Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.     

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.     

Ultrastructural characterization of somatic embryos regenerated from NaCl selected and nonselected calli ofDactylis glomerata

Summary Calli were induced from leaf expiants of aDactylis glomerata L. (orchardgrass) genotype which has a high capacity for somatic embryogenesis. After 7 months culture on SH medium containing NaCl, a line was selected which was tolerant to 200 mM NaCl. When both selected and nonselected calli were maintained for 56 days on media containing 0 to 300 mM NaCl, the selected line showed significantly higher regeneration capacity than nonselected calli when placed on media containing more than 50 mM NaCl. Ultrastructural features of control somatic embryos not exposed to the salt were compared to those from nonselected and selected embryos cultured on 200 mM NaCl medium. In the presence of NaCl there were changes in the appearance of cell walls and mitochondria, accumulation of lipids and a higher degree of vacuolation in cells of nonselected embryos compared to control and selected embryos.     

Effect of NaCl on biomass, protein and proline contents, and antioxidant enzymes in seedlings and calli of two Trigonella species

The effects of NaCl on growth, contents of proteins and proline, and activities of catalase, peroxidase and polyphenol oxidase were investigated in seedlings and calli of Trigonella foenum-graecum L. and T. aphanoneura Rech. f. Seeds and hypocotyl explants were cultured on Murashige and Skoog medium supplemented with 0, 50, 100, 150 and 200 mM NaCl. Seed germination and the fresh and dry mass of the seedlings decreased significantly under salinity. In both species significant increases in protein content of seedlings over that of control were observed at 150 and 200 mM NaCl. Protein content in calli decreased at 200 mM NaCl over that of control. Protein content was higher in seedlings than in calli at all NaCl concentrations. Conversely, proline content was lower in seedlings than in calli at all the tested NaCl concentrations. NaCl caused changes in the activities of peroxidase, catalase and polyphenol oxidase in seedlings and calli.     

Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).     

In vitro selection of salinity tolerant variants from triploid bermudagrass (<Emphasis Type="Italic">Cynodon transvaalensis</Emphasis> × <Emphasis Type="Italic">C. dactylon</Emphasis>) and their physiological responses to salt and drought stress

A protocol was established for in vitro selection of salinity tolerant somaclonal variations from suspension cultured calli of triploid bermudagrass cv. TifEagle. To induce somaclonal variations the calli were subcultured for 18 months and were then subject to three-round selections for salt-tolerant calli by placing on solid medium containing 0.3 M NaCl for 10 days followed by a recovery for 2 weeks. The surviving calli were regenerated on regeneration medium containing 0.1 M NaCl. Three somaclonal variant lines (2, 71, and 77) were obtained and analyzed. The selected somaclonal lines showed higher relative growth and less injury than TifEagle under salt stress, indicating that they increased salt tolerance. In addition, they had higher relative water content and lower electrolyte leakage than TifEagle after withholding irrigation, indicating that they also increased drought tolerance. The three somaclonal variant lines had higher proline content than TifEagle under normal growth condition. The line 71 had a higher K⁺/Na⁺ ratio, whereas the lines 2 and 77 had higher CAT activity under control and salt stress conditions, indicating that different mechanisms for salt tolerance might exist in these three lines.     

耐盐药蒲公英(Taraxacum officinale Weber)愈伤组织筛选及生理生化特性分析

为获得耐1.5% NaCl的药蒲公英(Taraxacum officinale Weber)愈伤组织, 以药蒲公英叶片外植体为材料诱导愈伤组织。以NaCl为选择因子, 从愈伤组织直接筛选。在选择培养基上, 大部分愈伤组织褐化死亡, 个别褐化死亡的愈伤组织周围有少量新的细胞团长出, 将其转接到新鲜的选择培养基上, 每3周继代一次, 经3个月继代筛选获得了耐1.5% NaCl的药蒲公英细胞团。以普通愈伤组织为对照, 发现随着NaCl浓度升高, 耐盐愈伤组织的相对生长率下降但显著高于对照; 且随着盐胁迫处理时间延长持续升高, 而普通愈伤组织对照几乎停止生长, 说明耐盐愈伤组织具有相对稳定的耐盐性。在蛋白水平上, 耐盐愈伤组织与对照愈伤组织差异明显, SDS-PAGE分析显示: 耐盐愈伤组织比对照多出一条34 kD大小的蛋白带, 且30 kD、18 kD左右的蛋白带明显上调。相同处理条件下耐盐愈伤组织脯氨酸的增加幅度高于对照。盐胁迫条件下, 耐盐愈伤组织的超氧化物歧化酶(Super oxidase dimutase, SOD)、过氧化物酶(Peroxidase, POD)和过氧化氢酶(Catalase, CAT)活性明显高于对照,且随着处理时间的延长和盐浓度的增加呈现升高的趋势, 而对照则呈现先升高后下降的趋势。结果说明耐盐愈伤组织一方面通过小分子有机溶质如脯氨酸的方式调节其渗透平衡, 另一方面还可通过提高抗氧化能力降低盐分造成的次级伤害。积累蛋白也可能是耐盐愈伤组织调节渗透平衡的一种方式。通过生理生化分析确定我们获得的耐盐愈伤组织为耐盐变异体。     

Selection and regeneration of <Emphasis Type="Italic">Vitis vinifera</Emphasis> Chardonnay hydroxyproline-resistant calli

Proline (Pro) accumulation protects plant cell under abiotic stress. Hydroxyproline (Hyp) as selection agent is a toxic analog of proline and promotes Pro overaccumulation. In this study, Chardonnay calli were firstly irradiated with different dosages of ⁶⁰Co and then cultured on a Hyp-added medium. Finally, some stable hydroxyproline-resistant (HR) calli were obtained. When calli were cultured on 4 mM Hyp medium for 7 days, intracellular Pro content of the HR calli was five times higher than that detected in the normal calli. The regeneration of HR calli into plantlets was much slower than that of normal ones. When cultured on woody plant medium (WPM) containing 10 mM NaCl for 14 days, HR plantlets still grew well with lower Pro than withered normal plantlets. qRT-PCR results of Pro biosynthesis-related genes in HR plantlets showed that three genes VvP5CS, VvOAT, and VvP5CDH were conducive for Pro accumulation. These results confirmed that HR plantlets acquired salt tolerance ability. We prospect that this procedure to obtain salt-tolerant plants may be valuable to breed programs and improve grapevine genotypes with increased tolerance to salt and other abiotic stresses.     

Production of sodium-chloride-tolerant Brassica juncea plants by in vitro selection at the somatic embryo level

Summary Somatic embryos, developed from hypocotyl segments of light-grown seedlings of Brassica juncea cv RLM198, were subjected to selection at varying concentrations of sodium chloride (NaCl). Plants were developed from proliferated somatic embryos selected on NaCl-containing medium. The selections were characterized for salt tolerance, esterase isozyme pattern, and proline accumulation. It has been found that: (i) selected tolerant lines showed better root growth, shoot growth, and fresh weight accumulation on salt-containing medium when compared to the control; (ii) salt tolerance was transmitted to the next generation in seed progeny of tolerant plants grown in the absence of exposure to salt; (iii) both the starting material and the tolerant selections accumulated proline, even when grown in salt-free medium. On salt-containing medium, however, the differences in accumulated proline between the control and tolerant lines became more pronounced, and (iv) the patterns of esterase isozymes of two tolerant selections were similar but distinctly different from that of the parental control.     

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.     

Adaptation of Tobacco Cells to NaCl

Cell lines of tobacco (Nicotiana tabacum L. var Wisconsin 38) were obtained which are adapted to grow in media with varying concentrations of NaCl, up to 35 grams per liter (599 millimolar). Salt-adapted cells exhibited enhanced abilities to gain both fresh and dry weight in the presence of NaCl compared to cells which were growing in medium without NaCl (unadapted cells). Tolerance of unadapted cells and cells adapted to 10 grams per liter NaCl was influenced by the stage of growth, with the highest degree of tolerance exhibited by cells in the exponential phase. Cell osmotic potential and turgor varied through the growth cycle of unadapted cells and cells at all levels of adaptation, with maximum turgor occurring at approximately the onset of exponential fresh weight accumulation.

Adaptation to NaCl led to reduced cell expansion and fresh weight gain, while dry weight gain remained unaffected. This reduction in cell expansion was not due to failure of the cells to maintain turgor since cells adapted to NaCl underwent osmotic adjustment in excess of the change in water potential caused by the addition of NaCl to the medium. Tolerance of the adapted cells, as indicated by fresh or dry weight gain, did not increase proportionately with the increase in turgor. Adaptation of these glycophytic cells to NaCl appears to involve mechanisms which result in an altered relationship between turgor and cell expansion.

     

Effect of salt stress on growth and osmotic regulation in Thellungiella and Arabidopsis callus

The response of Thellungiella (Thellungiella holophila) and Arabidopsis (Arabidopsis thaliana) callus to salt stress was investigated. The relative growth rate of Arabidopsis calli decreased with increased levels of NaCl. However, the relative growth rate of Thellungiella calli increased with higher levels of NaCl, reaching maximal growth at 100 mM NaCl, but then subsequently declined. A similar pattern of accumulation of proline, glycine betaine, and total flavonoid was observed; whereas, accumulation of treholase continued to increase with increasing NaCl levels in both Thellungiella and Arabidopsis calli. Overall, with increasing NaCl levels, accumulation of glycine betaine, total flavonoid, and treholase was higher in Thellungiella than in Arabidopsis calli; while, proline and sucrose contents were higher in Arabidopsis than in Thellungiella calli. These results indicated that compatible solutes were involved in the response of plant calli to salt stress, and that the halophyte Thellungiella and glycophyte Arabidopsis selected different compatible solutes to adapt to salt stress environments. X. Zhao and H. J. Tan have contributed equally to the paper.     

Isolation and Characterization of Sodium Chloride-Resistant Callus Culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were initiated from seedling root segments ofmungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851on modified PC-L2 basal medium. Growing cells were exposed toincreasing concentrations of NaCl in the medium. A concentrationof 300 mol m^–3 NaCl proved completely inhibitory to growthof the calli. On incubation for 25 d, cells which could toleratethis concentration of NaCl grew to form cell clones. Selectedclones were characterized with regard to their growth behaviour,K⁺, Na⁺ and free proline content when grown under stress aswell as on normal media and were compared with the normal sensitivecallus. The selected callus was capable of growing on mediumcontaining NaCl at the inhibitory concentration. The K⁺ contentof the selected callus was lower in the case of the NaCl mediumthan for the normal medium. However, the selected clones maintainedhigher K⁺ and Na⁺ levels, with increased salinization comparedwith the wild-type cells. Salt-selected cells accumulated higherlevels of free proline under NaCl stress compared to wild-typecells. Under normal conditions, however, the amounts of freeproline in selected and non-selected calli were comparable. Key words: Vigna radiata, callus culture, NaCl stress     

Tolerance of mannitol-accumulating transgenic wheat to water stress and salinity

Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress. Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity. Wheat (Triticum aestivum L. cv Bobwhite) was transformed with the mtlD gene of Escherichia coli. Tolerance to water stress and salinity was evaluated using calli and T(2) plants transformed with (+mtlD) or without (-mtlD) mtlD. Calli were exposed to -1.0 MPa of polyethylene glycol 8,000 or 100 mM NaCl. T(2) plants were stressed by withholding water or by adding 150 mM NaCl to the nutrient medium. Fresh weight of -mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of +mtlD calli was not affected by stress. In -mtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in +mtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in -mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 micromol g(-1) fresh weight in the callus and 0.6-2.0 micromol g(-1) fresh weight in the leaf) was too small to protect against stress through osmotic adjustment. We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.     

Selection of higher regenerative callus and change in isozyme pattern in rice (Oryza sativa L.)

Summary Calli were initiated from seedling roots in rice (Oryza sativa L. var. Tadukan) and subcultured at 45-day intervals on MS medium supplemented with 2 mg/l 2,4-D. Sectors of callus which differentiated shoot meristems (green spots) under the same 2,4-D concentration were selected from the calli subcultured 90 days after initiation. The selection was continued for about 2 years. Responses to 2,4-D between original and selected lines differed considerably, although differentiation was not generally seen in rice callus in the presence of 2 mg/l 2,4-D. After 180 days, calli of the selected line differentiated into numerous shoot-bud primordia and grew out new callus tissues under 2 mg/l 2,4-D concentration; the frequency of the differentiation exceeded 90%. On the other hand, no calli of non-selected line differentiated into shootbuds under 2 mg/l 2,4-D, and the frequency of the shootbud was only about 50% under lower 2,4-D concentration (0.1 mg/l). The pattern and activity of peroxidase isozyme varied markedly between calli of the selected and non-selected lines. First, two strong peroxidase bands which show fast mobility and one intermediate peroxidase band with slow mobility were detected only in the calli of selected line. Secondly, changes in band pattern of proteins separated by SDS-PAGE were observed. In the calli of selected line, there was a loss of the polypeptide bands with molecular weight of 24 and 42 K in the selected calli, but they were clearly present in the unselected line. The appearance of new peroxidase isozyme bands and loss of polypeptide bands, change in response to auxin and increased ability for shoot bud differentiation are closely correlated to each other.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Enhanced salt stress tolerance in transgenic potato plants (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) expressing a bacterial <Emphasis Type="Italic">mtl</Emphasis>D gene

Bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene was introduced into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were selected on a medium containing 100 mg l⁻¹ kanamycin and confirmed by polymerase chain reaction (PCR), Southern blotting, and RT-PCR analyses. All of the selected transformants accumulated mannitol, a sugar alcohol that is not found in wildtype potato. Experiments designed for testing salt tolerance revealed that there was enhanced NaCl tolerance of the transgenic lines both in vitro and in hydroponic culture. Compared to 0 mM NaCl, the shoot fresh weight of wildtype plants was reduced by 76.5% at 100 mM NaCl under hydroponic conditions. However, under the same condition, the shoot fresh weight of transgenic plants was reduced only by 17.3%, compared to 0 mM NaCl treatment. The improved tolerance of this transgenic line may be attributed to the induction and progressive accumulation of mannitol in the roots and shoots of the plants. In contrast to in vitro experiments, the mannitol content in the transgenic roots and shoots increased at 50 mM NaCl and decreased slightly at 75 and 100 mM NaCl, respectively. Overall, the amount of accumulated mannitol in the transgenic lines was too small to act as an osmolyte; thus, it might act as an osmoprotectant. However, the results demonstrated that mannitol had more contribution to osmotic adjustment in the roots (but not in shoots). Finally, we concluded that mtlD expression in transgenic potato plants can significantly increase the mannitol accumulation that contributes to the enhanced tolerance to NaCl stress. Furthermore, although this enhanced tolerance resulted mainly from an osmoprotectant action, an osmoregulatory effect could not be ruled out.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of salt tolerant cell lines in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Cell lines able to grow on media containing 50, 100, 150 or 200 mM NaCl were established from potato callus cultures by direct recurrent selection or gradual selection. In callus subjected to direct selection only small clusters of cells survived on medium with 150 or 200 mM NaCl, whereas on 100 mM small cell portions appear necrotic. When cell lines were obtained by successive subcultures on media with increased concentrations of NaCl, salt-tolerant calli were more compact and developed a greenish colour free from necrotic areas. The response of calli lines grown on media with NaCl was compared to control line. The NaCl-tolerant calli showed a decrease in relative growth rate and water content, with higher reductions in the 150 mM tolerant callus. Lipid peroxidation was increased in 50 mM and 100 mM NaCl-tolerant calli, while in 150 mM tolerant callus remained similar to 100 mM values. There was a significant increase in ascorbic acid content in 100 mM and 150 mM NaCl-tolerant calli as compared to the 50 mM, that was two-fold the value found in the control. Also, the contents of soluble and insoluble proteins increased in salt-tolerant lines. SDS-PAGE of soluble proteins showed the synthesis of specific polypeptides in the presence of NaCl in culture medium and the synthesis of a new polypeptide.     

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.     

In vitro selection and characterization of polyethylene glycol (PEG) tolerant callus lines and regeneration of plantlets from the selected callus lines in sugarcane (Saccharum officinarum L.)

A system for in vitro selection of drought tolerant callus lines in sugarcane was developed. High molecular weight PEG was used as selective agent. Selected callus line grew better than non-selected callus when grown on different concentrations of PEG. The activity of antioxidant enzymes like CAT, POX, APX and SOD were high in selected callus than in non-selected callus. Osmolytes like proline and ascorbic acid were at higher levels in selected callus than in non-selected callus, however at higher concentrations (20–30 %) of PEG, levels of proline and ascorbic acid decreased. The frequency of organogenesis and number of plantlets decreased in selected callus than in non-selected callus. The results can be used for in vitro screening and manipulations of sugarcane for improvement of drought tolerance