中华绒螯蟹血细胞原代培养条件的优化

比较了几种常见血细胞培养基(L-15、2×L-15、3×L-15、M199和RMPI-1640)对中华绒螯蟹(Eriocheir sinensis)血细胞原代培养中细胞形态以及存活率的影响,以及在筛选获得的最佳培养基中添加不同比例胎牛血清(FBS)(0%、5%、10%和15%),进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明,3×L-15培养基培养效果较好,所培养的细胞形态相对完整,数量较多,培养至96 h时血细胞存活率仍大于60%;而其他4种培养基效果较差,培养12 h存活率均低于50%,且细胞形态结构变化明显。以3×L-15培养基为基础,添加不同比例胎牛血清后发现,对细胞存活有显著影响,存活率明显降低。因此,不添加血清的3×L-15培养基对中华绒螯蟹血细胞的生长较为适宜。     

Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis

Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg^?1) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5?×?10⁷ cells ml^?1, and three subpopulations of hemocytes were distinguished as granulocytes (43.46?±?4.98%), semigranulocytes (31.04?±1.95%), and hyalinocytes (25.50?±4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg^?1, pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.     

Hemolymph analysis and evaluation of newly formulated media for culture of shrimp cells (Penaeus stylirostris)

Summary Creation of a shirmp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp,Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps, and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2×L-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2×L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2×L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2×L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by³H-thymidine uptake and³⁵S-methionine uptake assays. The ovary cells ofP. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2×L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.     

Hepatopancreas cell cultures from mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz’ L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2–3 d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

The effects of naphthalene on the ultrastructure of the hepatopancreas of the fiddler crab, Uca minax

The hepatopancreas of the red-jointed fiddler crab, Uca minax, is a bilateral evagination of the midgut, composed of numerous blind-ending tubules. Groups of these tubules empty into collecting ducts which join to form the main hepatopancreatic duct. Ultrastructural examination of tubules from the hepatopancreas of adult fiddler crabs revealed four major cell types, designated as E, R, B, and F cells. The E cells were found at the apex of the tubule and were assumed to serve as meristematic tissue. The R cells were most numerous and were scattered along the length of the tubule. Characterized by extensive smooth and rough endoplasmic reticulum and abundant lipid and glycogen reserves, the R cell was assumed to function in absorption and storage of the organic products of digestion. The B cells were recognized by the presence of a single, large apical vacuole that likely functioned in the secretion of digestive enzymes into the lumen of the hepatopancreas. The F cells, which contained extensive amounts of rough endoplasmic reticulum, were believed to be responsible for the synthesis of digestive enzymes. Electron microscopy of the hepatopancreas of crabs exposed to naphthalene for 5 days revealed that those cells with abundant membrane lipids (F cells) and abundant storage lipids (R cells) were most altered while those cells having little membrane or storage lipids (B and E cells) were only slightly altered. Furthermore, alterations in the F and R cells were not uniform along the length of the tubule, but increased in severity toward the proximal end.     

脂质营养对中华绒螯蟹幼体肝胰腺超微结构的影响

采用透射电镜技术研究了中华绒螯蟹（Eriocheir sinensis)各期幼体肝胰腺的超微结构,结果表明,蟹的肝胰腺腺管上皮由Ｅ,Ｆ,Ｂ和Ｒ４种细胞组成,其中Ｅ细胞为胚胎细胞,能分化成其他３种细胞;Ｂ,Ｒ和Ｆ细胞均呈,高柱状,腔面有发达的微绒毛,基底部有基膜,呈明显的极性分布;Ｂ细胞粗面内质网丰富,胞质中有１－２个大液泡,起分泌作用,属分泌液;Ｆ细胞内含发达的粗面内质网,还可见酶原颗粒;Ｒ细胞胞质中有丰富的滑面内质网、游离的核糖体和脂肪滴,主要起贮存养人的作用。细胞的连接有紧密连接和中间连接２种方式。与脂质营养缺乏时相比,脂质营养充足的幼体其肝胰腺超微结构有如下特点：Ｒ细胞质中有连多的脂肪滴,线粒体呈饱满的圆形或椭圆形,且膜未见有内陷或萎缩,滑面内质网膨胀成小泡状结构。     

Combined effects of sediment and lead (PbCl<Subscript>2</Subscript>) on the demography of <Emphasis Type="Italic">Brachionus patulus</Emphasis> (Rotifera: Brachionidae)

