Effect of culture medium and illumination on the acid invertase activity in callus of Medicago strasseri

The different acid invertase activity (total, soluble, wall-bound and extracellular) in calli induced on explants (cotyledon, petiole, hypocotyl and leaf) originated from Medicago strasseri seedlings were evaluated. In cultures subjected to 16 h photoperiod, the highest total, soluble and extracellular activities were found in calli from leaves cultured in medium 12 (MS with 0.01 mg·dm⁻³ (0.045 μM) of TDZ), elevated amounts of total and wall-bound invertase being found in calli induced on petioles in 12G medium (MS with 0.01 mg·dm⁻³ (0.045 μM) TDZ and 3.104 mg·dm⁻³ glycerol). In cultures maintained in darkness, the activity detected was lower than that observed in cultures under light conditions. The highest amounts of enzyme was bound in calli cultured on medium 12 (total and extracellular invertase) -leaves- and medium 12D (MS with 0.001 mg·dm⁻³ (0.0045 μM) TDZ) (soluble invertase) -using hypocotyls. In general, the different forms of invertase activity studied seem to appear in greatest amounts in calli induced under light conditions using leaves as explant and TDZ as growth regulator.     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

Influence of physical conditions of nutrient medium and sucrose on somatic embryogenesis of date palm

Shoot tips and leafy bud fragments removed from offshoots of adult date palms (Phoenix dactylifera L.) were cultured on a nutrient medium containing the Murashige and Skoog inorganic salts, 453 M 2,4-dichlorophenoxyacetic acid, 14.8 M N⁶-(2-isopentenyl)adenine and 3 g l^-1 activated charcoal to develop nodular callus after 8 months of culture. Callus was cultured in agar-solidified and stationary or shaken liquid media containing half-strength MS inorganic salts, 3 g l^-1 activated charcoal and different sucrose concentrations to study the influence of these factors on somatic embryogenesis. The best conditions for embryo development were culturing in liquid medium shaken at 100 rpm for a period of 2 weeks without sucrose, followed by culture on 3% sucrose.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP N⁶-(2-isopentenyl) adenine - MS Murashige & Skoog (1962) - rpm revolutions per minute     

Effect of temperature on carbohydrate content,ADP glucose pyrophosphorylase,and ATP- and PPi-dependent phosphofructokinase activity of potato tuber callus tissue

Callus derived from Lemhi Russet and Russet Burbank tuber tissue was incubated at 20°C and 30°C on a high sucrose medium for starch-formation and subjected to simulated storage and reconditioning treatments at 5°C and 25°C after transfer of the callus to a medium without a carbon source. Percent dry weight of callus from both cultivars averaged about 21% after starch formation and 5% after storage and reconditioning treatments. Total sugars were higher in callus incubated on the starch forming treatment. Lemhi Russet callus contained predominantly reducing sugars, while Russet Burbank callus contained mostly non-reducing sugars. Total sugar content was generally lower for both cultivars after the storage and reconditioning treatment in callus after starch formation at 20°C. Starch content was generally higher in Lemhi Russet tissue. After starch formation at 20°C Lemhi Russet had higher starch after the storage and reconditioning treatment than tissue from 30°C, while the opposite trend was found in Russet Burbank tissue. Total protein declined in Russet Burbank tissue during the storage and reconditioning treatment regardless of prior incubation conditions, while this decline only occurred in Lemhi Russet tissue initially incubated at 30°C during the starch formation treatment. In tissue of both cultivars, ATP- and PPi-dependent phosphofructokinase activities were inversely proportional to total sugar concentrations, while in RB callus ADP glucose pyrophosphorylase activities were proportional to starch content.Research Paper 91B1 of the Idaho Agricultural Experiment Station.     

Differences in invertase activity in embryogenic and non-embryogenic calli from Medicago arborea

The different invertase activities in embryogenic and non-embryogenic calli induced from explants (cotyledons, petioles, hypocotyls and leaves) obtained from Medicago arborea L. subsp. arborea seedlings were evaluated. Total invertase activity was lower in the calli with the greatest embryogenic capacity. The greatest fraction of this activity corresponded to soluble invertase. Wall-bound invertase showed maximum activity during the first two months of culture and the highest activities of this type were found in non-embryogenic calli. Extracellular invertase formed the smallest fraction of the total invertase activity evaluated. Acid and alkaline invertase activities were found in all calli but differences were detected between the embryogenic and non-embryogenic calli. In the former, the activity of both types of invertase exhibited a similar type of behaviour but different from that observed in the non-embryogenic calli. The calli with the greatest embryogenic capacity had very low levels of acid invertase and very high levels of the alkaline form. Soluble invertase – both acid and alkaline – accounted for the highest fraction after the first two months of culture and was present in lower amounts in the embryogenic than in the non-embryogenic calli. Regarding bound invertase, the highest production was seen to correspond to acid invertase. The extracellular invertase evaluated corresponded to the acid form since the alkaline extracellular invertase did not show any physiologically significant activity.     

培养条件对三七愈伤组织生长和皂苷积累的影响

以MS为基础培养基,改变激素配比、氮源和光照等因素,以分光光度法和HPLC法分析三七愈伤组织培养过程中皂苷含量的变化。结果表明:培养条件对三七愈伤组织中皂苷积累有一定影响,激素配比对愈伤组织中皂苷含量的影响最大,在0.5 mg·L-12,4-D+1.0 mg·L-16-BA组合下,培养物中总皂苷含量最多,达到4.72%±0.29%;在总氮量为60 mmol·L-1条件下,45 mmol·L-1KNO3+7.5 mmol·L-1NH4NO3(NO3-/NH4+=7∶1)时,愈伤组织皂苷含量最多,达到4.71%±0.17%;分别在1 000 lx和500 lx光强下每天光照12 h的愈伤组织,皂苷含量均低于黑暗培养的愈伤组织,三者皂苷含量分别为1.94%±0.31%、2.38%±0.12%和3.57%±0.27%,光照引起愈伤组织表面变绿及细胞分化,可能是抑制愈伤组织中皂苷合成与积累的主要原因;HPLC检测发现,三七愈伤组织和根中均含有Rg1、Re、Rb1及Rd四种皂苷,但栽培三七根含有R1皂苷,而三七愈伤组织中未检测到R1,其原因需要进一步研究。该研究结果为未来愈伤组织培养成为部分代替人工栽培生产三七天然产物的潜在途径提供了研究基础。     

Effects of carbohydrate addition on the induction of somatic embryogenesis in Hevea brasiliensis

Plant Cell, Tissue and Organ Culture (PCTOC) - The effect of different carbohydrates was tested on early somatic embryogenesis of Hevea brasiliensis. Sucrose was replaced with maltose, fructose or...     

Production of polysaccharides by Silene vulgaris callus culture depending on carbohydrates of the medium

Sources of carbohydrate nutrition such as sucrose, glucose, and galactose, with the exception of arabinose, were shown to influence positively callus growth and polysaccharide (pectin silenan and acidic arabinogalactan) biosynthesis. Galactose was found to cause a stimulatory effect on yield and productivity of arabinogalactan. Low concentrations of sucrose failed to support the cell growth and polysaccharide biosynthesis. Increasing sucrose concentrations led to biomass accumulation but failed to enhance efficiency of the substrate utilization. The optimal medium for the campion cell culture growth was found to be one containing 30 g/liter of sucrose or a mixture of sucrose with glucose (in 15 g/liter). Increasing sucrose concentrations in the medium from 30 to 100 g/liter failed to significantly influence the polysaccharide yields while the polysaccharide productivity per liter of the medium grew due to promotion of culture productivity in biomass. Variations of the carbon sources in the nutrient media were shown to influence insignificantly the biochemical characteristics of arabinogalactan and silenan while an increase in the sucrose concentration to 50-100 g/liter led to a diminution of the galacturonic acid content in silenan and to changes in contents of the neutral monosaccharide residues in silenan and arabinogalactan.     

