5 mM. This disparity is explained by the availability of substrate to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K _m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg protein. The interaction is verified through separation of the enzyme–membrane complex which shows nearly double the activity towards an emulsified soybean oil substrate (activity ratio of 5:3) when compared to the native enzyme.     

Sonication assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO₄ solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.     

Desulfonation of propanesulfonic acid by Comamonas acidovorans strain P53: evidence for an alkanesulfonate sulfonatase and an atypical sulfite dehydrogenase

Evidence is presented for the presence in propanesulfonate-grown Comamonas acidovorans strain P53 of a cytoplasmically located sulfonatase that does not sediment at 100,000 × g. This enzyme catalysed the sulfonate-dependent oxidation of NADH or NADPH, indicating a monooxygenase that effects the addition of molecular oxygen to C₃-C₆ 1-alkanesulfonates. Enzyme activity was proportional to protein concentration only above approximately 2 mg cytoplasmic fraction protein ml^–1, suggesting that the sulfonatase is a multicomponent enzyme, possibly comparable with methanesulfonate monooxygenase. Enzyme activity was strongly inhibited by divalent metal-chelating agents, but was insensitive to cyanide and azide. Sulfite released from sulfonates by Comamonas acidovorans was oxidized by an unusual sulfite dehydrogenase. This was purified approximately 230-fold and was shown to have a molecular mass of 74.4 kDa, comprising two or more subunits. The enzyme activity was specific in vitro for ferricyanide as an electron acceptor and, unlike other bacterial sulfite dehydrogenases, did not contain native cytochrome c or reduce added cytochrome c. It was a basic protein, insensitive to chloride and sulfate, and exhibited a K _m for sulfite of approximately 45 μM. Received: 19 May 1999 / Accepted: 3 September 1999     

Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid-cycling Brassica napus

 A simple method to induce somatic embryogenesis from seeds of rapid-cycling Brassica napus is described. Seedlings cultured on Murashige and Skoog (MS) basal medium produced somatic embryos directly on hypocotyls and cotyledons after 2 to 3 subcultures onto the same medium. A low pH of the medium (3.5–5) was more conducive to somatic embryogenesis than a higher pH (6 and 7). Embryogenic potential of the seeds was inversely correlated to seed age: about 41–68% of immature seeds between the ages of 14 and 28 days after pollination (DAP) formed somatic embryos compared to 0–11% of the seeds obtained 29–37 DAP. About 54% of the somatic embryos produced secondary embryos after subculturing onto the same medium. The embryogenic potential of the cultures has been maintained on MS basal medium for 2 years (12 generations) without diminution. Up to 75% of the secondary embryos developed into plantlets on MS medium enriched with 10^–6  M zeatin, and 40% of these produced flowers when transferred to an optimised flower-induction medium. Viable seeds were produced in self-pollinated in vitro flowers. Received: 15 February 2000 / Revision received: 18 July 2000 / Accepted: 19 July 2000