Sorbitol as the Primary Carbon Source for the Growth of Embryogenic Callus of Maize

The effects of various carbon sources on initiation and maintenance of embryogenic callus of maize (Zea mays L.) and on the regeneration of plants from embryogenic callus were studied. Growth of embryogenic callus tissue on media containing sucrose was typified by the subsequent growth of both embryogenic (regenerable) and nonembryogenic (nonregenerable) callus. Growth of embryogenic callus on sorbitol was unique among the carbon sources tested in that sorbitol supported the subsequent growth of only embryogenic callus. Further experiments demonstrated that embryogenic callus grown on sorbitol had a greater regenerative capacity (more plants produced per gram fresh weight of callus) than callus grown on sucrose. Sorbitol dehydrogenase was detected in embryogenic callus of maize at a specific activity roughly equivalent to that found in zygotic embryos of developing seeds. Nonembryogenic callus did not contain significant levels of sorbitol dehydrogenase activity.     

Limitation of tobacco callus tissue growth by carbohydrate availability

Growth rate, maximum dry weight yield, and carbohydrate utilization were measured with pith callus derived from Nicotiana tabacum L. var. Wisconsin No. 38. Maximum tissue dry weights increased as carbohydrate (either glucose or sucrose) in the medium was increased. The time at which maximum growth was obtained coincided with the time at which carbohydrate was exhausted from the medium. The addition of carbohydrate to the medium before the end of log phase growth supported that amount of additional growth which would have been obtained if all of the carbohydrate had been added to the medium prior to planting the tissue. Maximum obtainable dry weights at logarithmic growth rates greater than 0.16 doubling per day depended on the amount of carbohydrate in the medium and not on a particular hormonal regime.     

胡萝卜愈伤组织形成过程中激素和创伤诱导的钙调素水平的变化

CaM抑制剂TFP抑制了胡萝卜愈伤组织的形成。ELISA法测定CaM的动态表明,愈伤组织形成过程中,出现了两个CaM 的峰。第一个峰值在6h,可能是由创伤引起的,单位蛋白和单位鲜重的CaM 量都增加了1倍左右,第二个峰值可能是由激素引起的,从48 h以后开始增加,7天时单位鲜重的CaM 增加了4倍左右,单位蛋白的CaM 量也特异地增加了0.7倍,此结果亦为CaM 免疫荧光组织化学定位结果所证实。研究结果表明胡萝卜愈伤组织形成过程中激素与创伤效应和CaM 动态有关。     

Effects of ionizing radiation (60Co gamma rays) on growth and morphogenesis ofHaworthia mirabilis,haw. Callus tissue

Summary The effects of γ-radiation on growth and morphogenesis ofHaworthia callus in vitro were determined. The doses ranged from 100 to 5000 rads. Survival, growth pattern, growth rate, and differentiation of vegetative buds and roots in both irradiated and nonirradiated callus were compared. Growth data up to 24 weeks for irradiated and control cultures were analyzed. The dose range between 800 to 2500 rads produced compact callus as compared to the controls which were friable. After 12 weeks all control cultures differentiated vegetative buds with roots, whereas callus exposed to 800 to 2500 rads continued to grow with little or no organogenesis. However, it was observed that the wet and dry weights of callus receiving 1000 to 1500 rads ultimately exceeded those of nonirradiated controls.     

芦苇胚性愈伤组织的形成及植株的再生

以芦苇种子为外植体,其愈伤组织的诱导率最高。叶鞘和叶片不发生脱分化。培养基中最合适的蔗糖浓度为4%。维生素 B 类、肌醇对愈伤组织的生长起促进作用。而酵母提取物对愈伤组织的诱导和生长具有明显的抑制作用。这种抑制效应,将随酵母提取物浓度的提高而增大。愈伤组织的继代培养,随培养基中2,4-D 浓度的提高,其平均鲜重明显降低。脱分化培养基中2,4-D 浓度对胚性愈伤组织的诱导形成具有一定的相关性。胚性愈伤组织经30代继代培养依然具有90%的分化频率,只是每块愈伤组织的分化苗数减少。反之,非胚性愈伤组织则完全丧失形态发生的能力。对两类愈伤组织进行扫描电镜的观察,发现其表面结构有很大差异。其过氧化物酶、酯酶同工酶谱以及可溶性蛋白的含量均有明显的差别。     

