应用花药培养途径筛选小麦耐盐变异体

“京花1号”小麦的花药培养在含不同 NaCl浓度,附加 2mg/L 2,4-D和0.5mg/L KT的N_6培养基上,确定筛选浓度。0.3％、0.4％和0.5％ NaCl逆境下,接种经γ射线辐照的和未辐照的“京花1号”小麦花药,诱导愈伤组织,并在同样NaCl逆境下分化,获得6株花粉植株。其中经γ辐照,0.5％NaCl下获得的1株,得到1粒种子。经无盐条件下繁殖后,γH_3(照射第3代)种植在中等盐渍地,在0.29％含盐量土壤中生长正常。γH_4代按单株作进一步盐渍地、盐池和水培鉴定,有3个株行其耐盐力明显高于亲本“京花1号”。     

马铃薯耐盐性离体鉴定方法的建立及52份种质资源耐盐性评价

为探究马铃薯种质资源耐盐性强弱,采用添加NaCl的MS培养基模拟盐胁迫的方法,研究了不同盐浓度对4份不同熟性耐盐代表材料的株高、总生物量、芽鲜质量和生根率的影响,确定耐盐筛选适宜浓度为100 mmol/L。利用该浓度胁迫52份马铃薯种质,采用隶属函数和聚类分析的方法进行耐盐性鉴定,综合评价得到极端耐盐材料为陇薯5号和LZ111,极端盐敏感材料为青薯9号、陇薯8号、中薯14号和04P48-3。本研究建立的马铃薯耐盐性离体鉴定方法和筛选获得的不同耐盐种质,将为耐盐育种及机理研究奠定技术和材料基础。     

甘蓝型油菜苗期生长阶段对NaCl胁迫的生理响应

以甘蓝型油菜自交系2205(强耐盐型)、487(中耐盐型)和1423(敏盐型)为材料,采用土培+水培方法于五叶期研究了0(无盐胁迫,CK)、60 mmol·L-1(低盐)、120 mmol·L-1(中低盐)、180 mmol·L-1(中盐)、240mmol·L-1(高盐)NaCl胁迫后的叶片MDA、可溶性糖、甜菜碱和叶绿素含量的变化特征,为油菜耐盐性评价提供理论依据。结果显示:(1)叶片MDA含量在低盐和中低盐胁迫下降低且显著低于CK,在中盐和高盐胁迫下显著升高,并以品系2205含量最低且升幅最小,品系1423最高且升幅最大。(2)叶片可溶性糖含量在低盐和中低盐胁迫下减少(但2205在中低盐胁迫下显著高于CK),在中盐和高盐胁迫下升高且显著高于CK,并以品系2205含量最高增幅最大且显著高于品系487和1423,品系1423含量最低且增幅最小。(3)叶片甜菜碱含量随NaCl浓度升高增加,品系2205和487在中低以上盐浓度胁迫下显著升高,品系1423仅在高盐胁迫下显著升高,并以品系2205含量最高且增幅最大,1423含量最低且增幅最小。(4)叶片叶绿素含量在低盐和中低盐胁迫下显著增加,且以2205叶片叶绿素含量最高且增幅最大,但在中盐和高盐胁迫下显著减少,并以品系1423含量最低且降幅最高。研究表明,120mmol·L-1以下NaCl胁迫对油菜苗期生长可能有促进作用,180mmol·L-1以上NaCl胁迫则有明显抑制作用,且NaCl浓度越高油菜受伤害越重;油菜苗期生长阶段,NaCl胁迫浓度在120~180mmol·L-1时各生理指标发生显著变化,可能是鉴定耐盐性强弱的适宜浓度;综合分析认为,品系2205具有强的耐盐性,品系1423耐盐性最差,这与之前萌芽期和幼苗期鉴定结果一致。     

StP5CS基因转化结球甘蓝的研究

为研究StP5CS基因在结球甘蓝中的耐盐作用,以结球甘蓝下胚轴为外植体,采用农杆菌介导法将耐盐基因StP5CS和抗除草剂Bar基因导入结球甘蓝基因组中,在双丙氨膦的筛选下扩繁、生根,共获得了36株抗性植株。PCR扩增和Southern印迹杂交检测表明:目的基因StP5CS和Bar基因已经成功导入结球甘蓝基因组中。RT-PCR检测表明:StP5CS基因在转录水平也有表达。转基因植株耐盐试验结果显示:高浓度盐处理(400mmol/L NaCl)下,对照植株整株枯死,而转基因植株仍能正常生长;转基因植株的SOD活性、脯氨酸含量和相对膜透性均随盐浓度的升高呈上升趋势,均在400mmol/L NaCl处理下达到最大。结果表明转基因植株对高盐环境有一定的耐受性。     

红树林耐盐相关基因转化水稻的研究

运用农杆菌介导法将红树林耐盐相关基因mangrin转入粳稻品种‘日本晴’中,通过GUS基因检测愈伤组织转化率,确定农杆菌菌液浓度OD600为0.5,浸染时间30min,共培养时间3d为最佳转化体系;经潮霉菌筛选,获得抗性再生植株。通过PCR扩增检测、Southern blot分析和GUS基因活性检测,结果表明,mangrin基因整合到再生水稻的染色体DNA上。耐盐性测定结果表明,转基因植株在200mmol/L NaCl胁迫下,成活率保持在83.3%,株高增长20%~40%,mangrin基因能提高转基因水稻对盐胁迫的抗性。     

大穗结缕草幼苗耐盐生理机制及耐盐能力研究

以大穗结缕草为实验材料,采用不同质量分数的NaCl处理后,观察盐胁迫对其生长及生理生化指标的影响.结果表明:随着盐胁迫浓度增大,大穗结缕草幼苗株高、鲜重、干重都逐渐下降,而根容量和根冠比却逐渐上升;随盐胁迫浓度的增大,大穗结缕草幼苗叶片细胞质膜透性和MDA含量逐渐增加,脯氨酸含量持续升高;其多数生长和生理指标在≥2.0% NaCl盐胁迫浓度下与对照差异显著,且此时的脯氨酸含量是对照的10倍以上.研究发现,大穗结缕草幼苗地上部分对盐胁迫更敏感、受害更严重;通过体内脯氨酸积累来减轻渗透胁迫是其可能的耐盐生理机制;2.0%盐胁迫可能是大穗结缕草的最高耐盐浓度.     

拟南芥耐盐突变体stg2的筛选

采取在高盐平板上萌发的方法,对一个雌激素诱导激活型拟南芥突变体库进行了耐盐突变体的筛选,最终得到了2株稳定的耐盐突变体。本文中对其中的一株耐盐突变体,命名为stg2(salt tolerance during germination 2),进行了研究。遗传实验表明它的耐盐特性是受雌激素诱导的,是功能获得型的耐盐突变体。本实验中还探讨了stg2突变体的筛选过程及耐盐生理特点。     

NaCl胁迫对甘薯苗期生长、IAA代谢的影响及其与耐盐性的关系

随NaCl胁迫浓度的提高 ,甘薯苗期株高和叶面积生长受抑制 ;叶片IAA水平下降 ,而IAA氧化酶和POD活性提高 .不耐盐品种生长受抑制程度及IAA水平下降幅度均大于中耐盐品种和耐盐品种 ,叶片IAA氧化酶和POD活性上升的幅度大于中耐盐及耐盐品种 .同时浓度NaCl胁迫还引起了叶片绿原酸含量的增加 ,中耐盐品种栗子香和不耐盐品种胜利百号在 170mmol·L-1的NaCl浓度胁迫下 ,绿原酸含量上升达到最高值 ,耐盐品种徐薯 18绿原酸含量达到最高值的浓度是 2 5 5mmol·L-1,而后随NaCl胁迫浓度的提高 ,绿原酸含量均有下降 ,但仍高于对照 .     

NaCl胁迫对栓皮栎幼苗生长及其生理响应

为了研究栓皮栎幼苗期对盐胁迫的生理耐受特性,选择2年生栓皮栎实生幼苗为材料,在盆栽条件下,设置NaCl盐分梯度(0%、0.4%、0.6%、0.8%),系统测定分析栓皮栎幼苗在不同盐胁迫梯度和胁迫时间下的形态生长指标以及保护酶活性、脯氨酸含量、根系活力等各项生理指标。结果显示:(1)随着盐胁迫程度的加剧,栓皮栎幼苗各部分器官鲜重和干重以及株高、基径和一级侧根长均先升高后降低,而主根长度逐渐增加,叶片数逐渐减少,并在重度胁迫下达到显著水平。(2)随着盐胁迫时间的延长,栓皮栎幼苗的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性均呈先升高后降低的趋势,但变化时间和幅度不同;丙二醛(MDA)含量则日益增加,且胁迫度越大增加越显著。(3)栓皮栎幼苗叶片的渗透调节物质脯氨酸和可溶性蛋白含量在低盐胁迫下不断升高,高盐胁迫下先升高后降低;而可溶性糖的含量则随时间延长和胁迫加剧显著增加。(4)随NaCl浓度的提高,幼苗根系活力值先增后减,叶绿素a和叶绿素b含量均呈下降趋势。研究表明,在轻、中度NaCl胁迫下,栓皮栎幼苗通过提高保护酶活性、增加渗透调节物质等策略缓解盐胁迫伤害,表现出一定的耐盐潜力;而在高浓度盐分胁迫下幼苗受到伤害,自我调节能力降低,导致保护酶活性、可溶性蛋白含量和根系活力等下降。     

大蒜耐盐愈伤组织变异系的选择研究

用含有0.1%、0.3%、0.7%、1.0%、1.5%、2.0%NaCl的MS培养基,采用直接接种和逐级培养法,对5个不同生态型的大蒜品种进行耐盐愈伤组织细胞系的选择研究.结果表明:NaCl对大蒜外植体的出愈率有很大的影响,在0.7%的NaCl培养基上,已很难诱导出愈伤组织;愈伤组织直接在含有不同浓度NaCl培养基上选择表明,5个品种在低盐 <0.7% 胁迫下都有一定的耐盐性;经逐级耐盐选择后的愈伤组织,其耐盐能力有明显的提高;在1.0%的NaCl培养基上,5个品种愈伤组织的成活率在22.4%～38.8%之间,生长率测定表示发育良好;在1.5%和2.0%的NaCl培养基上也有部分愈伤组织成活,耐盐稳定性检验证明,此耐盐能力在一定时间内能够保持下去.     

In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants

A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased NaCl-tolerance in wheat (Triticum aestivum L. andT. durum desf.) through in vitro selection

Summary Callus cultures were initiated from immature embryos of oneTriticum aestivum and threeT. durum cultivars. Growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.5, and 0.7%) added to the culture medium during two subsequent subcultures (4 wk each). The growth rate of the calli was determined by the relative fresh weight callus growth (RFWCG). The callus growth of all investigated genotypes was slightly changed in the presence of 0.3 and 0.5% NaCl, but strongly inhibited by 0.7% NaCl. Selected NaCl-tolerant clones were isolated and plants were regenerated on MS-based regeneration medium without NaCl. The regeneration capacity of the selected calli was highly reduced compared to the control. The highest number of regenerants was scored for cv. Gladiator (T. aestivum). All regenerated plants were morphologically normal and many developed to maturity and set seeds. Seedlings from the R₁ generation of selected and control plants were treated with 0.5% NaCl in vivo in liquid cultures for 6 wk. Salt tolerance of the progenies of selected plants appeared in all cultivars, but those derived from calli grown on medium with 0.7% NaCl showed the highest survival rate.T. aestivum showed higher tolerance to NaCl salinity thanT. durum.     

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.     

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.     

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.     

Plantlet regeneration from a NaCl-selected salt-tolerant callus culture of Shamouti orange (Citrus sinensis L. Osbeck)

Plantlets were regenerated from a selected salt-tolerant cell line of Shamouti orange (Citrus sinensis L. Osbeck). Embryogenesis was carried out both in the presence and absence of NaCl, yielding green and white globular embryos, respectively. Greening could be induced subsequently and normal heart shape embryo development was obtained. Plantlet formation required exposure to kinetin prior to the introduction of the root-inducing hormone naphthalene acetic acid. This system differs from the designed protocol for plant regeneration from the salt-sensitive, i.e., unselected callus. It is concluded that NaCl interferes with the regeneration process, with embryogenesis and/or embryo development into plantlets. Its presence during callus growth probably changes the balance of the phytohormones which is later manifested in plant regeneration. Citrus salt-tolerant callus yields salt-tolerant embryos. Salt-tolerant calli derived from regenerated plantlets indicate acquisition of salt tolerance on the whole plant level.     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm^?1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

     

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。     

NaCl-tolerant plants of Poncirus trifoliata regenerated from tolerant cell lines

Summary Salt-tolerant cell lines of citrus rootstock (Poncirus trifoliata cv Pomeroy) were selected by subculturing embryo-derived calli on media containing sublethal concentrations of NaCl (5 and 10 g/l). Selected lines showed a normal growth in the presence of salt at the concentrations used for selection, and salt tolerance persisted after a passage on a salt-free media. Their K⁺ and Ca²⁺ content remained higher than in control cells for increasing NaCl concentration in the medium, suggesting a modification of cell membrane permeability as the main cause of NaCl tolerance. Shoots and plants regenerated from selected cell lines showed improved growth and salt tolerance. Calli induced from these plants tolerated a salt concentration of 10 g/l, indicating the persistance of the selected trait.