Oxidative metabolism and protoplast culture

Summary Barley leaf blade protoplasts accumulate malonaldehyde, a product of lipid peroxidation, during culture. In addition, glutathione levels fall after protoplast isolation and the proportion of glutathione in the oxidized state rises. These data indicate oxidative stress after protoplast isolation and during culture. The cause of this phenomenon is revealed by data showing that the activities of enzymes associated with antioxidative processes including glutathione reductase and ascorbate peroxidase decrease after barley protoplast isolation. In contrast, protoplasts isolated from suspension cultured cells of bromegrass and soybean exhibit little evidence for oxidative stress and increased activities of glutathione reductase and ascorbate peroxidase. We suggest that an antioxidative response is associated with mitosis and colony formation from protoplasts, as exhibited by bromegrass and soybean. Conversely, failure of an antioxidative response is associated with low viability and absence of mitosis, as in barley. Increased viability of barley leaf protoplasts cultured on feeder layer cells is correlated with increased glutathione content and higher glutathione reductase activity.     

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper

We studied the sequence of several metabolic reactions, representative for oxidative damage and protection, in primary leaves of Phaseolus vulgaris (cv. Limburgse vroege) as a function of root assimilation of a toxic sublethal Cu concentration (630 μ M ). A transient increase of products of membrane peroxidation was observed in the primary leaves during the period of Cu uptake. This rise was mainly due to the oxidizing properties of copper itself and not to a stimulation of the lipoxygenase (EC 1.13.11.12) activity. In our experimental conditions, membrane lipid peroxidation and K⁺-leakage were not directly related; during at least three days after Cu application to the roots, when products of lipid peroxidation were already detected in the leaf, permeability of the cytoplasmic membrane for K⁺ was improved. However, Cu stimulated the capacity of catalase (EC 1.11.1.6) and ascorbate peroxidase (EC 1.11.1.11). These enzymes protect the tissue against oxidative stress since at least the hydrogen peroxide content was significantly reduced. Superoxide dismutase (EC 1.15.1.1) was not involved in this defense mechanism.     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C₁, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO₃, an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O₂-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A₂, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.     

Arbuscular mycorrhizal influence on growth,photosynthetic pigments,osmotic adjustment and oxidative stress in tomato plants subjected to low temperature stress

The influence of the arbuscular mycorrhizal (AM) fungus, Glomus mosseae, on characteristics of growth, photosynthetic pigments, osmotic adjustment, membrane lipid peroxidation and activity of antioxidant enzymes in leaves of tomato (Lycopersicon esculentum cv Zhongzha105) plants was studied in pot culture under low temperature stress. The tomato plants were placed in a sand and soil mixture at 25°C for 6 weeks, and then subjected to 8°C for 1 week. AM symbiosis decreased malondialdehyde (MDA) content in leaves. The contents of photosynthetic pigments, sugars and soluble protein in leaves were higher, but leaf proline content was lower in mycorrhizal than non-mycorrhizal plants. AM colonization increased the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) in leaves. The results indicate that the AM fungus is capable of alleviating the damage caused by low temperature stress on tomato plants by reducing membrane lipid peroxidation and increasing the photosynthetic pigments, accumulation of osmotic adjustment compounds, and antioxidant enzyme activity. Consequently, arbuscular mycorrhiza formation highly enhanced the cold tolerance of tomato plant, which increased host biomass and promoted plant growth.     

Specific features of the ascorbate/glutathione cycle in cultured protoplasts

Protoplasts isolated from Nicotiana tabacum (L.) leaves were cultured for 6 days in liquid medium and some features of their antioxidant capacity were investigated. Ascorbate exported into the culture medium was oxidized non-enzymically, whereas important modifications of the enzymic scavenging activity were detected inside protoplasts. A new pool of isoenzymes, most of them with cytoplasmic characteristics, ensures the increased ascorbate peroxidase activity. The specific activity of other enzymes involved in the ascorbate/glutathione cycle, such as dehydroascorbate reductase and glutathione reductase, and of glutathione peroxidase were modified, resulting in cultured protoplasts with quantitative differences in antioxidant capacity compared to leaves. The hypothesis presented here suggests that the new scavenging system is related to differences in the compartment-specific accumulation of active oxygen species following protoplast isolation. Received: 8 December 1997 / Revision received: 27 March 1998 / Accepted: 10 April 1998     

Changes in peroxidation under conditions of 3,4-benzypyrene-induced carcinogenesis

The activities of enzymatic systems generating and destroying peroxides and the lipid peroxide content in neoplastic rat liver and 3,4-benzpyrene-induced sarcoma were studied. The tumour was characterized by high activity of glutathione peroxidase and low activity of catalase. No urate- and glycolate oxidases or ascorbat dependent peroxidation of lipids and lipid peroxides were found in the tumour. In the liver of neoplastic animals the activities of glutathione peroxidase and NADPH-dependent system of microsomal phospholipid peroxidation and the lipd peroxides content were increased, whereas the activities of catalase and urate oxidase were decreased.     

Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): Involvement of lipid peroxides in chlorphyll degradation

Germinating seedlings of mung bean ( Phaseolus vulgaris L. cv. K-16) were treated with different concentrations of cadmium acetate (10, 50 and 100 μ M ). Cd²⁺ lowered the chlorophyll and heme levels. The level of lipid peroxides were higher on day 3 than on day 6. However, Cd²⁺ treatment significantly enhanced the level of lipid peroxides. Similarly, a dose-dependent induction of lipoxygenase (EC 1.13.11.12) activity was observed with Cd²⁺ treatment. Further, the activities of antioxidant enzymes such as superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) were decreased. Our results suggest that lipoxygenase-mediated accumulation of lipid peroxides on the one hand and inhibition of free radical scavenging enzymes like superoxide dismutase and catalase on the other caused a pronounced reduction in the chlorophyll and heme levels of the seedlings. The experiments conducted on the effect of Cd²⁺ on dark-grown seedlings did not conform with the result of light-grown seedlings. Though chlorophyll and heme levels decreased in a dose-dependent manner, no accumulation of lipid peroxides was observed, suggesting that the inhibition of chlorophyll synthesis by Cd²⁺ is achieved both by reaction with constituent biosynthetic enzymes as well as peroxide-mediated degradation.     

Catalase Is Differentially Expressed in Dividing and Nondividing Protoplasts

Based on our previous results that peroxidase is induced in dividing tobacco protoplasts but it is not expressed in the nondividing grapevine (Vitis vinifera L.) protoplasts during culture (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270), we further tested the hypothesis that oxidative stress may be implicated in the recalcitrance of plant protoplasts. The expression of catalase, a major defense enzyme against cell oxidation, was studied during isolation and culture of mesophyll protoplasts from the recalcitrant grapevine and regenerating tobacco (Nicotiana tabacum L.). Incubation of tobacco leaf strips with cell wall-degrading enzymes resulted in a burst of catalase activity and an increase in its immunoreactive protein; in contrast, no such increases were found in grapevine. The cathodic and anodic catalase isoforms consisted exclusively of subunits [alpha] and [beta], respectively, in tobacco, and of subunits [beta] and [alpha], respectively, in grapevine. The catalase specific activity increased only in grapevine protoplasts during culture. The ratio of the enzymatic activities to the catalase immunoreactive protein declined in dividing tobacco protoplasts and remained fairly constant in nondividing tobacco and grapevine protoplasts during culture. Also, in dividing tobacco protoplasts the de novo accumulation of the catalase [beta] subunit gave rise to the acidic isoenzymes, whereas in nondividing tobacco and grapevine protoplasts, after 8 d in culture, only the basic isoenzymes remained due to de novo accumulation of the [alpha] subunit. The pattern of catalase expression in proliferating tobacco leaf cells during callogenesis was similar to that in dividing protoplasts. The different responses of catalase expression in dividing and nondividing tobacco and grapevine mesophyll protoplasts may indicate a specificity of catalase related to induction of totipotency.     

Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defense system

Seedlings of Indian mustard (Brassica juncea L.cv. RH-30) grown in controlled condition (irradiance 75 Wm(-2), RH 60-70% and temp. 25 +/- 2 degrees C) for 7d and watered with Hoagland's solution containing different level of NaCL (50-250 mmol/L NaCl) with or without putrescine (PUT, 0.1 mmol/L) were examined for PUT amelioration of NaCl induced inhibition in seedling growth by altering activity of antioxygenic enzymes and level of free radicals in the leaves. Salinity caused reduction in seedling growth and biomass accumulation was parallel to increased superoxide (*O2-), hydrogen peroxide (H2O2) levels, lipid peroxidation (MDA content) and electrolyte leakage in leaf tissues which were reversed significantly by PUT. The antioxygenic enzymes viz superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) were differentially altered, depending on salt level. PUT induction of enzyme was in the following order APX>GR>CAT>SOD>POD in leaf tissues of salt stressed seedlings. PUT increased the level of glutathione and carotenoids in leaf tissues. This finding suggests that PUT might be activating antioxygenic enzymes and elevating antioxidants there by controlling free radical generation, hence preventing membrane peroxidation and denaturation of biomolecules resulting into improved seedling growth under salinity.     

Tissue degradation and enzymatic activity observed during protoplast isolation in two ornamental <Emphasis Type="Italic">Grevillea</Emphasis> species

Summary Degradative changes in tissue during protoplast isolation were a contributing factor to low protoplast yields in the saltsensitive Grevillea arenaria (R. Brown) and the salt-tolerant Grevillea ilicifolia (R. Brown). Protein and malondialdehyde content decreased significantly during the protoplast isolation procedure. Acid and neutral proteases were identified, and high acid protease activities were correlated to low protoplast yields. Acid phosphatase, catalase, polyphenol oxidase and lipoxygenase activities increased in both Grevillea species with cell wall digestion. High activities of catalase and low levels of polyphenol oxidase were correlated with high protoplast yields. Levels of acid phosphatase and lipoxygenase were not good indicators of final protoplast yields. The addition of the anti-oxidant, reduced glutathione, and the acid protease inhibitor, pepstatin A, significantly increased protoplast yields. Strategies were identified to minimize deleterious degradative effects during the isolation of protoplasts, including strict pH control, testing a number of cell wall digestion enzymes, and the addition of anti-oxidative metabolites and protease inhibitors.     

Leaf senescence and lipid peroxidation: Effects of some phytohormones, and scavengers of free radicals and singlet oxygen

During in vitro senescence (chlorophyll loss) of oat ( Avena sativa L. cv. Victory) leaf segments and of leaf discs of Rumex obtusifolius L, the activity of catalase decreases and lipid peroxidation increases. The activity of superoxide dismutase (SOD) decreases in Rumex leaf discs but changes little in oat leaf segments. Kinetin treatment of oat leaf segments, and GA₃ treatment of Rumex leaf discs, inhibit decline in the enzyme activities and increase in the level of lipid peroxidation and strongly inhibit senescence. In either leaf tissue a treatment with ethanol or vitamin E (scavengers of free radicals) or with diphenylisobenzofuran (scavenger of singlet oxygen) results in a strong inhibition of lipid peroxidation and senescence, but does not affect much the decline in the SOD and catalase activities. It is concluded that, i) senscence-associated lipid peroxidation is induced by free radicals and singlet oxygen; and, ii) kinetin and GA₃ inhibit senescence mainly by a modulation of lipid peroxidation through maintaining high levels of such cellular scavengers as SOD and catalase.     

Contamination of oat mesophyll protoplasts by apoplastic polyamine oxidase

Mesophyll protoplasts isolated from peeled oat ( Avena sativa L. cv Victory) leaves with 1% (w/v) Cellulysin in 20 m M KPO₄, pH 5.5 and 0.6 M sorbitol retain about 6% of the polyamine oxidase (PAO, EC 1.4.3.4) activity of the whole peeled leaf. However, more than 99% of the oat leaf PAO activity is apoplastic and can be extracted by vacuum infiltration with 200 m M NaCl and this procedure extracts no activity for the cytoplasmic marker enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49). By these criteria we consider PAO in oat leaves to be totally apoplastic and PAO found in the isolated protoplast to be contamination. The degree of protoplast contamination by PAO depends on the pH and ionic strength of the isolating and washing medium. It can be eliminated by washing protoplasts in 0.6 M sorbitol with 100 m M KPO₄, pH 6.5. Pellets of lysed protoplasts incubated with dialyzed apoplastic enzymes in 5 m M KPO₄, pH 5.5 adsorb about 87% of the added PAO activity but only about 25% of the added peroxidase (EC 1.11.1.7) activity. The adsorbed activity can be solubilized from the pellet by extraction with 1 M NaCl. The results demonstrate that weakly ionically bound cell wall enzymes may contaminate protoplasts isolated and purified by conventional techniques.     

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid     

Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues

The effects of hyperoxia on the status of antioxidant defenses and markers of oxidative damage were evaluated in goldfish tissues. The levels of lipid peroxides, thiobarbituric acid reactive substances, carbonyl proteins and the activities of some antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of goldfish, Carassius auratus L., over a time course of 3-12 h of hyperoxia exposure followed by 12 or 36 h of normoxic recovery. Exposure to high oxygen resulted in an accumulation of protein carbonyls in tissues throughout hyperoxia and recovery whereas lipid peroxides and thiobarbituric acid reactive substances accumulated transiently under short-term hyperoxia stress (3-6 h) but were then strongly reduced. This suggests that hyperoxia stimulated an enhancement of defenses against lipid peroxidation or mechanisms for enhancing the catabolism of peroxidation products. The activities of principal antioxidant enzymes, superoxide dismutase and catalase, were not altered under hyperoxia but catalase increased during normoxic recovery; activities may rise in anticipation of further hyperoxic excursions. In most tissues, the activities of glutathione-utilizing enzymes (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) as well as glucose-6-phosphate dehydrogenase, were not affected under hyperoxia but increased sharply during normoxic recovery. Correlations between some enzyme activities and oxidative stress markers were found, for example, an inverse correlation was seen between levels of thiobarbituric acid reactive substances and glutathione-S-transferase activity in liver and catalase and glucose-6-phosphate dehydrogenase in kidney. The results suggest that liver glutathione-S-transferase plays an important role in detoxifying end products of lipid peroxidation accumulated under hyperoxia stress.     

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

Influence of Root Oxygen Deficiency on Photosynthesis and Antioxidant Status in Barley Plants1

Roots of barley plants (Hordeum vulgare L., cv. Alfa) were subjected to hypoxia for 120 h. By 72 to 120 h of soil flooding, a noticeable decrease in the rate of CO₂ assimilation and transpiration was observed. A drop in the activities of Rubisco and photorespiratory enzymes was found. We examined the changes in the activities of enzymes involved in the antioxidative system and stress markers related to membrane integrity, namely, lipid peroxidation and electrolyte leakage. Catalase and peroxidase activities were increased during the experiment, whereas superoxide dismutase activity drastically decreased.     

Induction of Male Sterility in Wheat by Meiotic-Stage Water Deficit Is Preceded by a Decline in Invertase Activity and Changes in Carbohydrate Metabolism in Anthers

Total peroxidase, NADH-peroxidase, ascorbate peroxidase, superoxide dismutase, and catalase activities were measured in tobacco (Nicotiana tabacum) leaves and in regenerating and nonregenerating protoplasts isolated from the same tissue and cultured for 2 weeks. The specific ranges of H2O2 concentration at which the enzymes scavenging the active forms of oxygen may efficiently operate and the activities of those enzymes were determined in an extract from tobacco leaves and in dividing and nondividing tobacco mesophyll protoplasts. The overall H2O2-scavenging enzyme activities were similar in both protoplast populations during the 2 to 3 d of culture. After 3 d, the regenerating protoplasts started to divide and both the antioxidant enzyme activities and the total peroxidase activity increased; in contrast, the viability and the H2O2-scavenging enzyme activities in nonregenerating protoplasts dramatically decreased. Surprisingly, the regenerative potentiality in dividing protoplasts was specifically correlated with a higher NADH-peroxidase activity, which resulted in a net H2O2 accumulation in the cells. Light, which causes the accumulation of active forms of oxygen in photosynthetic organelles, also stimulated catalase and ascorbate peroxidase activities in dividing protoplasts. We suggest that the localization of H2O2 rather than its absolute concentration might be responsible for oxidative stress and that controlled amounts of H2O2 are necessary to allow proper cell-wall reconstitution and the consequent cell division.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine