Somatic embryogenesis, plant regeneration and cyanogenesis in Manihot glaziovii Muell. Arg. (ceara rubber)

 The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3–6 mm long) adjacent to the apex in Murashige and Skoog medium containing 18 μm 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 μm 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10⁻⁴ m kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos. Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of linamarase activity were almost the same in leaves and stem tissues and very low in roots. Received: 19 April 1999 / Revision received: 11 August 1999 / Accepted: 17 August 1999     

Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time

 A long-term regeneration system for garlic (Allium sativum L.) clones of diverse origin was developed. Callus was initiated on a modified Gamborg's B-5 medium supplemented with 4.5 μM 2,4-D and maintained on the same basal medium with 4.7 μM picloram+0.49 μM 2iP. Regeneration potential of callus after 5, 12 and 16 months on maintenance medium was measured using several plant growth regulator treatments. The 1.4 μM picloram+13.3 μM BA treatment stimulated the highest rate of shoot production. Regeneration rate decreased as callus age increased, but healthy plantlets from callus cultures up to 16-months-old were produced for all clones. Regeneration of long-term garlic callus cultures could be useful for clonal propagation and transformation. Received: 24 September 1998 / Revision received: 27 January 1999 / Accepted: 26 February 1999     

Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria

 Stem segments of seedlings from two Alstroemeria breeding lines, cultured on media supplemented with 4 mg/l 2,4-dichlorophenoxyacetic acid and 0.5–1.0 mg/l 6-benzylaminopurine (BA), initiated soft callus, which became compact after subculture on a medium with only 0.5 mg/l BA. Friable embryogenic calli were initiated from compact callus on a medium supplemented with 10 mg/l picloram. Proembryos developed from friable embryogenic calli via embryos into plants after subculture on medium supplemented with 0.1 mg/l BA. The proembryos formed friable embryogenic calli again after culture on medium supplemented with 10 mg/l picloram. The total time needed to regenerate a complete plantlet from friable callus was approximately 6 months. This system for the production of embryogenic material is considered to have valuable applications for genetic transformation in Alstroemeria. Received: 22 April 1999 / Revision received: 16 July 1999 · Accepted: 20 July 1999     

Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999     

Repetitive somatic embryogenesis in carnation on picloram supplemented media

An efficient method of repetitive somatic embryogenesis and plant regeneration of two carnation cultivars (Sagres and Impulse) was established using a two-step protocol. In the first step, embryogenic tissue were induced from petal explants on MS culture medium containing 9% sucrose (w/v), 9 μM 2,4-D and 0.8 μM BA. In the second step, embryogenic tissue transferred onto the MS medium containing 3% sucrose supplemented with different concentrations of picloram (0.8, 2, 4, 8 and 16 μM) to produce primary somatic embryos. Precotyledonary clumps and cotyledonary somatic embryos were then isolated and subcultured onto the same media as the second step where they formed secondary somatic embryos in repetitive cycles. Cotyledony somatic embryos were converted into plantlets when they transferred onto the growth regulator-free half-strength MS medium followed by being acclimated to the greenhouse conditions.     

Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.)

Summary Genetic engineering of cereals currently depends on the use of tissue culture and plant regeneration systems. In wheat (Triticum aestivum L.), immature embryos are the most widely used explant to initiate cultures, but they are inconvenient due to their temporal availability and production requirements. Mature embryos are easily stored and are readily available as mature seeds. However, plant regeneration frequencies from cultures derived from mature embryos are generally low. This research was undertaken to improve callus induction and plant regeneration from wheat mature embryos of cultivar ‘Bobwhite’. The effects of four auxins [2,4-dichlorophenoxyacetic acid (2,4-D): 3,6-dichloro-o-anisic acid (dicamba); 4-amino-3,5,6-trichloropicolinic acid (picloram): and 2-(2-methyl-4-chlorophenoxy) propionic acid (2-MCPP)], and the effect of maltose vs. sucrose under filter sterilized and autoclaved conditions were evaluated. All auxin treatments resulted in callus induction except 2 MCPP. A highly significant effect of auxin type on both callus and plantlet production was detected, though interactions were observed. The effect of sugar type was dependent on the type of auxin used. Substitution of sucrose by maltose enhanced the regenration ability of callus from embryos cultured on media containing 2,4-D and picloram, but caused an opposite effect on media containing dicamba. Picloram significantly enhanced callus growth, however, embryogenic response and plant regenerability were low. Relative to 2.4-D, dicamba (18μM) resulted in a twofold increase in the number of plants regenerated per embryo and reduced the amount of time required for plant regeneration by 3–4 wk. Mention of a trademark or proprietary product does not constitute a guarantce or warranty by the University of Wisconsin and does not imply its approval to the exclusion of other products that may be suitable.     

