Organogenesis in pepper tissue cultures

Knowledge concerning in vitro growth and developmental responses of bell and chile peppers (Capsicum annuum L.) has been limited. Shoot and root organogenesis in cultures of seedling explants was restricted to primary cultures or those less than three months old under 12-and 16-h photoperiod at 25°C. Shoot organogenesis was extended to 5 months under continuous light at 25°C, and to 8 months under continuous light at 28.5°C. Murashige and Skoog basal media containing 0.05mg/l each of IAA and BA promoted shoot elongation and rooting of some explant sources, while 0.05-4 mg/l IAA with 10–50 mg/l BA promoted adventitious shoot bud formation. Glucose was superior to sucrose as the carbon source. Leaf discs collected from greenhouse-grown plants regenerated shoots for at least 2 months. Incubation environment, carbon source, explant source, growth regulator treatment and passage number were not independent variables as demonstrated by statistical analysis. The plant regeneration techniques described here have useful but limited applications, not extending to unorganized callus or cell suspension cultures.Journal article no. 1151 of the New Mexico Agricultural Experiment Station.     

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of spontaneous shoot regeneration on leaf stipules in relation to hyperflowering response in micropropagated strawberry plantlets

Summary Stipular bud (SB) formation occurred spontaneously on “Gorella” strawberry leaf stipules during proliferation phase on Boxus medium. These buds gave rise to normal shoots on the inner median zone between the stipule tips. Some stipular buds also have been observed on other parts of adaxial surface of the stipule. Stipular bud formation took place directly on the stipule without an intermediate callus. The first stage consisted of subspherical protrusions of small cells which progressively differentiated into shoots that were able to proliferate and to root. Scanning electron microscopy was used to examine the developmental stages of this adventitious organogenesis and to describe morphologic abnormalities, e.g., multiapex formation and fasciation. In vitro cloning of plantlets was made from axillary and SBs produced by the same tufts. The greater flowering abundance of stipular plants resulted in a drastic reduction of the commercial production and the fruit caliber when compared to axillary plants. However, the general phenotype of the mother cultivar (Gorella) was not affected in either case.     

Antagonistic Effects of Triazolo[4,5-d]pyrimidine and Pyridylurea Derivatives on Cytokinin-Induced Cytokinin Oxidase/Dehydrogenase Activity in Young Pea Plants

The effect of strong and weak cytokinin antagonists, belonging to the groups of triazolo[4,5-d]pyrimidines (TP), and pyridyl-phenylurea derivatives (PU), on cytokinin oxidase/dehydrogenase activity (CKX) in the tissues of young pea plants was studied. Tested anticytokinins, with the exception of the most efficient one – PU-1, were able to promote increased CKX activity in roots, when applied alone, but they had no significant influence on the enzymatic activity in leaves. N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (CPPU) provoked strong increase in CKX activity in roots, while in leaves considerable inhibition of enzymatic activity was observed. Different types of anticytokinins exhibited diverse preference towards taking off the action of purine and phenylurea cytokinins over CKX activity.     

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

Summary We report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N⁶-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl⁻¹ BA (8.8 and 17.7 μM) in combination with 0.5 mg l⁻¹ 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.     

Micropropagation of commercially important sugar beet cultivars

Plants have been regenerated from petiole explants of six elite sugar beet (Beta vulgaris L.) cultivars. Significant variation was found between the responses of the six cultivars to elevated temperature and the N⁶-benzyladenine concentrations evaluated. The importance of genotype in the regeneration of plantlets from sugar beet petiole explants is considered.     

Phytotoxic effects of Cu, Zn, Cd and Pb on in vitro regeneration and concomitant protein changes in Holarrhena antidysenterica

The nodal explants of in vitro shoots of Holarrhena antidysenterica L. were cultured on Murashige and Skoog's (MS) medium augmented with 15 μM N⁶-benzyladenine (BA) alone (control) or supplemented with different concentrations (1, 5, 10 and 20 mg dm⁻³) of CdCl₂, CuSO₄, Pb(NO₃)₂ and ZnSO₄. The maximum morphogenic response in terms of average shoot number (4.95 ± 0.17) was seen in control. ZnSO₄ proved to be less inhibitory in comparison to CuSO₄, Pb(NO₃)₂ and CdCl₂. None of the explants cultured on CdCl₂ containing medium induced multiple shoots. Maximum protein content [3.80 ± 0.04 mg g⁻¹(f.m.)] was observed in control and slightly less [3.50 ± 0.02 mg g⁻¹(f.m.)] in tissues exposed to 1 mg dm⁻³ of CuSO₄ and minimum [1.00 ± 0.02 mg g⁻¹(f.m.)] in Zn treated (20 mg dm⁻³) explants. SDS-PAGE analysis of the treated tissues revealed that two new polypeptides (29 and 20 kDa) in response to Cu and Zn treatment, respectively, have been synthesized.     

Organogenesis and plant regeneration in Taxus wallichiana (Zucc.)

We describe an efficient process for regeneration of Taxus wallichiana plants via shoot organogenesis from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown's basal medium supplemented with SH vitamin ((1/2) WPMSH), 0.5 mg l(-1) 6-benzyladenine (BA) in combination with 1.0-2.0 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) or alpha-Napthaleneacetic acid (NAA) produced two morphologically distinct types of calli-compact, green callus (CG) and compact, yellow (CY) callus after 4 weeks of culture. Optimum frequency (63%) of adventitious shoot bud induction was achieved in CG callus (3.0+/-0.67 shoot buds per gram of CG callus) when cultured on (1/2) WPMSH basal medium supplemented with 2.5 mg l(-1) BA after 4 weeks. The inclusion of 1% activated charcoal (AC) to (1/2) WPMSH basal medium (shoot elongation medium) led to maximum shoot elongation (2.15 cms). Microshoots rooted in high frequency (40%) in MS basal medium in which the concentration of nitrates was reduced to one-fifth the normal concentration after 4 months of culture.     

Micropropagation of Sicilian cultivars with an aim to preserve genetic diversity in hazelnut (Corylus avellana L.)

The use of a small number of cultivars in agriculture can lead to a loss of agrobiodiversity. Since in vitro techniques are valuable tools for conserving plant biodiversity, an efficient micropropagation protocol for four Italian hazelnut cultivars, ‘Carrello’, ‘Ghirara’, ‘Minnulara’, and ‘Panottara’, was developed. The highest axillary bud survival was obtained after decontamination with 40?min 1% sodium hypochlorite followed by 40?min 0.1% sodium merthiolate in ‘Minnulara’ and ‘Ghirara’, while the 35?+?35?min treatment was the best for ‘Carrello’ and ‘Panottara’. Shoot multiplication was higher in ‘Minnulara’ and ‘Ghirara’ when 6.6?μM N⁶-benzyladenine was used, even if some hyperhydric shoots were observed, while metatopolin was more effective in the other two cultivars. In vitro rooting, performed in ‘Carrello’ and ‘Panottara’, was higher with 17.6 than with 9.8 µM indole-3-butyric. Following in vitro root induction with 17.6 µM indole-3-butyric acid for 7 days, rooting and acclimatisation in greenhouse exceeded 85% for all four cultivars.     

Micropropagation of yellow cedar (Chamaecyparis nootkatensis)

Mature yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach) embroys were exposed to a range of N⁶-benzyladenine concentrations in a variety of culture media generally used for conifer caulogenesis. All seven media supported the induction of adventitious shoots but Schenk & Hildebrandt medium was the best. The best cytokinin level of N⁶-benzyladenine was 0.35 mg 1^-1. This resulted in an average of 4–5 large adventitious shoots per explant. Shoots arose primarily from the cotyledons regardless of whether they were in contact with the medium or not. Embryos from seeds stratified four weeks at 21°C and eight weeks at 5°C were more caulogenic than unstratified controls. An additional four weeks at 5°C caused a change in the pattern of shoot induction in that shoots arose from the hypocotyl as well as the cotyledons. Shoots elongated on basal Schenk & Hildebrandt medium. The best rooting response was obtained under non-sterile greenhouse conditions where approximately 60% of the shoots formed roots. Over a 12-month period the average shoot height ranged between 10–13.9 cm with a stem diameter of 2.29–2.68 mm. These propagules are still being grown under forest nursery conditions.     

Sugar-beet seed steep treatments to improve germination under cold,wet conditions

Modifying the present commercial sugar-beet steep treatment (12h in 0.2% thiram suspension, 25°C) to include an initial 2h steep in 0.3M hydrochloric acid, decreased fruit weight and cortex density and improved the performance of the inherently slower part of the population under cold, wet conditions. Adding gibberellins (GA_4/7) or an N-substituted phthalimide (AC 94377) to the thiram steep was also beneficial whereas kinetin of N⁶-benzyladenine gave no improvement. Germination was even more rapid and better synchronised following a 4-day seed advancement sequence, particularly when this started with the acid steep. Overall, it was possible to increase the proportion of seeds which gave a root or produced hypocotyls at least 2cm tall by 9% and 14% respectively; the thermal time needed for the 90th seed to germinate was reduced from 73 to 30 day degrees and synchrony could be improved at least two-fold.     

