Role of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus sylvestris L.)

Callus tissues originating from buds of mature Scots pine (Pinus sylvestris L.) trees exhibit the typical problem of browning, which leads to degeneration and death of the tissues. The effects of medium, origin (tree and location) and endophyte infection were studied on the browning and growth of bud-derived tissue cultures. The calli growing on medium with higher kinetin content and source of organic nitrogen, and originating from the southern location grew better and exhibited less browning. Endophytic microbial cells were detected in the brown callus tissues by transmission electron microscopy. The natural endophyte infection frequency of Scots pine buds was studied and found dependent on the tree, but not on the location. A well-growing, green callus line was artificially infected by an endophytic strain of Methylobacterium extorquens, and browning was not observed on solid media compared to the uninfected control clones of the same callus. However, suspension cultures started from the infected callus died faster than cultures started from the uninfected callus. The endophyte species composition and plant genotype together with tissue culture conditions are the key factors for gaining plant tissue cultures with high regeneration capacity.     

尾叶桉的不同类型愈伤组织芽分化与某些相关生理生化指标的关系

切取8d苗龄的尾叶桉幼苗下胚轴,培养7d后愈伤组织的诱导率达100%。8周后,愈伤组织分化成褐色、白色和浅绿色3种类型。其中只有浅绿色愈伤组织能分化出不定芽,分化率达57%。浅绿色愈伤组织的过氧化物酶（POD）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和多酚氧化酶（PPO）的活性最高。除SOD外,浅绿色愈伤组织的POD、CAT和PPO活性与其他两类愈伤组织相比具有显著性差异（P〈0.05）。H2O2和丙二醛含量则是褐色愈伤组织的最高。     

Changes in morphology and biochemical indices in browning callus derived from <Emphasis Type="Italic">Jatropha curcas</Emphasis> hypocotyls

Callus browning is a typical feature of callus cultures derived from the hypocotyl of Jatropha curcas. Brown callus results in decreased regenerative ability, poor growth and even death. In this study, we investigated the effect of browning on callus morphology and biochemical indices. Light microscopy and scanning electron microscopy showed striking differences in callus morphology. During browning, chlorophylls and carotenoids concentrations decreased steadily. Polyphenol oxidase (PPO) and peroxidase (POD) enzymatic activities patterns were similar during callus culture with a higher activity level at week 3 compared to week 2 or later weeks. Grey relation degree analysis indicated that PPO played a more important role than POD in enzymatic callus browning. Polyacrylamide gel electrophoresis results showed differences between browning and non-browning callus. Gas chromatography–mass spectrometry results showed that saturated and unsaturated fatty acid quantities differed significantly but there was little difference in fatty acid composition between non-browning and browning callus. Differences in 17, 18.4 and 25 kDa protein concentrations were also observed in browning and non-browning callus using sodium dodecyl sulfate–polyacrylamide gel electrophoresis.     

A single genetic locus in chromosome 1 controls conditional browning during the induction of calli from mature seeds of Oryza sativa ssp. indica

Being the crucial step for rice transgenic manipulation, callus culture from mature seeds is severely restricted by browning of induced calli, especially in the case of indica (Oryza Sativa L.) rice. Once this browning occurs, the callus will die and no embryonic calli can be obtained for regeneration. Here we report an induction procedure that overcomes callus browning was found. To clarify the inheritance pattern of callus browning, two reciprocal crosses F₂ and two backcrosses BC₁ were made between indica cultivar inbred lines 93-11 and YueTaiB (YTB) which produced normal and browning respectively in the same induction medium. The ratio of browning to normal in the reciprocal F₂ and backcross (BC₁) populations tested was approximately 1:3 and 1:1, respectively, these results indicate that callus browning is controlled by one single chromosomal locus which is tentatively named Ic1 (Induced callus 1). The genetic mapping of this locus was carried out using microsatellite markers (SSR) in a 216 extremely browning F₂ seed callus. The analysis of genetic linkage indicated that one single locus that mapped to chromosome 1 was correlated to callus browning, and the closest marker in this study was mapped within 1.9 cM from the target locus.     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Callus cultures from shoot tips of mature Scots pine ( Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non-embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non-embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean ( Vicia faba ) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS-PAGE and non-denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light-yellow non-embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.     

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

抗氧赖氨酸的芦笋细胞系(简报)

芦笋(石刁柏)栽培品种是较好的蔬菜国外已研究了它的试管苗繁殖技术和单倍体育种。其总氮含量之中,蛋白质氮占四分之三,非蛋白质氮占四分之一。Draper等(1983)曾观察到根癌农杆菌(Agrobateriumtu mefaciens)能附着于从石刁柏叶状枝分离出的细胞之细胞壁。我们设想,芦笋也可能是研究赖氨酸积累或Ti质粒转化单子叶植物的适宜材料。——     

Genotype and explant-type dependent morphogenesis and silicon response of common reed (Phragmites australis) tissue cultures

The effects of silicon on the growth and development of Phragmites australis (Cav.) Trin. Ex Steud. (common reed) stem nodal and root embryogenic calli were investigated. Silicon is considered to be a beneficial or quasi-essential nutrient for several Gramineaceous plants, including reed. Seven callus lines of four geographical locations (genotypes 1-4) within Hungary were investigated. Callus lines 1A, 2A and 3A were produced from stem nodal explants, while lines 1B, 2B, 3B and 4 were produced from roots. For the assay of silicon-dependent growth of callus lines of identical genotype but originating from different explants, we measured the increase of fresh weight of lines 1A and 1B. The studied developmental parameters were the increase of the number of somatic embryos (for callus lines 1A and 1B) and plant or root production from somatic embryos (for all genotypes/callus lines). Silicon was added to the culture medium as sodium silicate. In control cultures, plant or root regeneration from embryogenic calli was strongly genotype- and explant type-dependent. Stem nodal explants developed plants on regeneration medium in case of callus lines 2A and 3A, while line 1A produced roots only. All root derived calli developed roots on regeneration medium. Silicon stimulated the growth of both stem nodal and root calli (callus lines 1A, B) however, the concentration optima were different. Somatic embryogenesis of root calli, but not of stem nodal calli, was stimulated by silicate at low concentrations. However, for both of these callus lines, root development was stimulated by silicon. It had genotype-dependent influences on plant regeneration: while stimulation was observed in case of callus line 2A, inhibition occurred for line 3A. Root morphogenesis on calli was significantly influenced by silicon and depended on the callus line studied. Root production was stimulated on callus lines 1A, B and 2B, while in case of callus line 3B, it was significantly inhibited. The morphogenetic effects of Si were similar for different explants of the same geographical origin, i.e. plant or root production was similarly stimulated or inhibited by this element. We can conclude that the effects of Si on plant or root development depend on reed genotype used for callus induction. Its effect on growth and somatic embryogenesis depends on the explant type used for callus production. This is the first detailed report on the role of silicon in plant vegetative development and morphogenesis of a Gramineaceous plant.     

Enhanced proteolysis leads to pre-mature cell death under the influence of elicitor like mycelial components from karnal bunt (tilletia indica) pathogen in wheat callus cultures

Calli raised from mature embryos of susceptible wheat cultivar WH 542 were used in the present study as in vitro bioassay system to study the influence of disease determinant(s) of Karnal bunt (Tilletia indica), a semi-biotrophic fungal pathogen of wheat. Influence of elicitor and conditioned medium (CM) prepared from fungal cultures of T. indica was investigated on induction of programmed cell death (PCD). Induction of PCD was observed as hypersensitive response (HR) in terms of browning at localized regions of callus cultures and induction of proteolytic enzyme(s). Elicitor treated calli showed higher induction of protease activity than untreated and CM-treated cultures, which showed not much change in the activity. It was further substantiated by gel protease assay and activation of caspase-3 like protein(s) in callus cultures that clearly suggested the presence of signaling molecule(s) in the fungal elicitor preparation rather than in conditioned medium. This study further demonstrated that only elicitor preparation possesses such molecule(s), which might be cell wall bound components, rather than secretory in nature as CM was unable to induce PCD in wheat callus cultivars.     

Selection of higher regenerative callus and change in isozyme pattern in rice (Oryza sativa L.)

Summary Calli were initiated from seedling roots in rice (Oryza sativa L. var. Tadukan) and subcultured at 45-day intervals on MS medium supplemented with 2 mg/l 2,4-D. Sectors of callus which differentiated shoot meristems (green spots) under the same 2,4-D concentration were selected from the calli subcultured 90 days after initiation. The selection was continued for about 2 years. Responses to 2,4-D between original and selected lines differed considerably, although differentiation was not generally seen in rice callus in the presence of 2 mg/l 2,4-D. After 180 days, calli of the selected line differentiated into numerous shoot-bud primordia and grew out new callus tissues under 2 mg/l 2,4-D concentration; the frequency of the differentiation exceeded 90%. On the other hand, no calli of non-selected line differentiated into shootbuds under 2 mg/l 2,4-D, and the frequency of the shootbud was only about 50% under lower 2,4-D concentration (0.1 mg/l). The pattern and activity of peroxidase isozyme varied markedly between calli of the selected and non-selected lines. First, two strong peroxidase bands which show fast mobility and one intermediate peroxidase band with slow mobility were detected only in the calli of selected line. Secondly, changes in band pattern of proteins separated by SDS-PAGE were observed. In the calli of selected line, there was a loss of the polypeptide bands with molecular weight of 24 and 42 K in the selected calli, but they were clearly present in the unselected line. The appearance of new peroxidase isozyme bands and loss of polypeptide bands, change in response to auxin and increased ability for shoot bud differentiation are closely correlated to each other.     

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.     

Relationships among iron deficit-induced potato callus growth inhibition,Fe distribution,chlorosis, and oxidative stress amplified by reduced antioxidative enzyme activities

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H₂O₂) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.     

枸杞原生质体培养及高效成株体系的建立

由枸杞髓部组织诱导出胚性愈伤组织,并由此愈伤组织建立起稳定的细胞悬浮系。从悬浮细胞游离的原生质体在改良KM培养基(1.5 mg/L 6_BA,0.5 mg/L NAA和0.5 mg/L 2,4_D)中进行液体浅层培养,3～4 d后出现第一次分裂,第7 d统计分裂频率为50.3%,15 d左右可形成细胞团,3～4周后形成肉眼可见的愈伤组织,愈伤组织植板率为1.25%。将细胞团转移到液体分化培养基(MS+6_BA 1.5 mg/L+2,4_D 0.2 mg/L) 8～10 d可形成大量胚状体,及时将胚性愈伤组织块转移到固体分化培养基上(MS+6_BA 0.2 mg/L),可形成大量绿芽,分化率54.17%。绿芽在生根培养基（MS+NAA 0.2 mg/L）可形成完整植株,移栽后成活良好。     

Expression and Immunogenicity of Enterotoxigenic <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis> Heat-Labile Toxin B Subunit in Transgenic Rice Callus

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G_M1-ganglioside, a receptor for biologically active LTB, was confirmed by G_M1-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G_M1-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.     

Determination of phenolic content and antioxidant activity of extracts obtained from Rosmarinus officinalis' calli

Rosmarinus officinalis is widely found in the lands of Aegean and Mediterranean regions of Turkey. Stem explants of very young shoots were cultured in both woody plant medium (WPM) and Murashige and Skoog (MS) media supplemented with 7g/L agar, 30g/L sucrose, and 1 and 3mg/L naphthaleneacetic acid (NAA) for callus initiation. Induced calli were subcultured 4 times with intervals of 7-10 days. MS medium supplemented with 1mg/L NAA proved to be the best medium for the production of callus (65.0%) among the samples tested. The lyophilized calli were subjected to solvent extraction. Active constituents of 8 calli extracts were analyzed by HPLC, and rosmarinic acid (RA) was determined to be the primary compound. Calli cultivated in WPM supplemented with 1mg/L NAA and extracted at 50 degrees C, yielded the highest amount of RA (34.4mg/g dry weight). Moreover, antioxidant activity of calli extracts was determined using a number of in vitro assays, including total phenol assay, DPPH radical scavenging activity (RSA), and trolox equivalent antioxidant capacity (TEAC). On the basis of the current findings, we conclude that WPM supplemented with 1mg/L NAA yields higher phenolic content as well as higher antioxidant activity.     

Plant regeneration from protoplasts of Capsicum annuum

Leaf protoplasts were isolated from axenic shoot cultures of four varieties of Capsicum annuum (Americano, Dulce Italiano Florida Gynat and Nigrum) and a wild species C. chinense. Protoplasts of both species, cultured in KM8P medium and using agarose bead culture, entered division with the exception of the variety Nigrum. Cell colonies formed callus in agar-solified MS medium supplemented with zeatin and for C. annuum v. Dulce Italiano shoots were regenerated when protoplast-derived calli were transferred to MS medium with 6-BAP. Excised shoots were rooted on MS medium which lacked phytohormones.Abbreviations 6-BAP 6-benzylamino purine - MS Murashige and Skoog (1962) - KM8P Kao and Michayluk (1975) - SDS sodium dodecyl sulphate     

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D₂ ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.     

Callus production from protoplasts of Cyclamen persicum

Cyclamen persicum (Mill.) cv. Sierra Rose protoplasts were isolated from adventitious shoots regenerated in vitro from petiole and leaf explants with yields of 1.3 × 106 protoplasts/g f.wt. of tissue. Protoplasts were embedded within agarose lenses bathed in modified KM8p medium supplemented with 1.0 mg l-1 NAA, 0.1 mg l-1 2,4-D and 0.5 mg l-1 BA. Cell division was observed after 4–5 days. After 6 weeks calli had grown out of the lenses which were transferred to modified MS medium for further callus growth. The most rapid callus growth was on medium containing 0.1 mg l-1 NAA and 10 mg l-1 TDZ. This revised version was published online in June 2006 with corrections to the Cover Date.