两种生态型芦苇胚性悬浮培养物对渗透胁迫的生理响应Ⅱ.抗氧化酶类活性的变化

渗透胁迫处理下,沙丘芦苇和沼泽芦苇培养细胞的H2O2含量变化不大,MDA含量随时间延长而降低.抗氧化酶中,沙丘芦苇的SOD、CAT、POD和APx的活力均显著高于沼泽芦苇,显示出比沼泽芦苇更强的ROS清除能力.胁迫初期(O.5 h～1 d)沙丘芦苇细胞中主要表现出SOD、CAT活力的提高,胁迫2 d后4种酶活力均明显被诱导.两种生态型芦苇的PEG适应性培养物APx及SOD酶活力显著提高,POD活性仅沼泽芦苇中升高.提示SOD、CAT主要参与沙丘芦苇在渗透胁迫下的短期响应,而APx与长期响应有关.     

两种生态型芦苇胚性悬浮培养物对渗透胁迫的响应Ⅰ、生长及渗透调节物质的变化

在浓度为20％的PEG-8000介导的渗透胁迫下,沙丘芦苇的胚性悬浮培养物的生长受抑程度小于沼泽芦苇,相对细胞活力也较高,表现出比沼泽芦苇较高的耐渗透胁迫能力。对渗调物质含量的分析显示：两种生态型芦苇细胞中脯氨酸含量均随处理时间逐渐增高。沙丘芦苇的可溶性蛋白含量、蔗糖含量等表现出比沼泽芦苇更为显著的积累。两种生态型芦苇的渗透适应性细胞中均大量积累脯氨酸和糖类。说明有机渗透调节物质的积累对提高芦苇细胞抗渗透胁迫能力有显著贡献。     

水分胁迫引起的两种不同生态型芦苇的DNA损伤与修复

利用DNA解链荧光分析（FADU）法检测两种不同生态型芦苇（Phragmites communis T.)在PEG6000胁迫处理后的DNA损伤。结果表明：无论是20％还是30％PEG6000胁迫处理,耐旱性强的沙丘芦苇的DNA损伤都比耐旱性弱的沼泽芦苇较轻。利用不同浓度的二乙基二硫代谢氨基甲酸钠（DDC）、H2O2、FeSO4以增加芦苇的3种活性氧（O2^-、H2O2、OH）的实验也同样显示出沙丘芦苇抵抗水分胁迫引起的DNA损伤的能力较强。同时,当加入外源活性氧清除剂二甲基亚砜（DMSO）、抗坏血酸（Vc）时,水分胁迫处理的芦苇DNA损伤表现出不同程度的减轻。当PEG胁迫处理的芦苇复水后,DNA损伤随复水时间延长而逐渐减轻,但沙丘芦苇的DNA损伤修复较快而完全。实验初步证明：水分胁迫可引起植物体内DNA损伤且该损伤与活性氧有关,植物的抗旱性与DNA损伤及修复密切相关。     

菊芋悬浮细胞对盐胁迫的生理响应

为探讨菊芋（Helianthus tuberosus）悬浮培养细胞对盐胁迫的生理响应,在0、50、100、150、200 mmol·L^-1NaCl处理下测定了细胞的生物量、相对细胞活力、抗氧化酶活性以及过氧化氢、丙二醛、脯氨酸、可溶性糖、可溶性蛋白质的含量,测定了总酚含量并鉴别、定量分析了14种酚类化合物。结果表明：盐胁迫显著减弱了菊芋悬浮细胞的活力,抑制了细胞的增长,200 mmol·L^-1NaCl处理下细胞生长基本上停止。盐胁迫诱发了细胞氧化胁迫,丙二醛含量显著增加,抗坏血酸过氧化物酶、过氧化物酶活性以及总酚含量、部分酚类化合物含量均随NaCl浓度升高而增加,酚类物质和抗氧化酶系统共同参与了应对氧化胁迫的抗氧化作用。脯氨酸在菊芋悬浮细胞应对NaCl渗透胁迫的渗透调节作用中扮演了重要的角色,而可溶性糖发挥的作用不大。     

渗透胁迫和外源ABA对芦苇愈伤组织中3种保护酶活性的影响

PEG-6000渗透胁迫下水生芦苇胚性愈伤组织(WREC)和沙生芦苇胚性愈伤组织(SREC)的质膜相对透性增大,前者增幅明显大于后者;过氧化氢酶(CAT)和过氧化物酶 (POD)的活性升高显著,超氧化物歧化酶(SOD)活性则呈小幅度升高,SREC的3种保护酶活性升幅较大.胁迫同时施加外源ABA时, WREC的质膜透性明显降低,CAT和POD的活性则显著提高,但对SREC的影响不大.     