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Summary Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - CA casamino acids - B5CA B5 with 0.2% casamino acids - IBA indolebutryric acid; Picloram (4-amino-3,5,6-tricnloro-2-pyridinecarboxylic acid) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - BA 6-benzylaminopurine     

Organogenesis and tuberization in cultures of Dioscorea floribunda

Callus cultures were established from node and internode segments of Dioscorea floribunda Mart. & Gal. Both Murashige and Skoog's and modified White's medium supported callusing as well as organogenesis when supplemented with either 2,4-D or NAA in combination with BAP or Kn. On development of shoot primordia, calli were transferred to unsupplemented, half strength MS basal medium. This procedure led to the increase in formation of shoots. Several crops of shoots were obtained from single differentiating callus cultures by excising the shoots and subculturing the residual part. Seventy percent of plantlets survived rooting and transfer to soil.When they were maintained in half-strength MS basal medium and 0.5 mg1^-1 of NAA, 70% of plantlets formed aerial tubers at nodes. These tubers produced both roots and shoots and could be detached from the mother plant.     

Ce4+-stimulated ion fluxes are responsible for apoptosis and taxol biosynthesis in suspension cultures of Taxus cells

Ion fluxes across the plasma membrane activated by 1 mM Ce⁴⁺, cell apoptosis and taxol biosynthesis in suspension cultures ofTaxus cuspidata were studied. The extracellular pH sharply decreased upon the addition of 1 mM Ce⁴⁺, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular Ca²⁺ concentration decreased within the first 3 h after the addition of Ce⁴⁺, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a Ca²⁺-channel blocker indicated that the dynamic changes in extracellular pH and the Ca²⁺ concentration resulted from the Ce⁴⁺-induced activation of H⁺ uptake and Ca²⁺ influx across the plasma membranevia ion channels. A pretreatment of the ion channel blocker initiated Ce⁴⁺-treated cells to undergo necrosis, and the prior addition of the Ca²⁺-channel blocker inhibited Ce⁴⁺-induced taxol biosynthesis and apoptosis. It is thus inferred that H⁺ uptake is necessary for cells to survive a Ce⁴⁺-caused acidic environment and is one of the mechanisms of Ce⁴-induced apoptosis. Furthermore, the Ca²⁺ influx across the plasma membrane mediated both the Ce⁴⁺-induced apoptosis and taxol biosynthesis.     

Effect of a plant-derived smoke extract, N6-benzyladenine and gibberellic acid on the thermodormancy of lettuce seeds

The effects of a plant-derived smoke extract, BA and GA₃ on the thermodormancy of Grand Rapids lettuce seeds were studied. Thermodormant lettuce seeds treated either with BA, GA₃ or smoke extract alone did not germinate. Combinations of BA with smoke extract and BA with GA₃ were most effective in overcoming induced thermodormancy. GA₃ plus smoke did not break the induced thermodormancy. The effects of the different treatments on germination were concentration dependent. BA was most effective at 10^–5 to 10^–3 M in combination with smoke dilutions 1:5,000 to 1:1,000,000 in overcoming thermodormancy.Abbreviations BA N⁶-benzyladenine; - GA₃ gibberellic acid; - SM smoke extract     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Axillary shoot proliferation and in vitro flowering in an adult giant bamboo, Dendrocalamus giganteus Wall. Ex Munro

Summary Continuous axillary shoot proliferation and in vitro flowering were achieved using single node explants from a mature (over 70-yr-old) field clump of Dendrocalamus giganteus (giant bamboo). The shoots proliferated in a basal Murashige and Skoog medium with 6 mgl⁻¹ (26.6 μM) N⁶-benzyladenine (BA) and 2% sucrose. The rate of shoot proliferation gradually increased to over three-fold before in vitro flowering took place. In vitro flowering was not the expression of a species-specific mechanism believed to occur during gregarious flowering, as the mother clump did not flower. The rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation leading to build up, and release of stress in relation to flowering and its reversion are discussed.     

N6-methyl adenosine in siRNA evades immune response without reducing RNAi activity

Abstract

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N⁶-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N⁶-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.     

Production of taxol and related taxanes in Taxus brevifolia cell cultures: Effect of sugar

Summary Suspension cells of Taxus brevifolia were cultured as an alternative source of taxol and related taxanes. The effects of basal medium, sugar type, and sugar concentration on growth and taxane production were investigated. To induce taxol production, modification of medium composition was necessary. Fructose supported the best taxol yield and the use of high concentration of sugar was desirable. The maximum level of taxol obtained after 10 days of culture in an optimized condition was 1.43 mg/L, which was 0.013% as a specific content.