Callus Relative Growth Rate Research Articles

α-Tocopherol application as a countermeasure to UV-B stress in bread wheat (Triticum aestivum L.).

The source of energy for all photoautotrophic organisms is light, which is absorbed by photosynthetic processes and used to transform carbon dioxide and H2O into organic molecules. The majority of UV-B light (280 to 320 nm) is absorbed by stratospheric ozone layer, although some of it does reach at the Earth's surface. Because of the sedentary lifestyle of plants, this form of abiotic stress is unavoidable and can induce growth and even cell death. Ten-day-old calli generated from mature Kirik wheat embryos were subjected to UV-B radiation for 0, 2, 4, and 6 h to examine the function of exogenous α-tocopherol, a lipophilic antioxidant, in wheat tolerance to UV-B radiation stress. The calli were then moved to a callus medium containing α-tocopherol (0, 50, and 100 mg/l) and cultivated there for 20 days after being subjected to UV-B stress. For plant regeneration, embryogenic calli were put on a medium for plant regeneration after 30 days. The findings of this investigation demonstrated that an increase in UV-B exposure period resulted in a substantial drop in the relative growth rate of callus, the rate of embryogenic callus, the rate of responding embryogenic callus, and the number of plants in each explant. On the other hand, with the application of α-tocopherol, all these parameters improved, and the best result was observed in the application of 100 mg/l of α-tocopherol in terms of plant regeneration under UV-B stress.

Inducing Ni sensitivity in the Ni hyperaccumulator plant Alyssum inflatum Nyárády (Brassicaceae) by transforming with CAX1, a vacuolar membrane calcium transporter

AbstractThe importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were sub‐cultured three times on kanamycin‐rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0–500 µM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 µM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants.

In vitro micropropagation and allelopathic effect of lantana (Lantana camara L.)

The invasive plant, lantana (Lantana camara L.), is well known as a traditional medicinal plant and it may become important in the development of modern drugs. Lantana has long been touted as containing potent allelochemicals and in vitro-produced tissues may be appropriate sources for the production and isolation of bioactive compounds. In this research, effective techniques for shoot multiplication and root and callus induction were developed and the allelopathic efficiency of in vitro leaf and callus was examined. The optimized medium for shoot multiplication was Murashige and Skoog (MS) medium supplemented with 12.0–20.0 μM thidiazuron. For rooting, high root numbers were obtained on MS medium containing 5.0 or 10.0 μM 1-naphthalene acetic acid (NAA). In addition, the highest relative growth rate of callus was achieved when lantana leaf was cultured on NB medium (MS medium with 21.5 μM NAA and 22.5 μM N6-benzyladenine). For allelopathic effects, the results suggested high potential activity of lantana leaf and callus that was able to variably inhibit the seed germination and seedling growth of all four test species. Leaf and callus extract had no significant effect on the germination of Brassica campestris var. chinensis. Callus extract showed superior ability to suppress germination for Ipomoea aquatica Forsk. and Zea mays L. but inferior inhibition ability for Sorghum bicolor L. These results suggested that the extract of lantana in vitro leaf and callus will be an interesting natural source for further study to develop natural herbicides.

Optimization of induction, subculture conditions, and growth kinetics of Angelica sinensis (Oliv.) Diels callus.

Background:Angelica sinensis (Oliv.) Diels is an important traditional Chinese medicine, and the medicinal position is its root. This perennial herb grows vigorously only in specific areas and the environment. Tissue culture induction of callus and plant regeneration is an important and effective way to obtain large scale cultures of A. sinensis.Objective:The objective was to optimize the inductive, subculture conditions, and growth kinetics of A. sinensis.Materials and Methods:Tissue culture conditions for A. sinensis were optimized using leaves and petioles (types I and II) as explants source. Murashige and Skoog (MS) and H media supplemented with 30 g/L sucrose, 7.5 g/L agar, and varying concentrations of plant growth regulators were used for callus induction. In addition, four different basal media supplemented with 1.0 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D), 0.2 mg/L 6-benzyladenine (BA) and 30 g/L sucrose were optimized for callus subculture. Finally, growth kinetics of A. sinensis cultured on different subculture media was investigated based on callus properties, including fresh weight, dry weight, medium pH, callus relative fresh weight growth, callus relative growth rate (CRGR), and sucrose content.Results:MS medium supplemented with 5 mg/L α-naphthaleneacetic acid, 0.5 mg/L BA, 0.7 mg/L 2,4-D, 30 g/L sucrose and 7.5 g/L agar resulted in optimal callus induction in A. sinensis while petiole I was found as the best plant organ for callus induction. The B5 medium supplemented with 1.0 mg/L 2,4-D, 0.2 mg/L BA and 30 g/L sucrose displayed the best results in A. sinensis callus subculture assays.Conclusion:The optimized conditions could be one of the most potent methods for large-scale tissue culture of A. sinensis.

