Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl₂ at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl₂ at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl₂ and MgSO₄ were observed at a wide range of concentrations up to 400 mM. CaCl₂ was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.     

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

海桑属植物分子鉴定的ISSR分析

为准确鉴别海桑属植物,采用ISSR（inter-simple sequence repeat）分子标记技术,对6种海桑属植物(海桑、拟海桑、杯萼海桑、海南海桑、卵叶海桑及无瓣海桑)基因组DNA进行PCR扩增。建立了海桑属植物ISSR标记的标准程序和海桑属各物种的DNA指纹数据库。采用的11个引物共产生71个物种特征性标记,其中无瓣海桑23个,海南海桑16个,海桑15个,杯萼海桑6个,拟海桑6个,卵叶海桑5个。运用这些特征性标记,可迅速区分海桑属植物。研究表明ISSR技术是在分子水平上鉴定海桑属植物的一种行之有效的方法。     

The ectopic expression of apple <Emphasis Type="Italic">MYB1</Emphasis> and <Emphasis Type="Italic">bHLH3</Emphasis> differentially activates anthocyanin biosynthesis in tobacco

Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula?×?P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1?×?10⁷ protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG₁₅₀₀. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar.     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Protoplast isolation for species in the Chamelaucium group and the effect of antioxidant enzymes (superoxide dismutase and catalase) on protoplast viability

An effective protocol for protoplast isolation from young leaves and somatic embryogenic cells of species in the Chamelaucium group and the use of superoxide dismutase (SOD) and catalase (CAT) to enhance protoplast viability are described. Mesophyll protoplasts were isolated from young leaves of a white Geraldton waxflower (Chamelaucium uncinatum) line 583, using a mixture of 1% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, and 0.1% (w/v) pectolyase. Viability of isolated mesophyll protoplasts increased dramatically when SOD and CAT were added. The highest increase of 7.61-fold in viability and 4.34-fold of viable protoplast yield were achieved when a combination of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 was added to the enzyme mixture. Somatic embryogenic cell-derived protoplasts were isolated from embryogenic suspension cells of C. uncinatum line 583 when 1% (w/v) hemicellulase was added to a combination of 2% (w/v) cellulase R10, and 1% (w/v) macerozyme R10. Addition of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 to the enzyme mixture improved viability only slightly, to above 90%, but improved yield significantly (6.6-fold). This combination of enzymes was also used to isolate protoplasts from embryogenic suspension cells of Chamelaucium repens and from young leaves of C. uncinatum, Actinodium calocephalum, Verticordia etheliana, Verticordia grandis, Verticordia hughanii, and Verticordia mitchelliana successfully with viability >80% and viable yield >7?×?10⁵ cells g^?1 fresh weight (or per milliliter packed cell volume in the case of suspension cells).     

Plant regeneration from protoplasts in Indian local Coriandrum sativum L.: scanning electron microscopy and histological evidences for somatic embryogenesis

Coriandrum sativum L. is an annual herb belonging to the family Umbelliferae. It is used as a spice plant in Indian subcontinent and it has several medicinal applications as well. In this present article, an efficient plant regeneration protocol from protoplasts via somatic embryogenesis was established and is reported. This is the first ever protoplast isolation study in Indian local coriander in which plant regeneration was achieved. Hypocotyl-derived embryogenic callus was used as a source of protoplast. The embryogenic callus suspension was prepared by transferring tissues onto rotary-agitated liquid Murashige and Skoog, added with 1.0 mg l^?1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l^?1 KIN (6-furfurylaminopurine). The suspension was digested with enzymatic solutions and a combination of cellulase (2.0 %), pectinase (1.0 %), macerozyme (0.02 %) and driselase (0.50 %) induced maximum yield of protoplasts (34.25 × 10⁵). In 1.0 mg l^?1 2,4-D + 1.0 mg l^?1 KIN containing medium, protoplasts divided well and formed maximum number of microcolonies (14.30/test tube). The protoplast callus (PC) biomass grew well in solid medium. The protoplast embryogenic callus was rich in protein, proline and sugar compared to non-embryogenic PC. The protoplast originated callus later differentiated into somatic embryos. The somatic embryo morphology, scanning electron microscopy and histology of embryo origin and development were investigated and discussed in details in this present communication. In 1.0 mg l^?1 2,4-D + 0.5 mg l^?1 BA (6-Benzyladenine), maximum number of embryos were formed on microcallus (26.6/callus mass). The embryo matured and germinated into plantlets at a low to moderate rate, highest (31.3 %) embryo germination was observed in 1.0 mg l^?1 BA + 0.5 mg l^?1 α-Naphthalene acetic acid added medium. The entire process of regeneration took about 4–5 months’ time for recovering plantlets from protoplasts.     

An Efficient Protocol for Ulmus minor Mill. Protoplast Isolation and Culture in Agarose Droplets

We describe here an efficient and reproducible protocol for isolation and culture of protoplasts from Ulmus minor. Different sources of donor tissues were tested for protoplast isolation: callus and juvenile leaves from in vitro and greenhouse plants. Several combinations and concentrations of hydrolytic enzymes were used. Comparative tests between Cellulase Onozuka R10 and Cellulase Onozuka RS were made and the last one proved to be more efficient. Both the pectinases used, Macerozyme Onozuka R10 and Pectinase (Sigma^®), were efficient in protoplast isolation and there was no need for a more active pectinase. In vitro leaves proved to be the best source for protoplast isolation and produced an average of 3.96 × 10⁷ protoplasts per gram of fresh weigh. Elm mesophyll protoplasts were cultured using the advantageous method of agarose droplets and a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 10⁵ and 2 × 10⁵ protoplasts ml^?1). Protoplast division and evolution into colonies and microcalli was promoted in the agarose droplets plated at 2 × 10⁵ protoplasts ml^?1. Ten weeks after protoplast culture initiation a plating efficiency of 2.7% was attained and the bigger microcalli, with at least 0.5 mm diameter, were transferred to a solid medium previously used for the production of embryogenic callus.     

Plant regeneration from protoplasts of Solanum species with potential agricultural value (S. hjertingii,S. polyadenium,S. capsicibaccatum)

Protoplasts have been isolated from three tuber-bearing Solanum species, S. hjertingii, S. polyadenium and S. capsicibaccatum, that are sexually incompatible with S. tuberosum, but possess potentially useful characters. For isolating protoplasts from leaves of in vitro shoot cultures of S. hjertingii and S. capsicibaccatum growth was improved by including silver thiosulfate in the medium. However, for S. polyadenium, leaves of pot-grown plants were the best source for protoplasts. Following protoplast division and culture, plants were regenerated from protoplasts of each of the species. The pattern of chromosome variation in regenerants was similar to that observed for other diploid and tetraploid Solanum species. The results indicate that it should be possible to introduce the potentially useful germplasm from these wild species into somatic hybrids with S. tuberosum by protoplast fusion.Abbreviations STS silver thiosulfate - BAP benzylaminopurine - GA₃ gibberellic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid     

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.     

Suspension and protoplast culture of U.S. rice cultivars

Efficient protoplast culture and plant regeneration of five U.S. rice cultivars (Oryza sativa L.) - Mercury, Lacassine, Maybelle, Cypress, and Lemont - were obtained from suspension cells maintained in modified General Medium. Embryogenic suspension cells were developed from calli grown on the original callus induction medium for 10–20 weeks without subculture. Weekly subculture of the suspensions for five to eight weeks yielded cells suitable for protoplast isolation. After 2 weeks, rate of colony formation from protoplasts varied among the cultivars and ranged from 2.5 to 6.8%. Improvement of plating efficiencies to as high as 13.7% was obtained by conducting a second cycle of protoplast culture. A total of 525 plants were regenerated from the cultivars studied.Abbreviations BAP 6-benzylaminopurine - CH casein acid hydrolysate - MGM modified General Medium - Kin kinetin - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Stable,fertile somatic hybrids between <Emphasis Type="Italic">Sinapis alba</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> show resistance to <Emphasis Type="Italic">Alternaria brassicae</Emphasis> and heat stress

Wild relatives of Brassica are a rich reservoir of genes that are invaluable for the improvement of cultivated species. Sinapis alba is a close relative of crop Brassicas that possesses several desirable traits such as tolerance to Alternaria black spot disease, heat stress, insect pests and nematodes. This study is aimed at developing and characterizing hybrids between Brassica juncea and S. alba with the ultimate goal of transferring genes for tolerance to Alternaria brassicae and heat stress, the traits that are lacking in cultivated Brassica. We generated three hybrids between B. juncea and S. alba through protoplast fusion. The hybridity was confirmed through cytology and molecular markers. While two of the hybrids were symmetric, the third one was asymmetric and had greater resemblance to B. juncea. Hybrids showed some characteristic features of the parents and were fully male and female fertile and also set seeds upon back crossing with the parent species. In vitro leaf assay and field inoculation studies revealed that the hybrids are highly resistant to A. brassicae. Besides, hybrids set seeds at temperature of >?38 °C when parents failed to produce seeds indicating that hybrids possess heat tolerance. These stable hybrids provide a reliable genetic resource for transfer of genes from S. alba into cultivated Brassica species.     

Formation,regeneration, and transformation of protoplasts of Streptomyces diastatochromogenes 1628

Toyocamycin exhibits effective biological activities for use against plant pathogenic fungi thanks to its structural similarity to nucleoside. It has been recognized as a promising agricultural antibiotic utilized in controlling the occurrence of plant diseases. In our previous study, a strain that was isolated was identified and designated as Streptomyces diastatochromogenes whose major secondary metabolite was toyocamycin, but the production was largely limited. Protoplast transformation is a useful technique in the improvement of streptomycete. In this study, we optimized some key factors necessary for protoplast formation, regeneration, and transformation of S. diastatochromogenes. When mycelium was cultivated in CP medium with 1 % glycine, harvested at 48 h old, and then treated with 3 mg lysozyme/mL in P buffer for 1 h, the greatest regeneration frequency (42.5 %) of protoplasts was obtained. By using 1?×?10⁹/mL protoplasts with polyethylene glycol 1000 at a concentration of 30 % (w/v), the best performance of protoplast transformation efficiency was 4.8?×?10³/μg DNA transformants.     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis,S. macrocephala and S. scabra

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.Abbreviations BAP 6-benzylaminopurine - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalenacetic acid On leave from: Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brasil     

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10⁶ protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.     

The isolation of protoplasts of the fission yeastSchizosaccharomyces byTrichoderma viride and snail enzymes

The formation of protoplasts of the fission yeastsSchizosaccharomyces pombe andSchizosaccharomyces versatilis after the combined application of snail enzymes andTrichoderma viride enzymes in an osmotic stabilizer (0.4m KC1, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.     

Ostreopsis cf. ovata in the Gulf of Gabès (south-eastern Mediterranean Sea): morphological,molecular and ecological characterization

In the last few decades, the frequency of the toxic benthic dinoflagellate Ostreopsis cf. ovata proliferation has increased in the Mediterranean Sea. These blooms are associated with harmful effects on human health and the environment. The present work provides the first long term study on the spatio-temporal distribution of O. cf. ovata in relation to physical parameters in the Gulf of Gabès coastal waters (south-eastern Mediterranean Sea), as well as its morphological, molecular and physiological features. The strains of O. cf. ovata were identified morphologically by light and epifluorescence microscopy. The morphology and the size range of cultured strains were similar to those described regarding O. cf. ovata isolated from the Mediterranean Sea. The ultrastructural analysis of O. cf. ovata cells using the transmission electron microscopy showed the presence of numerous vesicles (VE) containing spirally coiled fibers (SCFs) connected to the mucus canal (CH). The phylogenetic tree based on the internal transcribed spacer region containing the 5.8S rDNA (ITS-5.8S rDNA) revealed that O. cf. ovata strains were placed into the Mediterranean/Atlantic clade. In addition, O. cf. ovata toxicity was evaluated by the mouse bioassay and a dose level ≥ 4 × 10⁴ cells was found to be lethal to mice. The examination of the O. cf. ovata occurrence in the Gulf of Gabès at a large temporal scale (1997–2012) revealed a clear seasonal pattern with dominance from midsummer (July) to late autumn (November). Furthermore, a positive correlation was found between the abundance of O. cf. ovata and salinity, whereas no correlation was found as regards temperature. The occurrence of O. cf. ovata was only detected at salinity above 35 and the highest concentrations were observed at 45. Laboratory experiments confirmed such a result and showed that isolated O. cf. ovata strains had optimal growth at salinity ranging between 35 and 45, with its peak at 40.     

A general method for the high-yield isolation of mesophyll protoplasts from deciduous tree species

High yields (1.2–8.0 × 10⁶ g fresh wt.^?1) of viable leaf mesophyll protoplasts have been isolated from a range of mature deciduous woody-plant species (Betula pendula, Alnus glutinosa, Salix caprea var. Kuroyanagi, S. alba var. Tristis, Populus Tacatricho (= P. tacamahacca × trichocarpa 32), Ulmus glabra var. camperdown), and juvenile glasshouse-grown material (B. pendula, B. pubescens, A. glutinosa). Protoplasts are only released if chopped leaf tissue is thoroughly washed prior to digestive enzyme addition. The nature of the washing requirement has been investigated and it has been demonstrated that water soluble compounds are released from chopped leaves which modify their cell-wall structure rendering them resistant to enzymic digestion. When analyzed by paper chromatography the leachate from B. pendula leaves was found to contain the hydroxycinnamic acids p-coumaric acid (PCA) and o-coumaric acid (OCA). Pre-incubation of B. pendula tissue (which is normally susceptible to enzymic digestion) in authentic samples of PCA and OCA prior to enzymic incubation, completely suppressed protoplast yields. The relevance of hydroxycinnamic acids to protoplast isolation and plant tissue culture is discussed.     

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×10⁶/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R₁ plants were self-pollinated and immature R₂ embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R₂-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.Abbreviations BC backcross - BMS Black Mexican Sweetcorn - 2,4-D 2,4-dichlorophenoxyacetic acid - PWS protoplast wash solution (0.2 M mannitol, 80 mM CaCl₂) - FDA fluorescein diacetate - ABA abscisic acid