Plant regeneration from explant and protoplast derived calluses of Medicago littoralis

Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l^–1 2,4-dichlorophenoxyacetic acid and 0.5 mg l^–1 N⁶-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N⁶-benzyladenine plus -naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine - IAA indole-3-acetic acid - BOA 1,2-benzisoxazole-3-acetic acid - KIN kinetin - MS Murashige & Skoog (1962) - GRFMS growth regulator free MS medium - B5 Gamborg et al. (1968) - RL Phillips & Collins (1979) - KM8 KM8P Kao & Michayluk (1975) - CPW Frearson et al. (1973) - f. wt fresh weight - FDA fluorescoin diacetate     

Interspecific hybridization of perennial Medicago species using ovule-embryo culture

Summary New interspecific hybrids between alfalfa (Medicago sativa L.) and several perennial Medicago species were obtained by embryo rescue techniques. The methodology, designated ovule-embryo culture, involved preculturing the fertilized ovule (10 to 20 days post-pollination) for a period of six to 12 days followed by excision and direct culture of the embryo. Placement of the hybrid embryo directly onto culture medium without the interim ovule culture was unsuccessful. Ovule culture to germination without removing the embryo also was unsuccessful. Ovule-embryo culture was essential for recovering interspecific hybrids between diploid alfalfa (2n=2x=16) and the following diploid (2n=2x=16) species: M. hybrida Traut., M. marina L., M. papillosa Boiss., M. rhodopea Velen. and M. rupestris M.B. In addition, trispecies hybrids between M. sativa x M. dzhawakhetica Bordz. F₁ hybrids (2n=3x=24) and either M. cancellata M.B. (2n=6x=48) or M. saxatilis M.B. (2n=6x=48) were obtained from ovuleembryo culture. Media manipulations using M. sativa x M. rupestris F₁ and first backcross generation embryos demonstrated the optimum concentration of 12.5 mM NH₄ ⁺ for successful embryo rescue; ammonium salt formulation (whether chloride, nitrate or sulfate) was not critical. From a few thousand crosses, hybrids between M. sativa and either M. rhodopea or M. rupestris were recovered relatively efficiently with 157 and 66 hybrids, respectively. However, only 13 hybrids between M. sativa and M. papillosa were obtained from more than 2,000 crosses, and just two hybrids each have been recovered from the combinations M. sativa x M. hybrida and M. sativa x M. marina from 2,000 to 3,000 crosses. The predominant chromosome number between diploid alfalfa and the other diploid perennial species was 2n=2x=16. Morphology of the hybrids was generally intermediate. Electrophoretic analysis of the F₁ hybrids and parental clones on uniform or gradient polyacrylamide gels demonstrated that peroxidase phenotypes could be used to confirm hybridity. For all interspecific combinations there was at least one peroxidase isozyme unique to the wild species that was present in the F₁ interspecific hybrid.     

Regeneration and characterization of plants obtained from anther cultures in Medicago sativa L

Summary The androgenic ability of four Medicago sativa L. genotype (Boynitza 5, Byala, 494, and 3815) was tested. Callus and organogenesis were induced in all lines studied. The percentage of anthers producing calluses and organogenesis showed wide variation (calluses—from 11% up to 77%; organogenesis—4.8% to 15.2%). It has been established that genotype, nutrient medium composition, and stage of pollen development considerably affected both callus production and organogenesis. Androgenesis in M. sativa could be achieved via callus and direct embryogenesis. About 500 morphologically different regenerants were obtained. Wide variability in chromosome number of regenerated plants was observed by cytological studies. Haploid, dihaploid, as well as mixoploid plants were obtained.     