Influence of osmotica and abscisic acid on triglyceride accumulation in peanut somatic embryos

Attempts were made to determine the influence of sucrose, mannitol, sorbitol and abscisic acid on accumulation of triglycerides in peanut (Arachis hypogaea L.) somatic embryos. The results revealed that 0.584 m sucrose in the medium produced increased triglycerides in the embryos compared to the control. At 0.730 m sucrose, embryos were necrotic although the triglyceride content was high. Sorbitol at 0.6 m or abscisic acid at 20 μm were effective in increasing triglycerides in the embryos. The increase in triglycerides on a fresh-weight basis observed with increasing concentration of osmoticium was not apparent when determined in terms of dry weight. However, an increase in triglyceride as percent fresh weight observed in the presence of 20 μm abscisic acid remained unaltered when determined in terms of percent dry weight. An increase in storage lipid did not improve conversion of peanut somatic embryos. Received: 4 April 1997 / Revision received: 15 February 1998 / Accepted: 2 March 1998     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis

 An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 μM 2,4-D and 1 μM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants. Received: 29 April 1999 / Revision received: 2 July 1999 · Accepted: 12 July 1999     

Formation of α-D-glucose-1-phosphate by thermophilic α-1,4-D-glucan phosphorylase

For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg⁻¹ and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to ¹³C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93. Received 12 May 1999/ Accepted in revised form 29 August 1999     

Regeneration from long-term embryogenic callus of the <Emphasis Type="Italic">Rosa hybrida</Emphasis> cultivar kardinal

Summary Media components used for three stages of development: (1) callus maintenance, (2) maturation of embryos, and (3) conversion of embryos to plants were shown to affect regeneration of plants for the commercially important red rose cultivar Kardinal. Embryogenic callus was maintained for 5yr on either Schenk and Hildebrandt’s basal salts medium (SH) supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or Murashige and Skoog’s basal salts medium (MS) supplemented with 18.1 μM dicamba and 0.46 μM kinetin. Maturation of embryos was three times higher using callus maintained on the SH medium supplemented with 2,4-D while conversion of cotyledonary-stage embryos to plants was significantly higher (10 times) using callus that had been maintained on MS medium with dicamba and kinetin. Maximum maturation (13.5%), and conversion (15.2%), occurred when callus was cultured on MS maturation medium without hormones. Cotyledonary-stage embryos cultured on MS conversion medium supplemented with abscisic acid (5–20 μM) produced plants that survived at a significantly higher rate (two times) in the greenhouse than when embryos were cultured without abscisic acid. The highest rate of plant regeneration occurred when embryogenic callus of ‘Kardinal’ was maintained on MS medium supplemented with dicamba and kinetin, maturation of embryos occurred on MS maturation medium without hormones, and conversion of cotyledonary-stage embryos occurred on MS conversion medium supplemented with abscisic acid.     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb

Friable callus was obtained from styles and flower pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige and Skoog (MS) media containing either 2 μm dicamba or 2 μm picloram. Cell suspension cultures were established by suspending the callus of L. longiflorum Snow Queen in liquid medium containing 2 μm dicamba. Through a purification process, a fine fast-growing cell suspension was obtained. This suspension was composed of a homogenous population of small dense cells, which tended to organise into embryo like structures (ELS). In liquid culture with the auxin dicamba, the ELS underwent continuous callus formation. When transferred to solidified hormone-free MS medium, the ELS germinated, forming complete plantlets. Histological investigation showed that in the ELS both shoot and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact somatic embryos. Received: 4 April 1997 / Revision received: 13 May 1997 / Accepted: 15 June 1997     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid

 Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance. The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates. Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999     

Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.)

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5–7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 μm 2,4-dichlorophenoxyacetic acid and 0.93 μm kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology. Received: 29 December 1997 / Revision received: 9 April 1998 / Accepted: 24 April 1998     

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Characteristics of dipeptide transport in pig jejunum in vitro

 Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-l-glutamine and glycyl-l-sarcosine (20 mmol · l⁻¹) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 ± 0.15 and 1.57 ± 0.20 μeq · cm⁻² · h⁻¹, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-l-sarcosine, a net flux rate for glycyl-l-sarcosine of 49.8 ± 6.7 nmol · cm⁻² · h⁻¹ was calculated. In mucosal uptake experiments, the apical influx of ¹⁴C-labelled glycyl-l-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-l-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut. Accepted: 30 June 1999