Callus induction in plantain (Plantago major L.) for in vitro production of mucilage

Plantago major is a medicinal herb belongs to the family Plantaginaceae. The effects of a range of plant growth regulators and different explants on mucilage production were examined. Root and leaf tissues were used as explants. Murashinge and Skoog (MS) medium supplemented with 2,4-D or Kinetin, at 0.01, 0.1 and 0.5 mg l -1 alone or in combination with IAA 1 mgl-1 for callus induction. Result of analysis of variance showed significant difference between explants for some traits included callus induction rate, fresh weight, and callus diameter. Also, significant differences were observed between media for callus induction, callus diameter, fresh weight, dry weight, relative water content of callus, mucilage harvest index and mucilage yield. The best response was observed from root segments on MS medium containing 0.8 mg/l 2,4,D and 0.1mg l -1 kinetin in some traits included callus diameter, fresh and dry weight, mucilage percentage and mucilage yield. Results revealed that the highest relative growth rate of callus was observed on media containing 1 mg l-1 2,4-D and 0.5 mg l-1 kinetin. MS medium supplemented with 2.5 mgl -1 2,4-D and 0.01 mg l -1 kinetin showed the highest harvest index of mucilage. Optimal medium for mucilage production was found to be MS medium containing 0.8 mgl -1 2,4-D and 1 mg l-1 kinetin and root as explants. Results of mean comparison for mucilage production showed that media containing 0.8 mg l -1 2,4-D and 0.1 mg l -1 kinetin and leaf as explant was produced higher mucilage than that of control plant.

Assessment of immature embryo culture to select for drought tolerance in bread wheat

In order to evaluate the response of bread wheat genotypes to callus induction and in vitro drought stress, an investigation was carried out as a factorial experiment with a completely randomized design and five replications. The results of analysis of variance indicated significant differences between the entries and stress levels for callus relative growth (CRG), callus relative growth rate (CRGR), callus growth rate (CGR), percentage of callus chlorosis (PCCH) and percentage of callus water content (PCWC) indicating the presence of genetic variability, different responses of genotypes to different drought intensities and invitro selection of droughttolerant genotypes. Mean comparisons between genotypes revealed that maximum CRG, CGR, CRGR, PCWG, PCCH and INTOL were attributed to genotypes 5, 16, 17, 2, 3 and 20 (drought tolerant), respectively. Graphic observation exhibited that indices of drought tolerance decreased with increase of PEG concentrations. Cluster analysis of genotypes (Ward’s method) based on CRG, CGR, CRGR, PCWG, PCCH and INTOL and subsequent discriminant analysis for confirming the number of clusters, grouped the genotypes into four different clusters. The first group included genotypes 1, 3, 6, 7, 12, 13 and 18, the second group included genotypes 2, 4, 8, 9, 10 and 11 and the third group consisted of genotypes 14, 16, 17, 19 and 20, while the genotype 15 formed the fourth group. Superior genotypes 2, 5 and 16 showed drought tolerance at the callus culture level together with their high potential for callus induction leaded us to the conclusion that a hybridization breeding program using these superior plant materials supplemented with in vitro selection for drought tolerance might be beneficial for improvement drought tolerance in bread wheat.

Effects of magnetic field on cell dedifferentiation and callus induction derived from embryo culture in bread wheat (Triticum aestivum. L) genotypes

Current study was conducted to investigation of effect of magnetic field on cell dedifferentiation and follow it callus induction derived from mature embryo culture in bread wheat genotypes. For this purpose, a factorial experiment based on completely randomized design was carried out with two wheat genotypes and three level of magnetic field strength (0.0, 8.8 and 17.6 Tesla) in three replications. Callus growth rate (CGR), relative growth rate (RGR), callus relative growth rate (CRGR), percentage of callus water content and percentage of callus induction traits were measured. To sum up, the results showed that differences between wheat genotypes and level of magnetic field strength were significant for some studied treats related to callus induction. The effect of magnetic field levels on CGR (from 0.181 to 0.175), RGR (from 1.442 to 0.655) and CRGR (from 0.052 to 0.022) were decreased with increment of magnetic field intensity.