Defined conditions for the initiation on growth of cotton callus in vitro I.Gossypium arboreum

Summary Defined in vitro conditions for callus initiation byGossypium arboreum L. were determined, and different tissues were evaluated as explant sources. Environmental conditions tested included light versus dark, and low light versus high light. Different nutrient media as well as carbohydrate sources were examined. Our data show that hypocotyl tissue was superior to cotyledon or leaf tissue as the explant source for callus proliferation; the Murashige-Skoog inorganic formulation with (in mg per 1) 100 myo-inositol, 0.4 thiamine·HCl, 2 indoleacetic acid (IAA), 1 kinetin, and 3% glucose solidified by agar was the best medium to initiate callus. Cultures with sucrose as a carbohydrate source browned rapidly. Callus proliferation was superior under high light (8000 to 9000 lux) conditions at 29±1°C. Various combinations of auxins and cytokinins were tested for their ability to improve callus proliferation and subsequent growth of subcultures. Although the MS medium containing IAA and kinetin was found superior for obtaining rapid proliferation of callus from hypocotyl explants, a second medium containing 2 mg per 1 naphthalenacetic acid (NAA) and 0.5 to 1 mg per 1 benzyladenine (BA) was found necessary for vigorous growth of subcultured callus. A MS medium with 5 to 10 mg per 1 {ie329-1} (2iP) and 1 mg per 1 NAA was also favorable for continued subculturing. Technical Article 12485 from the Texas Agricultural Experiment Station.     

Effects of in vitro tissue culture conditions and acclimatization on the contents of Rubisco, leaf soluble proteins, photosynthetic pigments, and C/N ratio

The levels of two subunits of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), total soluble proteins, carbon and leaf nitrogen content, and photosynthetic pigments in various plants (avocado, oak, olive, and strawberry) grown in vitro and ex vitro were analysed. Compared to ex vitro grown plants, micropropagated avocado, oak, and strawberry showed a markable decrease in large subunit Rubisco. However, the small subunit only decreased in strawberry and oak. Contrary to this, olive did not reveal any difference in the level of either subunit. The C/N ratio increased significantly in in vitro grown plants, except in the case of olive, where an opposite behaviour was found. Leaf chlorophyll concentration on unit mass basis was higher in all the in vitro plants than in those of greenhouse- grown plants. Only avocado plantlets showed a statistically significant decrease in total soluble proteins. Further, overall data suggest that in vitro cultural conditions have a species-specific influence on large and small subunits of Rubisco, independent of the protein, chlorophyll, or nitrogen level.     

Role of exogenous reduced nitrogen and sucrose in rapid high frequency somatic embryogenesis in Medicago sativa

The effect of exogenously supplied reduced nitrogen and sucrose on high-frequency somatic embryogenesis in petiole-derived tissue cultures of a diploid and a tetraploid regenerable clone of Medicago sativa ssp. falcata was investigated. There was an absolute requirement for ammonium during embryo induction and differentiation, with 5mM being the optimum for induction and 10–20 mM the optimum for differentiation of somatic embryos. Exogenous amino acids were not essential for differentiation and often even inhibitory, except 1 or 2 g/l casein hydrolysate or 4.4 mM glutamine with 3.1 mM proline which, under certain conditions, resulted in increases of 20–30% in the number of embryos obtained. High and low sucrose concentrations inhibited somatic embryogenesis and there was no reason to deviate from the 3% (0.088 M) sucrose level commonly used in plant tissue culture media. Selected clones from three M. sativa cultivars showed a response similar to the highly regenerable ssp. falcata clone F1.1.     

Cold acclimation and abscisic acid induced alterations in carbohydrate content in calli of wheat genotypes differing in frost tolerance

The effect of cold and abscisic acid (ABA) treatment on soluble carbohydrate content was compared in callus cultures of wheat genotypes differing in frost tolerance. The effect of 5A chromosome substituted from the frost tolerant to the sensitive on cold-induced carbohydrate accumulation was also determined. Following cold hardening, the increase in sucrose and fructan level in calli of tolerant varieties was significantly higher than those of the sensitive ones. In 5A substitution line higher sucrose and fructan content was detected than in recipient . Tendentiously, cold stress caused higher degree of changes in carbohydrate content than the exogenously applied ABA did. Comparing the accumulation pattern of the components of WSC measured in vitro to the previously published in vivo results it can be concluded that in the case of sucrose and fructans it was similar, while for the reducing sugars it was different. The regulatory role of chromosome 5A either in the development of freezing tolerance or carbohydrate accumulation was confirmed in dedifferentiated calli, as well.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Isolation and culture of Lens culinaris Medik. cv. Eston epicotyl protoplasts to calli

Protoplasts of Lens culinaris Medik. cv. Eston were isolated from epicotyl tissues of seedlings grown on Murashige & Skoog basal medium. For isolating the protoplasts, epicotyl tissues were digested for 12–14 h at 25°C in an isolation mixture (pH 5.7) containing 1% Cellulase RS, 0.5% Driselase, 0.25% Pectolyase Y23, 0.2M calcium chloride, 10 mM mannitol and 10 mM MES. Protoplasts were purified by flotation over 20% sucrose and washed with 0.2 M calcium chloride solution supplemented with 10 mM mannitol. Purified protoplasts were cultured at a density of 10⁵ ml^-1 in agarose (Seaplaque, 0.6%) blocks which were suspended in an identical but liquid KM8P culture medium lacking amino acids, ammonium nitrate, and coconut water but containing 0.35 M glucose and a growth regulator complement of either 2.2 M 2,4-dichlorophenoxyacetic acid (2,4-D), 2.7 M naphthaleneacetic acid (NAA), 2.3 M N-(2-furanylmethyl)-1H-purine-6-amine (kinetin), 2.2 M benzylamino purine (BAP), 2.3 M 2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol (zeatin), and 1.4 M gibberellic acid (GA₃), or 5.4 M NAA and 2.2 M each of 2,4-D and BAP. The osmotic potential of the liquid culture medium was gradually reduced over a period of 3 weeks by replacing the spent medium with a fresh medium containing 0.25, 0.1 and 0 M glucose at weekly intervals. About 6% of the dividing protoplasts developed into cell colonies after 3 weeks of culture at 25°C in diffuse light (10 E m^-2s^-1). In 35–42 days the microcolonies were about 1 mm in diameter and developed into calli on transfer to agar-solidified B5 medium supplemented with growth regulators used in the protoplast culture medium and 5 mM glutamine. Attempts to regenerate plants from protoplast-derived calli have so far been unsuccessful.Department of Applied Microbiology and Food Science, University of Saskatchewan     

Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia

Micropropagated shoots of Maytenus ilicifolia Mart. were obtained from axillary buds cultured in Murashige & Skoog medium supplemented with 13.3 M 6-benzyladenine (BA). Addition of 1.1 M 1-indole-3-acetic acid (IAA) to the medium increased shoot elongation. The number of shoots formed was influenced by BA concentration, degree of juvenility of the explant, and by bud explant position on the stem. Cultures of buds taken from stem parts located close to the shoot tip yielded more callus than shoots, whereas axillary buds at distant positions from the apical bud yielded more shoots.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic-acid     

Continuous long-term somatic embryogenesis in alfalfa

Summary Somatic embryogenic callus was induced on two induction media, B5h and SH4K. Embryos formed on the callus induced on B5h medium when the callus was still on the induction medium. On the other hand, embryos could not form on the callus induced on SH4K medium unless the callus was transferred to a growth regulator-free medium. Callus induced and maintained on B5h medium lost embryogenic capability quickly during the subculture. Callus induced and maintained on SH4K medium, however, consistently remained highly embryogenic. The callus mass showed steady increase during its maintenance on SH4K medium. The embryos induced on SH4K medium showed vigorous germination. Normal and fully fertile plants were recovered from the embryos developed from the callus maintained on SH4K medium.