We studied the response of Brachionus patulus to different concentrations of the heavy metal Pb in the presence and absence of sediments. We conducted acute (LC₅₀) and chronic (life table demography and population growth) toxicity tests using sediment levels of 0, 30 and 280 mg l⁻¹ (=0, 17 and 170 NTU) and Pb at 0, 0.06 and 0.6 mg l⁻¹. Experiments were conducted at 20 ± 1°C on a horizontal shaker and algal food (Chlorella vulgaris) was added at a density of 1.0 × 10⁶ cells ml⁻¹. The median lethal concentration (LC₅₀ ± 95% Confidence intervals) of PbCl₂ for B. patulus was 6.15 ± 1.08 mg l⁻¹. Age-specific survivorship and fecundity curves showed increase in turbidity level resulted in decreased survival and offspring production of the rotifers. Increase in Pb concentration too had a negative effect on the survival and reproductive output of B. patulus. Statistically, average lifespan, life expectancy at birth, gross and net reproductive rates and the rate of population increase were all significantly influenced by the concentration of Pb, turbidity level as well as the interaction of Pb concentration × turbidity level. Rotifers exposed to 170 NTU did not grow regardless of the heavy metal concentration in the medium. Similarly, B. patulus exposed to 0.6 mg l⁻¹ Pb did not survive beyond 10 days regardless of the turbidity level in the medium. The rate of population increase of B. patulus derived from the growth experiments was negative in all treatments containing Pb as low as 0.06 mg l⁻¹ or turbidity level as low as 17 NTU. In treatments containing Pb or sediments, there existed no relation between the egg ratio and the population density. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Temperature sensitivity in peroxisome assembly processes characterizes milder forms of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) contain various clinical phenotypes; Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), decreasing in the clinical severity in this order. We found that all IRD cell lines and some NALD lines belonging to several different complementation groups are temperature-sensitive in peroxisome assembly; that is, they lacked catalase-positive peroxisomes at 37°C, but do gain the peroxisomes at 30°C. We identified heterozygous mutations E55K/R119Stop in the PEX2 gene of an IRD patient of complementation group F. The E55K mutation was the direct cause of the temperature-sensitivity because similar phenotypes could be transferred to PEX2-defective CHO cells by transfecting the mutant gene. Thus, temperature-sensitive peroxisome assembly is representative of milder forms of PBDs. The main part of this study was published by Imamura et al. (1).     

Responses of nonstructural carbohydrates to shoot removal and soil moisture treatments in <Emphasis Type="Italic">Salix nigra</Emphasis>

Aboveground disturbances are common in dynamic riparian environments, and Salix nigra is well adapted with a vigorous resprouting response. Soil moisture stresses are also common, and S. nigra is flood tolerant and drought sensitive. The objective of this study was to quantify nonstructural carbohydrate (NSC) reserves in S. nigra following shoot removal and soil moisture treatments. NSC reserves provide energy for regeneration of shoot tissue until new functional leaves are developed. Three soil moisture treatments: well-watered (W), periodic flooding (F) and drought (D); and three shoot removal treatments: no shoots removed (R0), partial shoot removal (R1), and complete shoot removal (R2) were applied. Plants were harvested when new shoot development was observed (day 13). Statistical significance in the 3 × 3-factorial design was determined in two-factor ANOVA at P < 0.05. Both roots and cuttings were important reservoirs for NSC during resprouting response, with decreases in root (31%) and cutting (14%) biomass in R2 compared to R0. Rapid recovery of photosynthetic surface area (from 15 to 37% of R0) was found in R1. A clear pattern of starch mobilization was found in roots in R0, R1 and R2, with lowest root starch concentration in W, F higher than W, and D higher than F. Shoot starch concentration was lower in F and D compared to W in R0, however, in R1 shoot starch was reduced in W compared to F and D, possibly indicating reduced rates of translocation during soil moisture stress. Evidence of osmotic adjustment was found in roots and shoots with higher total ethanol-soluble carbohydrates (TESC) during soil moisture stress in F and D treatments. Total plant NSC pool was greater in F and D treatments compared to W, and progressively reduced from R0 to R1 to R2. Results indicated negative effects of drought, and to a lesser extent periodic flooding on resprouting response in S. nigra, with implications for reduced survival when exposed to combined stresses of aboveground disturbance and soil moisture.     

Improved conditions for culture of biosynthetically active cockroach corpora allata

Summary Currently, short-term culture of insect corpora allata is most often performed in TC199. We now show that L-15B, a medium widely used in arthropod tissue culture, is superior to TC199 for both short- and long-term culture of cockroach corpora allata. In 3-h and 48-h incubations, juvenile hormone biosynthesis by corpora allata from Diploptera punctata was significantly higher in L-15B than in TC199. In addition, in both media, corpora allata activity was significantly improved by flotation of glands at the medium surface. Characteristics of L-15B responsible for its superiority were examined by comparison of gland activities in several TC199 formulations that had been modified in different ways to be more similar to L-15B. Adjusting the osmotic pressure of TC199 (288 mOsm/l) to near that of L-15B (362 mOsm/l) and D. punctata hemolymph (360 mOsm/l) significantly improved gland activity during the second 12 h of a 36-h incubation. Increasing the concentrations of amino acids, sugars, and organic acids in TC199 to the same levels as in L-15B significantly improved gland activity during both the second and third 12-h intervals of a 36-h incubation. These results suggest that L-15B is superior to TC199 because L-15B is isoosmotic with D. punctata hemolymph and because L-15B, like cockroach hemolymph, contains a high level of organic constituents. It is therefore more appropriate to use L-15B than TC199 for short-term in vitro assays of juvenile hormone biosynthesis and for extended corpora allata culture.     

Separation and viability of gill and hepatopancreatic cells of a mangrove crab Ucides cordatus

The gills contain essential cells for respiration and osmoregulation, whereas the hepatopancreas is the site of digestion, absorption, and nutrients storage. The aim of this work was to separate and characterize gill and hepatopancreatic cells of the mangrove crab, Ucides cordatus. For gills, the methodology consisted of an enzymatic cellular dissociation using Trypsin at 0.5%, observation of cellular viability with Tripan Blue, and separation of cells using discontinuous sucrose gradient at concentrations of 10%, 20%, 30%, and 40%. The hepatopancreatic cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at the same concentrations above. For gills, a high cellular viability was observed (92.5±2.1%), with hemocyte cells in 10% sucrose layer (57.99?±?0.17%, *P?相似文献   

Improved hydrogen photoproduction regulated by carbonylcyanide <Emphasis Type="Italic">m</Emphasis>-chlorophenylhrazone from marine green alga <Emphasis Type="Italic">Platymonas subcordiformis</Emphasis> grown in CO<Subscript>2</Subscript>-supplemented air bubble column bioreactor

To develop an integrated process of CO₂-fixation and H₂ photoproduction by marine green microalga Platymonas subcordiformis, the impact of algal cells grown in CO₂-supplemented air bubble column bioreactor was investigated on H₂ photoproduction regulated by carbonylcyanide m-chlorophenylhrazone. Highest cell growth (3.85 × 10⁶ cells ml⁻¹), starch content (0.25 ± 0.08 mg per 10⁶ cells) and hydrogen production (50 ± 3 ml l⁻¹) were achieved at 3% CO₂-supplemented culture, which are respectively 1.4, 2.1, 1.5-fold of the air-supplemented culture. Improved H₂ production correlated well with the increase in starch accumulation. In this process, the algal cells have been recycled for stable H₂ production of 40–50 ml l⁻¹ over five cycles.     

Optimization of the isolation and cultivation of <Emphasis Type="Italic">Cyprinus carpio</Emphasis> primary hepatocytes

The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO₂), or L-15 (cultured without 5% CO₂) medium then cultured at 17, 27, or 37 °C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 × 10⁸ per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO₂) or L-15 (cultured without 5% CO₂). The optimum culture temperature was 27 °C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.     

A new quaternary photoluminescence enhancement system of Eu−N‐(o‐vanillin)‐1,8‐diaminonaphthalene−1,10‐phenanthroline−Zn and its application in determining trace amounts of europium and zinc

A new sensitive quaternary photoluminescence enhancement system has been successfully developed to determine trace amounts of Eu³⁺ and Zn²⁺. The photoluminescence intensity of Eu ? N‐(o‐vanilin)‐1,8‐diaminonaphthalene systems was greatly increased by the addition of specific concentrations of 1, 10‐phenanthroline and Zn²⁺. The excitation and emission wavelengths were 274 and 617 nm, respectively. Under optimal system conditions, the photoluminescence intensity showed a linear response toward Eu³⁺ in the range of 5.0 × 10^–6 ~ 2.0 × 10^–5 M with a limit of detection (= 2.2 × 10^–9 M) and the photoluminescence intensity of the system decreased linearly by increasing the Zn²⁺ concentration in the range of 5.0 × 10^–8 ~ 1.0 × 10^–6 M with a limit of detection (= 8.8 × 10^–11 M). This system was successfully applied for the determination of trace amounts of Eu³⁺ in a high purity La₂O₃ matrix and in the synthetic rare earth oxide mixture, and of Zn²⁺ in a high purity Mg(NO₃)₂ · 6H₂O matrix and in synthetic coexisting ionic matrixes. The energy transfer mechanism, photoluminescence enhancement of the system and interference of other lanthanide ions and common coexisting ions were also studied in detail. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Enhanced phytoremediation of cadmium and/or benzo(a)pyrene contaminated soil by hyperaccumlator Solanum nigrum L.

The role of same amendment on phytoremediating different level contaminated soils is seldom known. Soil pot culture experiment was used to compare the strengthening roles of cysteine (CY), EDTA, salicylic acid (Sa), and Tween 80 (TW) on hyperaccumulator Solanum nigrum L. phytoremediating higher level of single cadmium (Cd) or Benzo(a)pyrene (BAP) and their co-contaminated soils. Results showed that the Cd capacities (ug pot^?1) in shoots of S. nigrum in the combined treatment T_{0.1EDTA+0.9CY} were the highest for the 5 and 15 mg kg^?1 Cd contaminated soils. When S. nigrum remediating co-contaminated soils with higher levels of Cd and BAP, that is, 5 mg kg^?1 Cd + 1 mg kg^?1 BAP and 15 mg kg^?1 Cd + 2 mg kg^?1 BAP, the treatment T_{0.9CY+0.9Sa+0.3TW} showed the best enhancing remediation role. This results were different with co-contaminated soil with 0.771 mg kg^?1 Cd + 0.024 mg kg^?1 BAP. These results may tell us that the combine used of CY, SA, and TW were more useful for the contaminated soils with higher level of Cd and/or BAP. In the combined treatments of Sa+TW, CY was better than EDTA.     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible