A rapid screening technique for salt tolerance in rice (Oryza sativa L.)

An effecient, reproducible and simple mass screening technique for the selection of salt tolerant rice lines has been developed. Fourteen-day old seedlings raised in silica gravel culture were transplanted to foam-plugged holes in polystyrene (thermopal) sheets floated over 100 dm³ of nutrient solution in painted galvanised-iron growth tanks lined with plastic (120×90×30cm). Three days after transplanting, NaCl was added to salinize the medium in increments, at the rate of 25 mol m^-3 per 24 hours, up to the desired salinity levels which ranged from 50–200 mol m^-3 NaCl. Six plants of each line were transplanted and allowed to grow for 15 days after the maximum desired stress level was achieved in each case. Absolute shoot fresh and dry weights, as well as percent mortality, were used as criteria for assessing relative salt tolerance. Related studies were also conducted to standardize the technique. The validity of this technique was tested by conducting experiments in salinised soil (pot culture) and in salt-affected field where 9 rice lines were grown up to maturity and absolute paddy yield was considered as the criterion for salt tolerance. Salt tolerance behaviour of cultivars based on different selection criteria was compared. Good reproducibility of results among the three solution culture experiments and their close association with the results of pot culture and of salt-affected field study, authenticated the validity of this technique for practical purposes.     

Some important physiological selection criteria for salt tolerance in plants

Undoubtedly, plant breeders have made a significant achievement in the past few years, improving salinity tolerance in a number of potential crops using artificial selection and conventional breeding approaches, although molecular biology approaches are currently being intensively pursued for achieving this goal. However, most of the selection procedures used so far, were based merely on differences in agronomic characters. Agronomic characters represent the combined genetic and environmental effects on plant growth, and include the integration of the physiological phenomena conferring salinity tolerance. In fact, physiological criteria are able to supply more reliable information than agronomic characters. Although there are large numbers of reports in the literature mainly dealing with water relations, photosynthesis, and accumulation of various inorganic ions and organic metabolites in individual crops, there is little information available on the use of these attributes as selection criteria for improving salt tolerance through selection and breeding programs. In this review, the major adaptive components of salt tolerance such as osmotic adjustment, photosynthesis, water relations and ion relations are reviewed. In view of the complexity of salt tolerance and its great variation at intra-specific and inter-specific levels, it is difficult to identify single criteria, which could be used as effective selection targets. Rather it is most meaningful if physiological and biochemical indicators for individual species are determined rather than generic indicators.     

Submergence tolerance of rice – A new glasshouse method for the experimental submergence of plants

A new method is described for evaluation of submergence tolerance of rice ( Oryza sativa L.) plants. Responses of a range of cultivars corresponded with known differences in field performance. The method 1) allows fast and effective determination of submergence tolerance, 2) allows screening of many plants in a small glasshouse area, 3) provides for recovery of superior plants for seed collection, 4) allows manipulation of many environmental variables to mimic the natural submergence environment, and 5) uses simple, inexpensive, readily available equipment. Physiological studies performed with this method gave results similar to those obtained from field studies and showed that submergence tolerance increased in older plants; it decreased with increasing depth, increasing temperature and with high or low light levels. The system is ideal for the rapid evaluation of rice germplasm under controlled conditions and physiological studies on the mechanism of rice submergence tolerance.     

Expression of betaine aldehyde dehydrogenase gene and salinity tolerance in rice transgenic plants

Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase C BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite, Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived, 13 of which set seeds, and the frequency of seed setting was very low, only 10% . But the controls could not grow under the same condition. The results of BADH ac-tivity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.     

