Enhanced mitotic division of cultured Passiflora and Petuniaprotoplastsby oxygenated perfluorocarbon and haemoglobin

Cell suspension-derived protoplasts were cultured in (A) liquid medium, (B) medium overlaying oxygenated perfluoro-decalin (PFC), (C) medium containing 1:50 (v:v) of haemoglobin solution ( Erythrogen TM ), or (D) medium with 1:50 (v:v) Erythrogen TM overlaying oxygenated PFC. In Passiflora, mitotic division of protoplasts was increased (P < 0.05) by all treatments, with Erythrogen TM being the most effective (120% increase over control). For Petunia, treatment D induced maximum mitosis (140% over control), whilst Erythrogen TM alone produced a less pronounced (80% over control) increase.     

Salinity tolerance and antioxidant status in cotton cultures

This investigation focuses upon cell growth and antioxidant status in cultured cells of cotton (Gossypium herbaceum) cvs. Dhumad (salt-tolerant, TOL), H-14 (medium salt-tolerant, MED), and RAhs-2 (salt-sensitive, SEN) exposed to saline stress (50-200 mM NaCl). Mean (+/- SEM) callus fresh weight (f.wt.) and dry weight (d.wt.) gains were significantly (p <.05) greater on Murashige and Skoog (MS) [1]-based medium with 50 mM NaCl for the TOL cv. (62% and 16%, respectively) over NaCl-free controls (2020 +/- 45 and 166 +/- 4 mg, respectively); comparable differences were not observed for the MED cv. A significant (p <.05) decrease in mean f.wt. occurred with the SEN cv. exposed to 50 mM NaCl. For all cvs., there were (p <.05) reductions in mean f.wts. in medium with >or=100 mM NaCl. At 200 mM NaCl, mean f.wt. decreases were 52% (TOL), 89% (MED), and 91% (SEN), respectively. A strong correlation existed between antioxidant status and growth of cells with NaCl. Superoxide dismutase and glutathione reductase activities increased with increasing salinity in the TOL cv. to maximum values of 26.3 +/- 1.1 U mg(-1) protein and 1.05 +/- 0.01 AB(340 nm) min(-1) mg(-1) protein, respectively, at 150 mM NaCl; for the MED and SEN cvs., there were no changes in activities of these enzymes between control and salt treatments. Catalase activity decreased progressively with increasing salt concentration in all cvs. except for SEN with 100 mM NaCl, where mean catalase activity (1.75 +/- 0.04 AB(240 nm) min(-1) mg(-1) protein) was greater (p <.05) than control (1.13 +/- 0.08). Overall, cultured cotton cells provide an experimental system for investigating the role of antioxidants in salt tolerance at the cellular level.     

Strategies for promoting division of cultured plant protoplasts: synergistic beneficial effects of haemoglobin (Erythrogen) and Pluronic F-68

Novel approaches, involving supplementation of aqueous culture medium with haemoglobin solution (Erythrogen), in the presence or absence of the copolymer surfactant, Pluronic F-68, have been evaluated to facilitate cellular oxygen availability to promote mitotic division. Cell-suspension-derived protoplasts of albino Petunia hybrida cv. Comanche were cultured for up to 45 days in KM8P medium containing 1:50–1:500 (vol:vol) Erythrogen. The mean initial protoplast plating efficiency after 9 days with 1:50 Erythrogen (18.5%) was significantly greater (P<0.05) than in untreated controls (11.3%). Supplementation of culture medium with 1:50 Erythrogen, together with 0.01% (wt/vol) Pluronic F-68, increased the mean plating efficiency after 9 days (24.4%) by 92% (P<0.05) over the control (12.7%). These treatments also produced increases in biomass of protoplast-derived cells up to 2.5-fold greater than control (P<0.01) over 80 days. Gassing the medium, containing 1:50 Erythrogen, with carbon monoxide abolished the increase in plating efficiency. There was no additional benefit of gassing Erythrogen-supplemented medium with 100% oxygen. The synergistic, beneficial effect of Erythrogen and Pluronic F-68 on protoplast division has implications for plant biotechnology utilising protoplasts. Received: 24 May 1996 / Revision received: 12 July 1996 / Accepted: 15 August 1996     

Ovarian response to PMSG treatment in ewes immunized against oestradiol-17 beta

The effects of active immunization against oestradiol-17 beta on the ovarian response to pregnant mare serum gonadotrophin (PMSG) was investigated in Merino ewes. Immunized (79) and control (41) ewes were synchronized with intravaginal sponges, given either 750 or 1500 i.u. PMSG and then mated to rams or inseminated laparoscopically with fresh diluted semen. All control ewes mated naturally exhibited oestrus and 40 out of 41 control ewes ovulated. The ovulation rate was higher in the controls receiving 1500 i.u. PMSG than in those ewes which received 750 i.u. PMSG (10.2 v. 3.3). Immunization against oestradiol-17 beta resulted in antibody titres varying from 100 to more than 100 000 in plasma taken 1-4 days after mating. The ovarian response increased significantly in the lowest titre group (100-1000) in conjunction with stimulation with 1500 i.u. PMSG. In these ewes the ovulation rate increased over controls (16.7 v. 10.2) as did the total ovarian response, which includes follicles greater than 10 mm diameter (22.3 v. 11.1). The total ovarian response was also increased in those ewes given 750 i.u. PMSG which had titres in the 1000-10 000 and 10 000-100 000 range, but this was not accompanied by significant increases in the ovulation rate. In general, the higher titre levels (greater than 1000) were correlated with decreases in the proportion of ewes showing oestrus and ovulating and in the embryo recovery rate. The 1500 i.u. PMSG treatment group with the highest titres (greater than 10 000) also showed a significant drop in the ovulation rate as compared to the 1500 i.u. PMSG controls.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

In vivo and in vitro studies on sex steroid binding protein (SBP) regulation in rainbow trout (oncorhynchus mykiss): Influence of sex steroid hormones and of factors linked to growth and metabolism

The respective roles of sex steroids and hormones related to growth and metabolism, on SBP regulation have been studied in rainbow trout. In vivo, oestradiol (E2) supplementation induces a slow but significant increase of plasma SBP concentration. Testosterone or cortisol injections have no effect. In vitro, the steroid binding protein that accumulates in incubation medium of hepatic cell primary cultures has been characterized and found to be similar to blood SBP. Its production is increased by addition of E2 (maximum: + 300%). This effect develops slowly over several days of culture and is dose dependent; as little as 1–10 nM E2 is effective.

Recombinant rainbow trout GH (rtGH)—0.01 to 1 μg/ml—also increases SBB accumulation as compared to control cells and seems to maintain SBP production over culture duration. In preliminary experiments, (1) insulin-like growth factor (IGF) and SBP concentrations were found to change inversely after a 4 days stimulation with increasing concentrations of GH; (2) recombinant human IGF₁ (250 ng/ml) tended to be inhibitory when SBP production was expressed per mg of total cellular protein, and a micromolar concentration of bovine insulin was clearly inhibitory.

Other hormones tested in vitro: triiodothyronine (10–1000 nM), thyroxine (100 nM), 17,20β-dihydroprogesterone (10–2000 nM), and testosterone (1–1000 nM) did not influence SBP concentration in hepatic cells culture media.     

Preventive and curative effects of Achyranthes aspera Linn. extract in experimentally induced nephrolithiasis

The present study was undertaken to evaluate the efficacy of Achyranthes aspera in preventing and reducing the growth of calcium oxalate stones in ethylene glycol induced nephrolithiatic model. Hyperoxaluria was induced in rats using ethylene glycol (EG, 0.4%) and ammonium chloride (1%) for 15 days and was then replaced with EG (0.4%) only. Upon administration of cystone (750 mg/kg body wt.), aqueous extract of A. aspera (500 and 1000 mg/kg body wt.), levels of renal injury markers (lactate dehydrogenase and alkaline phosphatase) were normalized with a decrease in serum urea and serum creatinine. Concurrent treatment reduced changes in the architecture of renal tissue and also decreased the size of crystals thereby helping in quick expulsion of the crystals. The present results indicated that Achyranthes aspera had an ability to maintain renal functioning and reduced renal injury.     

In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180.

Withania somnifera is a medicinal plant used in the treatment of a variety of ailments in the Ayurvedic system. Alcoholic extract of the root of the plant was injected(ip) at daily doses of 200 to 1000 mg/kg body wt for 15 days starting from 24 hr after intradermal inoculation of 5 x 10(5) cells of S-180 in BALB/c mice. Solid tumor growth was monitored for 100 days. Doses of 400 mg/kg and above produced complete regression of tumor after an initial growth, the percentage of complete response (CR) increasing with increasing drug dose. A 55% CR was obtained at 1000 mg/kg drug administration, but this dose also produced some mortality among the animals. A significant increase in the volume doubling time and growth delay was seen when the drug dose was increased from 500 to 750 mg/kg body wt, but further increase in drug dose to 1000 mg/kg did not produce any significant increase in these responses. Cumulative doses of 7.5 to 10 g at daily doses of 500 or 750 mg/kg seems to produce a good response in this tumor.     

Verticillium lecanii Spore Formulation Using UV Protectant and Wetting Agent and the Biocontrol of Cotton Aphids

Verticillium lecanii spores (10⁸ spores ml⁻¹) suspended in 1% (w/v) montmorillonite SCPX-1374 and 1% (w/v) of the wetting agent, EM-APW#2, which is a polyoxyethylene, had approx. 80% survival after exposure to UV-C for 30 min and about 93% after exposure to UV-B for 6 h. In greenhouse testing, cotton aphid densities increased 14-fold over their initial density in 15 d without spore application. However, initial cotton aphid densities were decreased by 60% of the initial level when plants were treated with the spore formulation.     

Modulation of oxidant lung injury by using liposome-entrapped superoxide dismutase and catalase

Increased cellular generation of partially reduced species of oxygen mediates the toxicity of hyperoxia to cultured endothelial cells and rats exposed to 95-100% oxygen. Liposomal entrapment and intracellular delivery of superoxide dismutase (SOD) to cultured porcine aortic endothelial cells increased the specific activity of cellular SOD up to 15-fold. The liposome-mediated augmentation of SOD activity persisted in cell monolayers and rendered these cells resistant to oxygen-induced injury in a cell SOD activity-dependent manner. Addition of free SOD to culture medium had no effect on cell SOD activity or resistance to oxygen toxicity. SOD and catalase-containing liposomes injected i.v. into rats increased lung-associated enzyme specific activities two- to fourfold. Liposome entrapment of both SOD and catalase significantly increased the circulating half-lives of these enzymes and was critical for prevention of in vivo oxygen toxicity. Free SOD and catalase injected i.v. in the absence or presence of control liposomes did not increase corresponding lung enzyme activities or survival time in 100% oxygen. These studies show that O2- and H2O2 are important mediators of oxygen toxicity and that intracellular delivery of oxygen protective enzymes can reduce tissue injury owing to overproduction of partially reduced oxygen species.     

Effects of nitrate and oxygen on photoautotrophic lipid production from Chlorococcum littorale

Effects of oxygen and nitrate on fatty acid/lipid production from a highly CO(2)-tolerant microalgal species Chlorococcum littorale were examined under photoautotrophic conditions of 295 K, a light intensity of 170 μmol-photon m(-2) s(-1), a bubbling CO(2) concentration of 5% (v/v) and bubbling oxygen concentrations to be volumetrically adjusted by mixing oxygen gas with inert nitrogen gas at concentrations ranging from 0% to 95% (v/v). The results showed that maximum fatty acid content reached ca. 34 wt.% under oxygen-freely bubbling conditions and this value decreased to be ca. 20 wt.% when air-like oxygen concentration of 20% was chosen. This means that degree of the accumulation strongly depended on the level of bubbling oxygen concentrations, which can be a crucial factor after nitrogen depletion in the photoautotrophic culture system. TLC-FID/FPD analyses showed that triglycerides were found to be a dominant lipid class for this accumulation.     

Biomass, litterfall and decomposition rates for the fringed rhizophora mangle forest lining the Bon Accord Lagoon, Tobago

The mangrove forest that fringes the Bon Accord Lagoon measures 0.8 km(2) and is dominated by red mangrove (Rhizophora mangle). This forest forms the landward boundary of the Buccoo Reef Marine Park in Southwest Tobago, and is part of a mangrove-seagrass-coral reef continuum. Biomass and productivity, as indicated by litterfall rates, were measured in seven 0.01 ha monospecific plots from February 1998 to February 1999, and decomposition rates were determined. Red mangrove above-ground biomass ranged between 2.0 and 25.9 kg (dry wt.) m(-2). Mean biomass was 14.1+/-8.1 kg (dry wt.) m(-2) yielding a standing crop of 11 318+/-6 488 t. Litterfall rate varied spatially and seasonally. It peaked from May to August (4.2-4.3 g dry wt. m(-2) d(-1)) and was lowest from October to December (2.3-2.8 g dry wt. m(-2) d(-1)). Mean annual litterfall rate was 3.4+/-0.9 g dry wt. m(-2) d(-1). Leaf degradation rates ranged from 0.3% loss d(-1) in the upper intertidal zone to 1% loss d(-1) at a lower intertidal site flooded by sewage effluent. Mean degradation rate was 0.4+/-1% loss d(-1) . The swamp produces 2.8 t dry wt. of litterfall and 12 kg dry wt. of decomposed leaf material daily. Biomass and litterfall rates in Bon Accord Lagoon were compared to five similar sites that also participate in the Caribbean Coastal Marine Productivity Programme (CARICOMP). The Bon Accord Lagoon mangrove swamp is a highly productive fringed-forest that contributes to the overall productivity of the mangrove-seagrass-reef complex.     

产尿激酶原CHO工程细胞无血清培基的研究

在分析产尿激酶原CHO工程细胞对培基中氨基酸和糖利用的基础上,对DMEM：F12(1：1)进行初步优化。采用正交实验设计建立了无血清培基11G—SG—SFM和11G—SF—SFM。用11G—SG—SFM悬浮培养11G细胞,细胞增殖速率与含5％小牛血清DMEM: F12或CHO-s-SFM相当。用11G—SE—SFM培养11G细胞,细胞增殖缓慢,但有利于提高11G细胞表达pro—uK的水平,pm—UK的表达水平比含5％小牛血清的DMEM：F12培养提高80％左右。     

Improved survival of Lactobacillus paracasei NFBC 338 in spray-dried powders containing gum acacia

AIMS: To assess the protective effect of gum acacia (GA) on the performance of Lactobacillus paracasei NFBC 338 during spray-drying, subsequent storage and exposure of the culture to porcine gastric juice. METHODS AND RESULTS: For these studies, Lact. paracasei NFBC 338 was grown in a mixture of reconstituted skim milk (10% w/v) and GA (10% w/v) to mid log phase and spray-dried at outlet temperatures between 95 and 105 degrees C. On spray drying at the higher air outlet temperature of 100-105 degrees C, the GA-treated culture displayed 10-fold greater survival than control cells. Probiotic lactobacilli in GA-containing powders also survived dramatically better than untreated cultures during storage at 4-30 degrees C for 4 weeks. A 20-fold better survival of the probiotic culture in GA-containing powders was obtained during storage at 4 degrees C while, at 15 and 30 degrees C, greater than 1000-fold higher survival was obtained. Furthermore, the viability of probiotic lactobacilli in GA-containing powders was 100-fold higher when exposed to porcine gastric juice over 120 min compared with the control spray-dried culture. CONCLUSIONS: The data indicate that GA has applications in the protection of probiotic cultures during drying, storage and gastric transit. SIGNIFICANCE AND IMPACT OF THE STUDY: Gum acacia treatment for the manufacture of probiotic-containing powders should result in more efficient probiotic delivery to the host gastrointestinal tract.     

Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.     

Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor

The increase in the concentration of atmospheric carbon dioxide is considered to be one of the main causes of global warming. As estimated by the Intergovernmental Panel on Climate Change (IPCC) criteria, about 10-15% of the gases emitted from the combustion coal being in the form of carbon dioxide. Microalgae and cyanobacteria can contribute to the reduction of atmospheric carbon dioxide by using this gas as carbon source. We cultivated the Scenedesmus obliquus and Spirulina sp. at 30 degrees C in a temperature-controlled three-stage serial tubular photobioreactor and determined the resistance of these organisms to limitation and excess of carbon dioxide and the capacity of the system to fix this greenhouse gas. After 5 days of cultivation under conditions of carbon limitation both organisms showed cell death. Spirulina sp. presenting better results for all parameters than S. obliquus. For Spirulina sp. the maximum specific growth rate and maximum productivity was 0.44 d(-1), 0.22 g L(-1)d(-1), both with 6% (v/v) carbon dioxide and maximum cellular concentration was 3.50 g L(-1) with 12% (v/v) carbon dioxide. Maximum daily carbon dioxide biofixation was 53.29% for 6% (v/v) carbon dioxide and 45.61% for 12% carbon dioxide to Spirulina sp. corresponding values for S. obliquus being 28.08% for 6% (v/v) carbon dioxide and 13.56% for 12% (v/v) carbon dioxide. The highest mean carbon dioxide fixation rates value was 37.9% to Spirulina sp. in the 6% carbon dioxide runs.     

Development of bovine IVF oocytes cultured in medium supplemented with a nitric oxide scavenger or inhibitor in a co-culture system

Bovine IVF oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with either a nitric oxide (NO) scavenger, hemoglobin (Hb, 1 microg/mL) and/or a NO synthesis inhibitor, L(omega)-nitro-L-arginine methyl ester (L-NAME, 1 or 1000 nM) in a cumulus-granulosa cell co-culture system. In Experiment 1, a total of 1,675 cumulus-oocytes complexes was collected for 7 mo and cultured to the blastocyst stage in mBECM with or without Hb after IVM and IVF. There were significant (P<0.0024) model effects of Hb addition and month of oocyte collection on embryo development. A significant (P<0.0023) monthly variation was detected in all developmental stages. However, addition of Hb to mBECM consistently enhanced embryo development to the blastocyst stage over all months. No statistical differences were found in the interaction between Hb addition and month except for the cleavage rate. Overall, a greater percentage of oocytes developed to the 8-cell (P<0.0459), 16-cell (P<0.001), morula (P<0.0013) and blastocyst (P<0.0024) stages after the addition of Hb. In Experiment 2, addition of L-NAME to mBECM supplemented with Hb did not further stimulate prehatched development. In conclusion, the promoting effect of Hb on in vitro development of embryos is highly repeatable over an extended period of time.     

Antioxidants and antioxidant enzymes protect against pulmonary oxygen toxicity in the rabbit

Two major lines of defense exist against oxidant lung injury: tissue antioxidants and antioxidant enzymes. We studied pretreatment with the antioxidants, vitamin E and butylated hydroxyanisole (BHA), and the antioxidant enzymes, superoxide dismutase (SOD) and catalase, in rabbits exposed to 100% O2 for 48 h. BHA (200 mg/kg ip) or vitamin E (50-100 mg/kg po) were given for 2 or 3 days, respectively, before O2 exposure. Combined therapy with polyethylene glycol- (PEG) conjugated SOD (12 mg/kg) and catalase (200,000 U/kg) was given intraperitoneally 1 h before and 24 h after beginning 100% O2. Hyperoxia significantly increased the pulmonary content of malondialdehyde, indicating enhanced lipid peroxidation. One hundred percent O2 also increased lung weight gain and alveolar-capillary permeability to aerosolized 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA, 500 mol wt) and fluorescein isothiocyanate-labeled dextran (7,000 mol wt). Pretreatment with vitamin E, BHA, or the combination of PEG-SOD and PEG-catalase prevented the increase in malondialdehyde, lung weight gain, and alveolar-capillary permeability caused by hyperoxia. These results indicate that augmenting either tissue antioxidants or antioxidant enzymes can prevent the pulmonary injury caused by 48 h of 100% O2 in rabbits.     

2,4-D Hyper Accumulation Induced Cellular Responses of <i>Azolla pinnata</i> R. Br. to Sustain Herbicidal Stress

In the present experiment with ongoing concentration (0 µM, 100 µM, 250 µM, 500 µM and 1000 µM) of 2,4-D, the responses of Azolla pinnata R.Br. was evaluated based on cellular functions. Initially, plants were significantly tolerated up to 1000 µM of 2,4-D with its survival. This was accompanied by a steady decline of indole acetic acid (IAA) concentration in tissues with 78.8% over the control. Membrane bound H⁺ -ATPase activity was over expressed within a range of 1.14 to 1.25 folds with activator (KCl) and decreased within a range of 57.3 to 74.6% in response to inhibitor (Vanadate) application. With regards to IAA metabolism, plants recorded a linear increase with wall bound oxidase activity up to maximum concentration of 2,4-D. The variations were more moderated when wall bound IAA-oxidase recorded a linear increase proportionate to the 2,4-D concentrations. This was more extended with the presence of different isoforms of IAA-oxidase which was much more pronounced with distinct polymorphisms of expressed proteins, however, not independent to the 2,4-D concentrations. Polyamines like spermine, spermidine and putrescine (spm, spd and put) were not consistent in concentration with the dosages of 2,4-D. Besides these, plants were induced to apoplastic NAD(P)H oxidase activity maximally by 1.6 folds under 500 µM 2,4-D over control. Still, putrescine responded more or less consistently and recorded maximally 11.9 folds at 500 µM 2,4-D as compared to the control. NAD(P)H oxidase activity recorded maximally 1.6 folds against control and remain consistent throughout the concentrations of 2,4-D. GPX along with APX were more linear in responses through the concentration of 2,4-D except CAT as compared to control. On enzymatic antioxidative activity, peroxidases (GPX and APX) were overexpresed in a similar manner except for catalase with a non-significant rise. In stabilization of cellular redox, glutathione reductase attended maximum value by 2.45 folds at 1000 µM evidenced with significant variations in protein polymorphism. The sensitivity of 2,4- D also appeared in Azolla with a maximum loss of nucleic acids as documented by the comet assay. Moreover, the Azolla might have some DNA damage protective activity as evident using frond extract with plasmid nick assay. Therefore, Azolla plants with its cellular responses is evident to sustain against the 2,4-D herbicidal stress and may be granted in bio remediation process for the contaminated soil.