In vitro selection of resistant mutant garlic lines by using crude pathogen culture filtrate of Sclerotium cepivorum

In vitro treatment of tissue cultures of two garlic cultivars with crude culture filtrates from Sclerotium cepivorium was used to select somaclonal variants with enhanced resistance to white rot disease. The crude culture filtrate had a negative effect on callus induction and growth. It also inhibited the differentiation of adventitious buds. The inhibitory effects were greater at higher culture filtrate concentration. With increase in concentration of crude pathogen culture filtrate, rate of callus induction, callus survival, relative growth rate of callus, callus differentiation and number of divided shoots per callus decreased. The inhibitory effect for all the parameters was more visible when crude pathogen culture filtrate was used at 50% concentration or more. However, the performance of disease resistant cultivar Hangzhong Red Skin was better than Gailiang (disease susceptible cultivar) at higher concentrations of crude pathogen culture filtrate. Some somaclonal lines and plantlets (predominantly from Hangzhong Red Skin) that showed resistance to Sclerotium cepivorum were obtained at 50%concentration level of crude pathogen culture filtrate.

Relationship between Callus Growth and Mineral Nutrients Uptake in Salt-Stressed Indica Rice Callus

ABSTRACT One month old rice calli were exposed to 0, 50, and 100 mol m−3 sodium chloride (NaCl) in the liquid LS basal medium supplemented with 2.5 mg L−1 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L−1 kinetin. Callus relative growth rate (RGR; fresh) of both cultivars indicated a progressive decrease; however, callus dry weight increased as the NaCl level increased in the culture medium. Salinity stress increased the callus sodium (Na+), manganese (Mn2+), and magnesium (Mg2+) contents while potassium (K+), calcium (Ca2+), and iron (Fe2+) contents decreased. ‘Basmati-385’ showed less reduction in callus RGR, K+, and Ca2+ contents and a larger increase in callus dry weight, Na+, Mn2+, and Mg2+ contents as compared to ‘Basmati-Karnal’. However, the reverse was true for Fe2+ contents. K+/Na+ and Ca2+/Na+ ratios generally decreased under salt stress. Overall, reduction in callus relative growth rate was found to be inversely correlated with decrease in K+, Ca2+, and Fe2+ uptake and directly correlated with increased Na+ and Mg2+ concentration in callus tissue.

Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes

One-month old calli of two indica rice genotypes, i.e., Basmati-370 and Basmati-Kashmir were subjected to two iso-osmotic concentrations (−0.57 MPa and −0.74 MPa) created with 50 and 100 mol m−3 NaCl or 10 and 18% solutions of PEG-8000. Both genotypes tolerated only low levels of stress and showed severe growth suppression at −0.74 MPa. The degree of stress tolerance of both genotypes was greater for PEG induced stress than for NaCl induced stress. The relative growth rate of callus was reduced under both stresses, however, the reverse was true for callus dry weight. Sodium (Na+) content of the callus tissue was increased only under NaCl induced stress. Salt induced stress reduced K+ and Ca2+ contents, but the PEG induced stress increased them. Higher levels of stress increased the proline content many folds with more increase being under PEG stress than that under NaCl. Water and osmotic potentials of the callus tissue decreased, whereas turgor potential increased under both abiotic stresses. Overall, Basmati-370 was more tolerant to both NaCl and PEG induced stresses than Basmati-Kashmir, because of less reduction in growth and more dry weight. Moreover, Basmati-370 accumulated higher amounts of cations, free proline, and maintained maximum turgor as compared to Basmati-Kashmir. In conclusion, at cellular level, mechanism of NaCl induced osmotic stress tolerance was found to be associated with more ionic accumulation of inorganic solutes and that of PEG induced osmotic stress tolerance with the accumulation of free proline, as an important osmolyte in the cytosol.

Effects of salinity on growth and compatible solutes of callus induced from Populus euphratica

Abstract The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l−1 (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l−1 (2.7 μM) 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l−1 (1.1 μM) BA and 0.5 mg l−1 NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l−1 NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.