Plant growth‐promoting endophytic bacteria augment growth and salinity tolerance in rice plants

• Salt stress negatively affects growth and development of plants. However, it is hypothesized that plant growth‐promoting endophytic bacteria can greatly alleviate the adverse effects of salinity and can promote growth and development of plants. In the present research, we aimed to isolate endophytic bacteria from halotolerant plants and evaluate their capacity for promoting crop plant growth.
• The bacterial endophytes were isolated from selected plants inhabiting sand dunes at Pohang beach, screened for plant growth‐promoting traits and applied to rice seedlings under salt stress (NaCl; 150 mm ).
• Out of 59 endophytic bacterial isolates, only six isolates, i.e. Curtobacterium oceanosedimentum SAK1, Curtobacterium luteum SAK2, Enterobacter ludwigii SAK5, Bacillus cereus SA1, Micrococcus yunnanensis SA2, Enterobacter tabaci SA3, resulted in a significant increase in the growth of Waito‐C rice. The cultural filtrates of bacterial endophytes were tested for phytohormones, including indole‐3‐acetic acid, gibberellins and organic acids. Inoculation of the selected strains considerably reduced the amount of endogenous ABA in rice plants under NaCl stress, however, they increased GSH and sugar content. Similarly, these strains augmented the expression of flavin monooxygenase (OsYUCCA1) and auxin efflux carrier (OsPIN1) genes under salt stress.
• In conclusion, the pragmatic application of the above selected bacterial strains alleviated the adverse effects of NaCl stress and enhanced rice growth attributes by producing various phytohormones.

     

In vitro screening for increased drought tolerance in rice

Summary In vitro screening at the cellular level was performed with mature seed-derived callus from five rice varieties, viz. IR 18351-229-3, IR 3185-6-3-3-2, SR 26-B, Nona Bokra, and C 14-8 of diverse geographical origin and with differential drought resistance at the in planta level. Callus was induced from mature seeds on Murashige and Skoog medium supplemented with 2.0 mgl⁻¹ (9 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0, 10.0, and 15.0 gl⁻¹ of high molecular weight polyethylene glycol (PEG, 6000) as stressing agent to create chemical drought. Simultaneous efforts were also made to assess the effects of chemical drought in altering morphogenetic response in different varieties under in vitro culture. Seed germination was almost unaffected in SR 26-B and C 14-8, unlike in other varieties where germination was seriously affected. In general, seed germination was found to be decreased in three genotypes, viz. IR 18351-229-3, IR 3185-6-3-3-2, and Nona Bokra, with increased PEG concentrations. All genotypes displayed callus induction percentage in decreasing order with increased PEG concentrations supplemented in the callus induction medium (CIM), except SR 26-B and Nona Bokra. Callus induction was found to be more on CIM fortified with 5.0 gl⁻¹ PEG. In general, embryogenic callus induction and plantlet regeneration was found to be indirectly proportional to increased PEG concentrations used in CIM. Considering all characters, C 14-8 was found to be most appropriate in developing drought-tolerant lines under in vitro culture conditions followed by SR 26-B and Nona Bokra. A number of putative drought-tolerant plants were developed in C 14-8, SR 26-B, Nona Bokra, and IR 18351-29-3, and forwarded for field evaluation. In the majority of the progenies, a monogenic inheritance pattern for the drought tolerance character was observed.     

Effects of maternal salinity on salt tolerance during germination of Suaeda aegyptiaca,a facultative halophyte in the Arab Gulf desert

Suaeda aegyptiaca is a facultative halophyte found in saline and non‐saline habitats of the Arab Gulf desert, which produces small‐sized undispersible seeds. The interactive effects of maternal salinity and other environmental conditions, such as salinity, light and temperatures, that are prevailing during seed germination have received little attention for a facultative halophyte. This study tested the effects of maternal salinity on salt tolerance during seed germination of S. aegyptiaca under different light and temperature regimes. Seeds collected from both saline and non‐saline habitats of the United Arab Emirates (UAE) were germinated in 0, 50, 100, 200 and 400 mM NaCl, and incubated at 15/25°C, 20/30°C and 25/35°C in both 12‐h light/12‐h dark regimes and continuous darkness. Generally, seeds of the non‐saline habitat were 56% heavier and attained greater germination at the lower temperatures than seeds of the saline habitat. Seeds of the saline habitat germinated better in saline solutions at higher temperatures and in light. Germination was faster for seeds of the saline habitat than for seeds of non‐saline habitats. Germination recovery after transfer to distilled water was significantly greater for seeds from the non‐saline habitat, compared with seeds from saline habitats. Recovery was greater at lower and/or moderate temperatures, compared with at higher temperatures. Germination was significantly faster during recovery, compared with in the saline solutions. The study indicates that the maternal effect of salinity was confounded with the seed‐size effect and it cannot be conclusively confirmed.     

生物炭调控盐胁迫下水稻幼苗耐盐性能

土壤盐渍化降低土壤生产力.探索生物炭对盐胁迫下水稻幼苗耐盐性能的影响,对调控盐渍区水稻生产潜力具有重要意义.本研究通过生物炭介入盐胁迫稻田土壤的盆栽试验,调查了生物炭对盐胁迫下土壤环境和水稻幼苗耐盐性能的影响.盐胁迫设置4个水平,分别为0 g NaCl·kg-1土(S0),1 g NaCl·kg-1土(S1),2 g ...     

Screening plants for salt tolerance by measuring K+ flux: a case study for barley

Development of salt-tolerant genotypes is central both to remediation of salinity-affected land and to meet increasing global food demand, which has been driving expansion of cropping into marginal areas. The bottleneck of any breeding programme is the lack of a reliable screening technique. This study tested the hypothesis that the ability of plants to retain K⁺ under saline conditions is central to their salt tolerance. Using seven barley cultivars contrasting in salt tolerance (CM72, Numar, ZUG293, ZUG95, Franklin, Gairdner, ZUG403), a comprehensive study was undertaken of whole-plant (growth rate, biomass, net CO₂ assimilation, chlorophyll fluorescence, root and leaf elemental and water content) and cellular (net fluxes of H⁺, K⁺, Na⁺ and Ca²⁺) responses to various concentrations of NaCl (20–320 m m ). Na⁺ selective microelectrodes were found to be unsuitable for screening purposes because of non-ideal selectivity of the commercially available Na⁺ LIX. At the same time, our results show very strong negative correlation between the magnitude of K⁺ efflux from the root and salt tolerance of a particular cultivar. K⁺ efflux from the mature root zone of intact 3-day-old seedlings following 40 min pretreatment with 80 m m NaCl was found to be a reliable screening indicator for salinity tolerance in barley. As a faster and more cost-effective alternative to microelectrode measurements, a procedure was developed enabling rapid screening of large numbers of seedlings, based on amount of K⁺ leaked from plant roots after exposure to NaCl.     

Expression of a calcineurin gene improves salt stress tolerance in transgenic rice

Calcineurin is a Ca²⁺- and calmodulin-dependent serine/threonine phosphatase and has multiple functions in animal cells including regulating ionic homeostasis. We generated transgenic rice plants that not only expressed a truncated form of the catalytic subunit of mouse calcineurin, but also were able to grow and fertilize normally in the field. Notably, the expression of the mouse calcineurin gene in rice resulted in its higher salt stress tolerance than the non-transgenic rice. Physiological studies have indicated that the root growth of transgenic plants was less inhibited than the shoot growth, and that less Na⁺ was accumulated in the roots of transgenic plants after a prolonged period of salt stress. These findings imply that the heterologous calcineurin plays a significant role in maintaining ionic homeostasis and the integrity of plant roots when exposed to salt. In addition, the calcineurin gene expression in the stems of transgenic plants correlated with the increased expression of the Rab16A gene that encodes a group 2-type late-embryogenesis-abundant (LEA) protein. Altogether our findings provide the first genetic and physiological evidence that expression of the mouse calcineurin protein functionally improves the salt stress tolerance of rice partly by limiting Na⁺ accumulation in the roots.     

Mechanism of salt tolerance in wild rice (Oryza coarctata Roxb)

Summary Oryza coarctata, a highly salt-resistant wild rice species, is commonly found on the banks of coastal rivers in India. This species can also withstand saline water (20 to 40 dSm⁻¹ E.C) submergence for quite a long period. It was revealed thatO. coarctata has some special unicellular salt hairs (trichomes) on the adaxial surface of the leaves, by which they efficiently maintain a low concentration of toxic salts in the plant tissue. Sodium and chloride were the dominant ions in the excreted material but they also excrete potassium, magnesium and calcium. With the increase in soil salinity sodium, magnesium and chloride excretion increased.O. coarctata maintained the optimum mineral concentration in its tissues. Maximum accumulation of potassium was observed in the leaves. With the increase in salt stress total biomass production and osmotic potential increased over control but there was no change in the moisture percentage of leaves.     

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.     

Engineering salt tolerance in plants

Recent progress has been made in the identification and characterization of the mechanisms that allow plants to tolerate high salt concentrations. The understanding of metabolic fluxes and the main constraints for the production of compatible solutes (i.e. feedback inhibition and the limitation of substrate supply) open up the possibility of genetically engineering entire pathways that could lead to the production of osmoprotectants. This, together with the identification of the different sodium transporters (in particular vacuolar and plasma membrane Na(+)/H(+) antiporters) that could provide the needed ion homeostasis during salt stress, opens the possibility of engineering crop plants with improved salt tolerance.     

Receptor‐like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants

Receptor‐like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine‐rich repeats was cloned and characterized in rice (Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn²⁺, and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter‐GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H₂O₂ treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock‐out mutants sik1‐1 and sik1‐2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1‐overexpressing plants. Also, the accumulation of H₂O₂ in leaves of OsSIK1‐overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3′‐diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system.     

A rice jacalin‐related mannose‐binding lectin gene,OsJRL, enhances Escherichia coli viability under high salinity stress and improves salinity tolerance of rice

• Salinity, which is one of the most common abiotic stresses, may severely affect plant productivity and quality. Although plant lectins are thought to play important roles in plant defense signaling during pathogen attack, little is known about the contribution of plant lectins to stress resistance.
• We cloned and functionally characterized a rice jacalin‐related mannose‐binding lectin gene, OsJRL, from rice ‘Nipponbare’. We analyzed the expression patterns of OsJRL under various stress conditions in rice. Furthermore, we overexpressed OsJRL in Escherichia coli and rice.
• The cDNA of OsJRL contained a 438 bp open reading frame, which encodes a polypeptide of 145 amino acids. OsJRL was localized in the nucleus and cytoplasm. Real time PCR analyses revealed that OsJRL expression showed tissue specificity in rice and was upregulated under diverse stresses, namely salt, drought, cold, heat and abscisic acid treatments. Overexpression of OsJRL in E. coli enhanced cell viability and dramatically improved tolerance of high salinity. Overexpression of OsJRL in rice also enhanced salinity tolerance and increased the expression levels of a number of stress‐related genes, including three LEA (late embryogenesis abundant proteins) genes (OsLEA19a, OsLEA23 and OsLEA24), three Na⁺ transporter genes (OsHKT1;3, OsHKT1;4 and OsHKT1;5) and two DREB genes (OsDREB1A and OsDREB2B).
• Based on these results, we suggest that OsJRL plays an important role in cell protection and stress signal transduction.

     

转OsCDPK7基因水稻的培育与耐盐性分析

以4℃处理的水稻品种辽盐241植株叶片总RNA为模板, 用基因特异引物通过RT-PCR扩增出1 700 bp的OsCDPK7基因。该基因序列比已报道的基因序列(GenBank登录号：AB042550)缺失了26个氨基酸, 而丝氨酸/苏氨酸蛋白激酶活性中心和钙结合结构域完整, 具备钙依赖的蛋白激酶活性。构建了由组成型启动子E12调控的OsCDPK7基因植物表达载体, 利用农杆菌介导法转化水稻, 经Km筛选及Southern杂交验证, 获得10株转基因植株。耐盐性分析表明：OsCDPK7基因的组成型表达提高了T₂代转基因植株的耐盐性, 部分转基因水稻在0.2 mol/L NaCl培养基中能够萌发; 幼苗期水稻经0.4 mol/L NaCl浇灌10 d, 去除胁迫后能恢复正常生长; 而对照在以上情况下均不能萌发和恢复。结果表明, 利用植物信号转导过程中的调控因子能够提高转基因作物的耐盐性。然而, 在不同耐性的转基因植株中, OsCDPK7基因的表达有一定的差异。     

Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants

Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.     

Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH₄ ⁺ and Na⁺ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.