河西走廊不同生态型芦苇对干旱和盐渍胁迫的响应调节

长期稳定的自然变异植物种是研究植物适应逆境机制的理想实验体系。就河西走廊芦苇不同生态型的划分、4种生态型的光合响应调节、抗氧化保护机制、膜流动性修饰机制和渗透调节机制中的响应特征、胁迫诱导的特异表达成分等作了概述。     

渗透胁迫和外源ABA对芦苇愈伤组织中3种保护酶活性的影响

PEG-6000渗透胁迫下水生芦苇胚性愈伤组织（WREC）和沙生芦苇胚性愈伤组织（SREC）的质膜相对透性增大,前者增幅明显大于后者;过氧化氢酶CAT）和过氧化物酶（POD）的活性升高显著,超氧化物歧化酶（SOD）活性则呈小幅度升高,SREC的3种保护酶活性升幅较大,胁迫同时施加外源ABA时,WREC的质膜透性明显降低。CAT和POD的活性则显著提高,但对SREC的影响不大。     

柽柳组培苗抗氧化酶及渗透调节物质对NaCl胁迫的响应

以柽柳种子为材料,利用组培方法建立其无菌离体培养体系而获得组培苗。向培养基(MS+1.0 mg/L6-BA+0.1mg/L NAA)中添加不同浓度NaCl(0、50、80、100、120、150和200mmol/L)建立胁迫条件,测定各浓度NaCl胁迫下柽柳组培苗抗氧化酶活性、渗透调节物质含量、质膜透性及超氧阴离子(O-·2)生成速率等的变化,探讨柽柳组培苗应对NaCl胁迫的生理机制。结果显示:(1)随着NaCl胁迫浓度的增加,柽柳组培苗超氧化物歧化酶(SOD)和过氧化物酶(POD)活性表现出先增加后降低的趋势,并均在100mmol/L NaCl的胁迫下达到最大值,而抗坏血酸过氧化物酶(APX)活性则表现出逐渐下降的趋势;(2)随着NaCl胁迫浓度的增加,组培苗体内有机渗透调节物质游离脯氨酸(Pro)、可溶性蛋白(Pr)和可溶性糖的含量逐渐升高且显著高于同期对照,在200mmol/L浓度下分别比对照显著增加53.77%、47.38%和67.65%;(3)组培苗过氧化氢(H2O2)、超氧阴离子(O-·2)生成速率、丙二醛(MDA)含量以及细胞质膜透性也随着NaCl胁迫浓度的增加均表现出逐渐增加的趋势且显著高于同期对照,在200mmol/L浓度下分别比对照显著增加36.42%、58.71%、82.60%和42.54%。研究表明,柽柳组培苗抗氧化酶系统在低浓度NaCl(≤100mmol/L)胁迫下表现出良好的活性氧清除活性,而其体内渗透调节物质在各浓度胁迫下均发挥着积极的调节作用,从而使柽柳表现出较强的耐盐性。     

水分胁迫对2种生态型芦苇（Phragmites communis）的可溶性蛋白含量、SOD、POD、CAT活性的影响

研究利用2种抗旱性迥异的芦苇为材料,用PEG6000进行水分胁迫处理,结果表明,抗旱性强的沙丘芦苇（沙芦）的可溶性蛋白含量明显低于沼泽芦苇（水芦）,约为水芦的1／5。但是,在受到20％PEG胁迫时,沙芦的可溶性蛋白含量有所上升,水芦的则稍微下降,在30％PEG胁迫时,水芦的可溶性蛋白含量显著下降,而沙芦则先升后降。沙芦的3种自由基清除酶（SOD、POD、CAT）的活性显著高于水芦。受到水分胁迫后,2种芦苇的SOD、POD、CAT活性或升或降。但是,无论在20％还是30％PEG胁迫条件下,相对水芦而言,沙芦都保持较高的自由其清除酶活性,从而保证其较强的自由基清除能力,减轻自由基对植物细胞生物大分子如DNA、蛋白质、脂肪酸的伤害,维持细胞正常的生命活动,这是沙芦抗旱性强的基础。     