Long-term culture modifies the salt responses of callus lines of salt-tolerant and salt-sensitive tomato species

Summary The changes in the salt responses induced by long term callus culture were studied in leaf callus tissues of the cultivated tomato species ( Lycopersicon esculentum Mill.) and its wild salt-tolerant relative L. pennellii (Correll) D'Arcy. The salt-tolerant wild species was able to adapt very quickly to the salt medium: after only three subcultures in salt medium (175 mmol/L NaCl) the callus relative growth rate (RGR) and the cell volume were higher in the salt medium than in the control. I n the cultivated species, two phases or steps were distinguished. In the first, callus RGR and cell volume tended to decrease with the subculture, both in the control and in the saline media (100 mmol/L NaCl), although the growth reduction was higher in stressful medium; this step seems to correspond to an adaptive period. In the second step, which would correspond to the phase following the adaptive period, callus RGR and cell volume increased as subculture advanced. In both species, the different growth changes along the subcultures had an influence on the degree of salt tolerance. Furthermore, the salt responses varied according to the procedence of calli (from control or saline media). In any case, the salt tolerance of the wild species was much higher than that of the cultivated species. The possibilities of using tissue cultures as tools to obtain salt-tolerant lines, as well as to evaluate the salt tolerance, are discussed.

NaCl Responses in Tomato Calli and Whole Plants

Summary The comparative salt responses between calli and whole plants were studied in the cultivated tomato species ( L. esculentum Mill.) and its wild salt-tolerant relative L. pennellii (Correll) DArcy. Based on the reduction in both the callus relative growth rate and plant growth, L. pennellii was more salt-tolerant than L. esculentum , which indicates a positive correlation between the salt responses at both organizational levels. The changes induced by salinity in water contents and osmotic potential (ψ s ) were different in calli (initiated from leaves) and in leaves of whole plants. In calli, both the water contents and ψ s decreased with salinity in the two tomato species, with the reductions being higher in the salt-treated calli of L. esculentum than in those of L. pennellii . In contrast to the callus response, the leaf water contents increased with salinity in both species, especially in the wild species. Moreover, a higher ψ s reduction as well as a higher solute contribution to the osmotic adjustment were observed in NaCl-treated leaves of L. pennellii than in those of L. esculentum . In both species, the accumulation patterns of Na + were quite similar in calli and leaves when the contents were calculated on a dry weight basis, with the Na + accumulation being greater in the wild species. However, the Na + accumulation was even slightly higher in salt-treated calli of L. esculentum when the contents were calculated on a tissue water basis. The different physiological responses found at both organizational levels were due to the different water uptake capacities of calli and leaves in saline medium; thus, the higher ψ s reduction and the higher Na + accumulation found in the L. esculentum calli were related to the higher callus dehydration by salinity, which did not occur in leaves of whole plants. Notwithstanding, the higher K + levels as well as the higher K + /Na + selectivity were shown at both organizational levels, although the greatest differences were found at the whole plant level.

In vitro selection of wheat callus tolerant to high levels of salt and plant regeneration

Embryogenic calli isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The effect of NaCl on the selected and unselected cell lines has been investigated. The results indicated that the relative growth rate of callus decreased as the concentration of NaCl increased in both callus lines. The selected callus line gave a higher growth weight in the presence of NaCl in the medium and was highly significant as compared with unselected callus line across medium protocols in all wheat cultivars. The dry weight of both kinds of callus lines of all wheat cultivars increased markedly with increasing NaCl concentration in most cases. The Na+ and Cl- contents of both callus lines were increased with increasing salinity levels while K+ content was decreased. The selected callus line of each cultivar at the same salinity level produced significant amounts of Na+, K+ and Cl- higher than the unselected callus line in most salinity levels. However, the unselected callus lines of the cultivars Giza-157 and Sakha-90 at the same salinity level produced significant amounts of K+ higher than the selected callus line in most salinity levels. The proline content of both kinds of callus lines for all wheat cultivars was increased with increasing salinity level. However, the selected callus line gave a significantly higher proline content than the unselected callus line in all wheat cultivars at the same Salinity level. Results from the in vitro selection for NaCl tolerance showed that the stepwise method of increasing NaCl in the medium was more effective for plant regeneration than other methods.