Embryogenic Callus Formation and Plantlet Regeneration of Piragmites communis

The present paper reports the embryogenic callus formation and plantlet regeneration of Phragmites communis. The results have been obtained as follows: The efficiency of callus induction was much higher, if reed seeds were used as explants. No dedifferentiation was observed by using leaf sheath and leaf blade as explants. The optim, um concentration of sucrose was 4% in medium. VB group and inositol had beneficial effects on callus growth. But yeast extract inhibited callus induction and callus growth markedly. For this inhibited reaction, the higher concentration, the more obviously the callus growth was inhibited. Higher levels of 2,4-D had unfavourable effects on callus growth in callus subculture. The concentration of 2,4-D in dedifferentiation medium had relation to embryogenic callus formation. Embryogenic callus had higher frequency of differentiation for long-term subculture. On the other hand, nonembryogenic callus most often lost their morphogenetic competence. Authors found that the surface structure of the two types of calluses was different by means of observation by scanning electron microscope. The peroxidase and the esterase isoenzyme pat- terns, as well as the soluble protein of both types of calluses were different too.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

Chemical constituents of cultured cells of Euphorbia tirucalli and E. millii

We induced calluses from two Euphorbia species and analyzed the lipids and pigments of their cells. Growth was promoted when malt extract was added to the medium for callus induction. The lipid constituents of both E. tirucalli and E. millii calluses were the same; sitosterol, stigmasterol, campesterol, palmitic acid and linoleic acid. In addition, an anthocyanin, cyanidin glycoside, was isolated from callus that had been induced from E. millii leaves cultured on medium containing 0.1 ppm 2,4-d.     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

Callus Induction,Plant Regeneration and an Assessment of Cytological Variation in Regenerated Plants of Lolium multiflorum L.

Callus cultures were initiated from roots, apical meristem tips and leaf explants of several genotypes of Lolium multiflorum L. (Italian Ryegrass). Genotypes were selected which showed a high frequency of callus initiation and from which plants could be regenerated. Plants could be routinely produced from root-derived callus of only one of the genotypes tested. The selected genotypes were still amenable if the temperature and concentration of 2,4-D in the medium were altered. Increase in temperature caused callus from one genotype to give rise to more albino regenerants. Callus formation and plant regeneration occurred at a higher frequency from diploid than tetraploid explants. All regenerants from the diploid cultures had the 2n = 2x = 14 chromosome number whereas plants regenerated from callus derived from tetraploid cultures lost up to 3 chromosomes.     

Effects of Pluronic F-68 on callus growth and protoplast plating efficiency of Solanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of callus and protoplasts from Solanum dulcamara L. have been studied. Growth of callus was stimulated by addition of 0.1% (w/v) commercial grade Pluronic to culture medium, whereas lower concentrations (0.01% w/v) had no corresponding effect. In contrast, higher concentrations (1.0% w/v) of Pluronic inhibited callus growth. The mean plating efficiency of protoplasts grown at different densities (15 days after plating) was increased up to 26% following culture with 0.1% (w/v) Pluronic, while 0.01% (w/v) Pluronic was ineffective. Mean protoplast plating efficiency decreased by up to 32% following culture with 1.0% (w/v) Pluronic.     

Regulating plant tissue growth by mineral nutrition

The objective of this study was to determine if the growth of sweet orange (Citrus sinensis (L.) Osbeck cv. ‘Valencia’) nonembryogenic callus could be regulated and controlled via the mineral nutrient components of the medium. The 14 salts comprising Murashige and Skoog (MS) basal medium were subdivided into five component groups. These five groups constituted the independent factors in the design. A five-dimensional hypervolume constituted the experimental design space. Design points were selected algorithmically by D-optimality criteria to sample of the design space. Growth of the callus at each design point was measured as % increase of fresh weight at 14 d. An analysis of variance was conducted and a response surface polynomial model generated. Model validation was conducted by mining the polynomial for design points to two regions—“MS-like” growth and MS + 25% growth and comparing callus growth to predicted growth. Five of the eight selected MS-like points and three of the six MS + 25% growth points validated, indicating regions within the design space where growth was equivalent to MS, but the salt combinations were substantially different from MS, and a smaller region where growth exceeded MS by greater than 25%. NH₄NO₃ and Fe were identified as important factors affecting callus growth. A second experiment was conducted where NH₄NO₃ and Fe were varied, thus creating a two-dimensional slice through the region of greatest callus growth and provided increased resolution of the response.     