松嫩平原两种趋异类型羊草对干旱胁迫的生理响应

采用盆栽实验,对灰绿和黄绿两种趋异类型羊草（Leymus chinensis(Trin.)Tzvel.）幼苗期用聚乙二醇（PEG）胁迫进行干旱生理指标测定,结果表明,在干旱胁迫下,灰绿型羊草和黄绿型羊草的电解质外渗率、脯氨酸、植株含水率、植株鲜重、SO4^2-、NO3^-之间均有显著或极显著差异,灰绿型羊草叶片电解质外渗率和叶绿素含量的b值均大于黄绿型羊草,而植株含水率的b值则小于黄绿型羊草,灰绿型羊草脯氨酸含量均大于黄绿型羊草,因子分析表明,脯氨酸是两种趋异类型羊草干旱胁迫生理反应的主要因子,两种趋异类型羊草在生理响应中均体现出耐旱能力,但灰绿型羊草的耐旱能力比黄绿型更强,两种趋异类型羊草在耐旱生理上有明显分化。     

Plant regeneration from callus cultures in two ecotypes of reed (Phragmites communis Trinius)

Summary Different ecotypes of reed (Phragmites communis Trinius) provide an ideal resource for studies on plant environmental adaptations and presence of genes relating to stress resistance. Dune reed is a drought-tolerant reed ecotype growing in the desert regions of north-west China. In this work, in vitro culture systems of dune reed and local swamp reed (as control) were established by optimizing the culture conditions for each of them. Bright yellow calluses were induced on a Murashige and Skoog medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.4 μM naphthaleneacetic acid and 2.2μM benzyladenine. Benzyladenine promoted callus induction, but was not required for callus maintenance. Four types of callus have been identified from each of the reed ecotypes. Two types of callus, i.e. type A (formed normal green shoots) and type C (formed albino plants), were both found as embryogenic calluses. The optimal concentrations of 2,4-D to maintain embryogenic callus were 2.3–4.5 μM for dune reed and 9.0–13.5 μM for swap reed. Plant regeneration was achieved from types A and C callus in a hormone-free medium. The embryogenic calluses of swamp reed have been maintained for over 2 yr and still retain their strong embryogenic potential; however, those of dune reed gradually lost their embryogenic potential after only 7 mo. of culture. Regenerated plants from the two reed ecotypes showed, after a growth season, similar morphology and the same chromosome number (2n=8x=96, octoploid) as the wild plants.     

不同生境两种生态型芦苇的抗氧化系统

以分布于甘肃临泽平川乡的两种芦苇生态型——水生芦苇(水芦)和重度盐化草甸芦苇(盐芦)叶片为材料,研究了其抗氧化系统的特征。结果表明,与水芦相比,盐芦中未出现活性氧和MDA(丙二醛)的积累,抗氧化酶SOD(超氧化物歧化酶)、CAT(过氧化氢酶)、POD(过氧化物酶)和APX(抗坏血酸过氧化物酶)的活性显著升高。总抗坏血酸和类胡萝卜素含量在两种生态型芦苇中没有差异,但还原型抗坏血酸和总谷胱甘肽含量在盐芦中显著升高。而且,盐芦的LOX(脂氧合酶)活性比水芦低。这些结果表明,盐芦中有效的抗氧化防御系统对抵抗盐渍胁迫起着重要的作用。此外,盐芦中高活性的Ca^2 -和Mg^2 -ATPase对细胞中过多离子的转运以及避免离子毒害起着重要的作用。     

Up-regulation of glutathione metabolism and changes in redox status involved in adaptation of reed (Phragmites communis) ecotypes to drought-prone and saline habitats

The glutathione (GSH) metabolic characteristics and redox balance in three ecotypes of reed (Phragmites communis), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), from different habitats in desert regions of northwest China were investigated. The DR possessed the highest rate of GSH biosynthesis and metabolism with the lowest levels of total and reduced GSH and its biosynthetic precursors, gamma-glutamylcysteine (gamma-EC) and cysteine (Cys), of the three reed ecotypes. This suggests that a higher rate of GSH biosynthesis and metabolism, but not GSH accumulation, might be involved in the adaptation of this terrestrial reed ecotype to its dry habitat. The HSMR shared this profile although it exhibited the highest reduced thiol levels of the three ecotypes. Two key enzymes in the Calvin-cycle possessing exposed sulfhydryl groups, NADP(+)-dependent glyceraldehydes-3-phosphate dehydrogenase (G3PD) and fructose-1,6-bisphosphatase (FBPase), and other two key enzymes in the pentose-phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGD), had very similar activities in the three reed ecotypes. Compared to the SR, the DR and HSMR had higher ratios of NADPH/NADP+ and NADH/NAD+, indicating that a more reduced redox status in the plant cells might be involved in the survival and adaptation of the two terrestrial reed ecotypes to long-term drought and salinity, respectively. These results suggest that changes of GSH metabolism and redox balance were important components of the adaptation of reed, a hydrophilic plant, to more extreme dune and saline habitats. The coordinated up-regulations of the rate of GSH biosynthesis and metabolism and reduction state of redox status of plant cells, conferred on the plant high resistance or tolerance to long-term drought and salinity.     

Nitric oxide protects against polyethylene glycol-induced oxidative damage in two ecotypes of reed suspension cultures

Dune reed (DR) is the more tolerant ecotype of reed to environmental stresses than swamp reed (SR). Under osmotic stress mediated by polyethylene glycol (PEG-6000), the suspension culture of SR showed higher ion leakage, and more oxidative damage to the membrane lipids and proteins was observed compared with the relatively tolerant DR suspension culture. Treatment with sodium nitroprusside (SNP) can significantly alleviated PEG-induced ion leakage, thiobarbituric acid reactive substances (TBARS) and carbonyl contents increase in SR suspension culture. The levels of H(2)O(2) and O(2)(-) were reduced, and the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were increased in both suspension cultures in the presence of SNP under osmotic stress, but lipoxygenase (LOX) activity was inhibited. 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific Nitric oxide (NO) scavenger, blocked the SNP-mediated protection. Depletion of endogenous NO with PTIO strongly enhanced oxidative damage in DR compared with that of PEG treatment alone, whereas had no effect on SR. Moreover, NO production increased significantly in DR while kept stable in SR under osmotic stress. Taken together, these results suggest that PEG induced NO release in DR but not SR can effectively protect against oxidative damage and confer an increased tolerance to osmotic stress in DR suspension culture.     

Growth and activities of enzymes of primary metabolism in batch cultures of Catharanthus roseus cell suspension under different pCO2 conditions

In vitro enzyme activities of glycolysis, pentose-phosphate pathway and dark CO₂ fixation were assayed in batch cultures of heterotrophic Catharanthus roseus cells under various gassing rates and partial pressures of carbon dioxide. Detrimental effects of low pCO₂ culture conditions on the growth characteristics could be linked to marked changes in levels of enzymes of primary metabolism during growth. The enzyme levels observed during the early stages of growth were found to be more stable when a constant pCO₂ (20 mbar) was maintained and enabled exponential growth to be reached more rapidly.The importance of carbon dioxide as a conditioning factor of the culture medium is discussed.     

四种生态型芦苇叶中离子分布对生境的生理适应

采用X射线微区分析技术 ,测定了 4种生态型芦苇 (Phragmitesaustralis (CaV .)Trin .exSteud .)叶的表皮泡状细胞、叶肉细胞和叶脉维管束鞘细胞离子的含量。结果表明 :沼泽芦苇的鞘细胞内 ,K 、Na 、Ca2 、Mg2 和Cl-分布均较叶肉细胞和泡状细胞高。沙丘芦苇的泡状细胞中Ca2 分布较叶肉细胞和鞘细胞高 ,而Mg2 在其叶肉细胞 ,以及K 、Na 和Cl- 在其鞘细胞内分布均较高。在轻度盐化草甸芦苇的叶肉细胞内分布较多的Na 和Mg2 ,而在鞘细胞内K 、Ca2 和Cl- 的分布均较叶肉细胞和泡状细胞为高。重度盐化草甸芦苇的泡状细胞内Na 和Mg2 的分布较多 ;同样 ,在叶肉细胞中K 、Ca2 和Cl- 的分布也较多。最后 ,讨论了上述各种离子在不同生态型芦苇叶内分布的状况 ,以及与其环境适应的生理意义。     

Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by micropro-jectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclo     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.