Short-term in vitro storage of t Miscanthus x ogiformis Honda 'Giganteus' as affected by medium composition,temperature, and photon flux density

Miscanthus x ogiformis Honda 'Giganteus' shoot cultures were stored in vitro on proliferation or rooting medium for up to 27 weeks at temperatures of 8, 12, 16, or 20 °C and photosynthetic photon flux densities of 5, 10, or 20 μmol m⁻² s⁻¹. Plants survived storage much better on rooting medium than on proliferation medium. Plants stored on rooting medium for 1 week survived well when survival was assessed immediately after storage or after 14 days of acclimatization, but had the lowest survival 28 days after transplantation. With increasing storage period on rooting medium increasing survival was found 28 days after transplantation. This was probably a result of the development of rhizomes and/or roots during storage. Best survival was observed at 20 μmol m⁻² s⁻¹ and a temperature of 8-16 °C. Increasing the temperature to 25 °C during the last week of storage improved survival considerably. Root formation was slow at 8 °C, but after 27 weeks of storage the rooting percentage was the same at all storage temperatures. An increasing number of shoots per plant 28 days after transplantation was found with increasing PPFD during storage.Miscanthus shoot cultures can be stored in vitro for at least 27 weeks with limited losses when stored on rooting medium at 20 μmol m⁻² s⁻¹, a temperature of 16 °C, and given a 1-week end-of-storage treatment of 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Salt tolerance in rice in vitro: Implication of accumulation of Na+, K+ and proline

The implication of accumulation of both inorganic (Na⁺, K⁺) and organic (proline) solutes were evaluated in unadapted and NaCl-adapted callus of a salt-sensitive (Basmati 370) and a salt-tolerant (SR-26B) cultivar of rice (Oryza sativa L.) after a NaCl shock. Accumulation of Na⁺,K⁺ and/or proline in callus was co relatable and the relative presence of these components in tissues after shock treatment was found to be important factors to support differential regrowth capacities of the shock treated calluses. Presence or retention of K⁺ in rice callus was a key factor for salt tolerance as it was observed to be positively correlated with growth in both the varieties. The results indicated that K⁺ was the first candidate to counteract the negative water potential of outside milieu, while proline was probably the last metabolic device that rice calluses opted for when exposed to salt stress.     

Changes in polyamines,proline and ethylene in sunflower calluses treated with NaCl

Responses of sunflower tissues to NaCl stress were studied in control (C), salt-stressed (S) and salt-adapted (T) calluses in terms of proline, polyamines and ethylene content for a period of 21 days. Salt-adapted calluses showed their adaptation to salinity by growing in the medium with 175 mM NaCl, at a similar rate than C calluses on medium without salt. Proline concentration was 27 times higher in salt-adapted calluses compared to control calluses at time 0, but salt stressed calluses (S calluses) were able to increase proline by day 21, demonstrating that proline was not just an osmoregulator but might be involved in other responses in sunflower salt-stressed calluses. Putrescine (Put) was the most abundant polyamine in C calluses at time 0, while spermidine (Spd) was the main polyamine in salt tolerant (T) calluses. Ethylene increased in C calluses until day 14, decreasing thereafter. In salt-adapted calluses, ethylene increased significantly over the concentration in C and S calluses by the end of the experiment. In control calluses, the highest level of total polyamines and the lowest of ethylene was found on day 21, while T calluses synthesized the highest ethylene level and had the lower polyamines level by this time. It seems that in salt-adapted calluses ethylene was related to stress tolerance and in salt sensitive tissues (S calluses), ethylene formation was related to senescence. The present data suggests a close relationship between proline, polyamines, ethylene and salt-stress tolerance in sunflower dedifferentiated tissues.     

Salt stress in cultured rice cells: effects of proline and abscisic acid

Abstract. The presence of 1 and 10 mol m⁻³ proline in media containing 100 and 200 mol m⁻³ of NaCl, had little effect on the growth of salt-adapted callus of rice. However, in such callus proline accumulation was stimulated by 10 mol m⁻³ proline in the presence of 100 mol m⁻³ NaCl. On the other hand, with 100 mol m⁻³ NaCl, both 1 and 10 mol m⁻³ proline significantly increased both the growth and proline content of salt-unadapted callus. On replacing NaCl with KCl (100 and 200 mol m⁻³), growth of saltadapted as well as unadapted callus was inhibited, but the presence of 10 mol m⁻³ proline had an ameliorating effect. Abscisic acid (ABA) supressed the growth of both salt-adapted and unadapted callus of rice in the absence of salt stress. ABA inhibited the growth of callus adapted to and grown in 100 and 200 mol m⁻³ of NaCl or when it was replaced by equimolar concentrations of KCl. Growth of 100 mol m⁻³ NaCl adapted cells was inhibited when they were transferred to a medium containing 200 mol m⁻³ of NaCl, but in the presence of ABA it was stimulated. ABA increased the growth of unadapted cells when subjected to different salts. Also, ABA accelerated the adaptation of cells exposed to salt but not to water deficits imposed by nonionic solutes.     