Ethylene Production by Tobacco (Nicotiana tabacum) Callus

Tobacco callus cultures grown on defined agar-solidified media produced ethylene in differing amounts, which were related to cultural treatment and age of the callus. There was a close correlation between the rate of ethylene production and growth. In darkness, maximal rates occurred in the third week of growth with ethylene production in the range of 750 nl (callus piece)^?1 d^?1 (fr. wt. = 1.5 g), and in the light, maximal rates occurred in the first week of growth, 200 nl (callus piece)^?1 d^?1 (fr. wt. = 200 mg). Growth was also correlated with ethylene production when the latter was altered by exposure of the callus to inhibitors of ethylene synthesis, L-canaline, benzyl isothiocyanate, and 3,5-diiodo-4-hydroxy-benzoic acid. No correlation was found following treatment with AgNO₃, a presumptive inhibitor of ethylene action. The inhibition of growth and ethylene production by L-canaline was partially reversed by gassing the cultures with ethylene (1 μl/1). A mercuric perchlorate sink had no significant effect on growth. A possible relationship between ethylene evolution and growth is discussed.     

Callus culture from hypocotyls of Kosteletzkya virginica (L.) seedlings

It was shown that callus established from Kosteletzkya virginica (L.) Presl. (Malvaceae) can grow in salinities higher than 200 mM NaCl if previously accomodated stepwise. Callus lines developed from seedlings of different harvests or of the same harvest at different times, all showed the same pattern of growth and sensitiviy to salinity. The absorption of Na⁺ into the callus increased with increasing external NaCl concentration. In the callus, Na⁺ was apparently distributed outside and inside a cellular membrane (possibly the plasmalemma). This membrane was, apparently, capable of regulating the Na⁺ concentration in the protoplast. Outside this membrane Na⁺ accumulated to concentrations higher than in the external growth medium. Exogenously supplied proline or glycine-betaine did not affect the growth of the callus. Externally applied ABA stimulated growth under saline conditions and increased the accumulation of proline. Growth and proline content were positively correlated in callus exposed to salinity, but in the presence of ABA they were negatively correlated. ABA was involved in both growth and proline accumulation, but there was no clear relationship between these two effects. Both ABA and proline, if added to the growth medium, improved the appearence of the callus.Abbreviations ABA abscisic acid - B₅ Gamborg's medium - BA benzylalanine - 2,4-d 2,4-dichlorophenoxy acetic acid - FW fresh weight - G B₅ medium without growth regulators - GH B₅ medium supplemented with growth regulators - NAA naphthalene acetic acid - PGR plant growth regulators - Q _T total amount of a certain ion in the tissue - Q _s amount of the ion that has leaked out - QAC Quaternarty Ammonium Compounds - RGR mean relative growth rate - W₁ and W₂ fresh weight at times t₁ and t₂     

Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.)

Summary The insecticidal effectiveness of a δ-endotoxin Cry protein from Bacillus thuringiensis in non-regenerable callus of a commercial Gossypium hirsutum L. variety was investigated. Two transgenic callus types were generated. The first callus type harbored the cry1A(c) gene and the hygromycin B phosphotransferase hpt selectable marker gene. The second callus type, the transgenic control, carried the marker genes β-glucuronidase (GUS) and hpt. Growth and survival rates of three major cotton moth species, Pectinophora gossypiella, Helicoverpa armigera, and Spodoptera littoralis, were examined with aseptic neonates reared on callus. Normal larval development occurred in all species supplied with non-transgenic callus, but insects died, or their growth was severely restricted, when reared on transgenic callus harvested from hygromycin B-supplemented medium. Development of larvae on transgenic control and on non-transgenic callus became very much alike after the transgenic control tissue had been subcultured on a hygromyein B-free medium for about 100 d prior to the insect-callus bioassay. Accordingly, for detection of Bt toxin activity without the interference of the influence of hygromycin B on insects, cry1A(c) callus was infested with insects after it had been propagated for more than 100 d on a medium free of the antibiotic. Under these experimental conditions all P. gossypiella and H. armigera, and most S. littoralis neonates died, and the growth (e.g., weight increment) of S. littoralis survivors was markedly impeded by cry1A(c) callus. Three new findings emerge from this study: first, P. gossypiella, a pest feeding in the field on bolls only, can be grown in vitro on cotton callus; second, in a host which is recalcitrant in terms of plant regeneration, the biological potency of an insectdetrimental transgene can nevertheless be evaluated by generating a transgenic host callus and conducting in vitro transgenic callus-insect assays; and third, our results suggest that hygromycin B is toxic to lepidopteran larvae.     

Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus

The effect of two different media on the growth, metabolism and viability of Scots pine ( Pinus sylvestris ) callus cultures was studied. Inorganic nitrogen in the culture media (modified MS) was in the form of either KNO₃ or NH₄NO₃. The cultures were started from buds of mature Scots pine. Growth was poor on the medium with KNO₃, but this compound had a noticeable effect on the metabolism of the callus, which was reflected in alterations in protein and polyphenol synthesis and the pH of the culture medium. Although the fresh mass, water content and viability of the callus decreased when KNO₃ was the exclusive inorganic nitrogen nutrient, protein synthesis was more abundant. Electrophoretic analyses indicated alterations in the patterns of soluble proteins and purified glycoproteins. Phenylalanine ammonia-lyase (EC 4.3.1.5) activities were high in all the calluses, and concentrations of condensed tannins and their precursors, catechins, were higher than in intact buds. The role of inorganic nitrogen nutrition in the deterioration of tissues is discussed on the basis of the effect of ammonium on the metabolism of pine callus.     

Tetrapyrrole Accumulation in in vitro Selected Somaclones of Acifluorfen-Tolerant Solanum ptycanthum Dun

Acifluorfen-tolerant callus lines of Solanum ptycanthum were isolated by stepwise selection. Growth of unselected lines was completely inhibited at 0.5 µM acifluorfen, while some selected lines grew at 8 µM acifluorfen. Twenty-two of 25 acifluorfen-tolerant callus lines regenerated shoots. Acifluorfen-tolerant S. ptycanthum callus lines differed in protoporphyrin IX content ranging from 2.0 to 43.5 nmole per 100 mg protein. As the concentration of acifluorfen increased, the amount of protoporphyrin IX accumulated increased. These results indicated that the possible site of action of acifluorfen was protoporphyrinogen oxidase which might be the molecular target of the herbicide within plant cell.     

Metal tolerance in tissue cultures of anthoxanthum odoratum

Tissue cultures were initiated from one non-tolerant (S20) and two zinc and lead tolerant (T92 and T94) clones of Anthoxanthum odoratum. Growth of callus from the non-tolerant clone was reduced by the presence of zinc, lead, copper and nickel, whereas callus from the two tolerant clones showed no reduction of growth in the presence of zinc and lead but growth was reduced by copper and nickel. The specificity of metal tolerance shown by the parental material was maintained in the callus. Tolerant and non-tolerant callus accumulated similar amounts of zinc and lead.     

High frequency production of embryos from liquid flask cultures of oilseed rape

As part of the program to scale-up the production of artificial seeds of winter oilseed rape, Brassica napus ssp. oleifera, we established a liquid flask culture system that enables the high frequency production of freely suspended embryos. As many as 4000 embryos could be obtained from 1 mL packed-cell-volume of cells. For initiation of liquid flask cultures, four different types of callus tissues were used. Among them, the most embryogenic cell suspension cultures were obtained from spontaneous callus developed on the surface of secondary embryos precultured in medium supplemented with 2,4-dichlorophenoxyacetic acid (4.52 muM) and kinetin (0.46 muM) (type B callus). Growth curves of the cell suspension were determined and the cell suspension was able to grow in medium without plant growth regulators. Embryos were observed to developed directly from the cells without going through an obvious callus phase. When subcultured to agar medium containing 44.38 muM benzylaminopurine, about 43% of the embryos developed into plants. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 231-238, 1997.     

Stimulation of growth factor synthesis in skin wounds using tissue extract (G-90) from the earthworm Eissenia foetida

Growth factors are biologically-active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. In the case of wound healing, a proper wound dressing is needed to cover the wound area, protect the damaged tissue, and if possible to activate cell proliferation and stimulate the healing process. In this study we examined the efficacy of a glycolipoprotein tissue homogenate extract from Eisenia foetida (G-90) to activate signal transduction pathways, leading to wound healing. We measured the activation of EGF and FGF in healthy skin, in wounds with physiological healing and in wounds treated with G-90. The activation of EGF and FGF was measured during the first 24 h of wound healing under both physiological conditions and treatment with G-90. In both cases an increased concentration of EGF and FGF was observed 6 h after wounding. In comparison with healthy skin, the concentration of EGF increased 10-fold and FGF five-fold in wounds treated with G-90 (10 ng ml(-1)). Healing in physiological conditions resulted in a two-fold increase of EGF and 1.5-fold of FGF.