Effects of iso-osmotic NaCl and mannitol on growth, proline content, and antioxidant defense in Mammillaria gracilis Pfeiff. in vitro-grown cultures

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K⁺ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na⁺ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na⁺ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.     

Cell growth and water relations of the halophyte,Atriplex nummularia L., in response to NaCl

Summary Growth reduction or cessation is an initial response of Atriplex nummularia L. cells to NaCl. However, A. nummularia L. cells that are adapted to 342 and 428 mM NaCl are capable of sustained growth in the presence of salt. Cells that are adapted to NaCl exhibit a reduced rate of division compared to unadapted cells. Unlike salt adapted cells of the glycophyte Nicotiana tabacum L., A. nummularia L. cells do not exhibit reduced rate of cell expansion after adaptation. However, the cell expansion rate of unadapted A. nummularia L. cells is considerably slower than that of unadapted glycophyte cells and this normally low rate of cell expansion may contribute to the enhanced capacity of the halophyte to tolerate salt. Turgor of NaCl adapted cells was equivalent to unadapted cells indicating that the cells of the halophyte do not respond to salt by osmotic over adjustment as reported for the glycophyte tobacco (Binzel et al. 1985, Plant Physiol. 79:118–125).     

NaCl alleviates polyethylene glycol-induced water stress in the halophyte species Atriplex halimus L

Atriplex halimus L. is a C4 xero-halophyte species well adapted to salt and drought conditions. To collect information on the physiological impact of low salt levels on their water-stress resistance, seedlings were exposed for 6 d to nutrient solution containing either 0% or 15% polyethylene glycol 10,000 (PEG), in the presence or in the absence of 50 mM NaCl. Similar experiments were performed with one PEG-resistant and one PEG-sensitive selected cell line exposed for 50 d to 0% or 15% PEG on standard Linsmaier and Skoog (LS) medium, on LS medium supplemented with 50 mM NaCl, or on Na+-free medium. NaCl mitigated the deleterious impact of PEG on growth of both whole plants and PEG-sensitive cell lines and improved the ability of stressed tissues to perform osmotic adjustment (OA). Water stress reduced CO2 net assimilation rates quantified in the presence of high CO2 and low O2 levels (A), stomatal conductance and transpiration, but NaCl improved water use efficiency of PEG-treated plants through its positive effect on A values, especially in young leaves. PEG increased the internal Na+ concentration. The resistant cell line accumulated higher concentration of Na+ than the PEG-sensitive one. The complete absence of Na+ in the medium endangered the survival of both cell lines exposed to PEG. Although Na+ by itself contributed only for a small part to OA, NaCl induced an increase in proline concentration and stimulated the synthesis of glycinebetaine in response to PEG in photosynthetic tissues. Soluble sugars were the main contributors to OA and increased when tissues were simultaneously exposed to PEG and NaCl compared with PEG alone, suggesting that Na+ may influence sugar synthesis and/or translocation.     

Adaptation versus shock response to polyethylene glycol-induced low water potential in cultured potato cells

We compared long-term adaptation versus short-term or shock response of potato ( Solanum tuberosum ) cells to polyethylene glycol (PEG)-induced low water potential. Potato cells, which were allowed to adapt gradually to a decreasing water potential, were able to grow actively in a medium containing 20% PEG. In contrast, no appreciable gain in dry weight was observed in potato cells shocked by abrupt transfer to the same medium. PEG-adapted cells were also salt-tolerant, as they were able to proliferate in a medium supplemented with 200 m M NaCl. No visible ultrastructural changes of mitochondria or proplastids were observed in adapted cells at values of low water potential (about −2.0 MPa), which caused membrane disruption and appearance of lipid droplets in unadapted cells. ABA cellular content increased 5-fold in PEG-shocked cells but no significant increase was found in PEG-adapted cells. The intracellular content of free proline increased 12.5 times over the basal level in PEG-adapted cells and 6.5 times in PEG-shocked cells. As shown by in vivo protein labeling, shock conditions strongly inhibited protein synthesis, which was completely recovered in PEG-adapted cells. Osmotin, a protein associated with salt adaptation in tobacco, was constitutively expressed at a high level in PEG-adapted cells and accumulated in PEG-shocked cells only three days after the transfer in a medium supplemented with 20% PEG. Proline and osmotin accumulation were coincident with the increase in cellular ABA content in PEG-shocked cells, but not in PEG-adapted cells. These data suggest that this hormone is mainly involved in shock response rather than long-term adaptation.     

Growth Yields and Maintenance Coefficients of Unadapted and NaCl-Adapted Tobacco Cells Grown in Semicontinuous Culture

Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y_g) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y_g (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.     

Rice callus growth, proline content and activity of proline and IAA oxidase under the influence of hydroxyproline and NaCl

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth, proline content and activity of proline and IAA oxidases of the cultured tissues were determined at the end of 0, 2, 4, and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing hydroxyproline and NaCl showed increased dry weight and proline content as compared to NaCl stressed calli. The levels of proline and IAA oxidases were also low in the hydroxyproline resistant calli.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

NaCl-Dependent growth, ion content and regeneration of calluses initiated from the mangrove plant,Bruguiera sexangula

Calluses initiated from leaves and seedlings of the mangrove,Bruguiera sexangula, were isolated from the original tissues and subcultured. Effects of NaCl on growth and ion content of each callus were measured. The growth rate of calluses derived from leaves (leaf callus) gradually decreased as the NaCl concentration in the medium increased, while that of calluses derived from seedlings (seedling callus) was highest in the medium containing 100 mM NaCl. Concentrations of Na and Cl in both calluses increased with increasing the NaCl concentration in the culture medium. The concentration of K of leaf calluses greatly decreased at 300 mM NaCl, while the K concentration of seedling calluses decreased only slightly and remained relatively high even in the presence of 300 mM NaCl. Transient treatment of leaf calluses with media containing high concentrations of NaCl frequently induced regeneration of adventitious tissues.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

卫星搭载亚麻后代中PEG和NaCl

把空间生物学和细胞工程相结合,通过组织培养技术对其离体筛选,得到抗1.2% NaCl和35% PEG的愈伤组织。将所得抗性系愈伤组织在2.0 mg/L 6-苄基氨基嘌呤、0.5 mg/L吲哚乙酸的MS培养基上分化得到完整的植株。抗性系能在胁迫条件下保持高的生长速度和高效的脯氨酸合成能力。表明空间诱变与组织培养相结合有望可成为筛选抗胁迫变异系的有效途径。     

卫星搭载亚麻后代中PEG和NaCl抗性系的初步筛选

把空间生物学和细胞工程相结合,通过组织培养技术对其离体筛选,得到抗１．２％ＮａＣｌ和３５％ＰＥＧ的愈伤组织,将所得抗性系愈伤组织在２．０ｍｇ／Ｌ６－苄基氨基嘌呤、０．５ｍｇ／Ｌ吲哚乙酸的ＭＳ培养基上分化得到完整的植株。抗性系能在胁迫条件下保持高的生长速度和高效的脯氨酸合成能力,表明空间诱变与组织培养相结合有望可成为筛选抗胁迫变异系的有效途径。     

Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress

The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (−28 bar) or 428 millimolar NaCl (−23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl₂ extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium.     

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Alleviation of Seed Dormancy in the Desert ForbZygophyllum simplexL. from Pakistan

Zygophyllum simplexL. is a succulent annual that grows on thecoastal and inland saline flats around Karachi, Pakistan. Theseeds are moderately salt tolerant during germination. GerminationofZygophyllum simplexseeds under various salinity, proline,betaine, GA and kinetin treatments was determined. Proline (0.1and 1 mM) and betaine (0.1 and 1 mM) alleviated the innate dormancyof seeds, and germination reached 60–70% compared to 12%in the control set. At low salinity compatible osmotica alleviatedsome effects of salinity, but at higher NaCl concentrationsboth proline and betaine were ineffective. Gibberellic acid(0.3 and 3 mM) and kinetin (0.05 and 0.5 mM) substantially alleviatedboth innate as well as salinity-induced seed dormancy. At highersalinity (125 mM), low concentrations of kinetin (0.05 mM) andhigh concentrations of GA (3 mM) were more effective. GA completelyalleviated the effect of salinity at all concentrations used. Betaine; desert; dormancy; forb; GA; germination; halophyte; kinetin; proline; seeds; Zygophyllum simplex     

Rapid osmotic adjustment by a succulent halophyte to saline shock

The objective of this research was to measure the short term osmotic adjustment of Salicornia europaea L. ssp. rubra (A. Nels) Breitung when suddenly exposed to 100 millimolar NaCl. Plants were grown hydroponically, shocked with 100 millimolar NaCl added to the culture solution, and stem tips analyzed for free inorganic ions and small organic molecules at intervals up to 72 hours. In the first 2 hours, the calculated leaf osmoticum showed a net increase of 158.8 millimolar most of which was free Mg²⁺ (+135.3 millimolar). Total sugars increased almost 5-fold by the 6th hour, enough to provide sufficient osmoticum for the cytoplasm if only partially confined there. By 24 hours, all measured osmotica had decreased except Na⁺, Mg²⁺, Cl⁻, and proline, with the net increase being 208 millimolar. By 72 hours, there was a net gain of 356 millimolar in osmotica of the stem tips, due to Na⁺ (+233.3 millimolar), Cl⁻ (+306.7 millimolar), and a small increase in sugar and proline (+3.5 millimolar), with all other osmotica decreasing in concentration. Compatible osmotica did not change sufficiently to account for osmotic balance between vacuole and cytoplasm; consequently, there must have been a reapportionment of osmotica within the cell in the short time duration of this experiment.