The influence of phytohormones on growth,organ differentiation and fructan production in callus of Symphytum officinale L.

Summary Callus derived from Symphytum officinale L. regenerants was cultured in the presence of various phytohormones. The growth rate of callus was stimulated by all phytohormones at various concentrations. With 1-naphthaleneacetic acid no organ differentiation could be observed. With indole-3-butyric acid at low concentrations only roots were formed, whereas 6-benzylaminopurine, kinetin and zeatin at various concentrations induced either root or shoot formation or the simultaneous regeneration of both. Minor amounts of fructans were formed at high 6-benzylaminopurine-, zeatin- and at all indole-3-acetic acid-concentrations. The concentration of 1-naphthaleneacetic acid had no influence on the fructan content. Highest rates of fructan synthesis occurred at low zeatin-concentrations up to 1.5 mg/l. Only zeatin at all concentrations induced the synthesis of polyfructans, whereas appreciable amounts of oligofructans were formed under the influence of all other phytohormones.Abbreviations NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - DP degree of polymerisation - GLC gas liquid chromatography - TLC thin layer chromatography     

The Effect of 6-Benzylaminopurine on the Starch Metabolism of Stellaria media

The effect of 6-benzylaminopurine (BAP) was studied by measuring the amylase activity of leaf extracts and the starch content of chloroplasts in Stellaria media-plants cultivated for five days in solutions containing different quantities of BAP. Small amounts of BAP decreased amylase activity in weak-growing plants. In strong-growing ones the effect was variable. However, in all experiments very high amount of this growth regulator caused a great increase in amylase activity which was accompanied by a relatively high concentration of starch in the chloroplasts. The induction of amylase activity was thus not high enough to diminish the starch content. This is supposed to be due to the lack of a sink organ since the growth of roots and shoots was greatly inhibited in concentrated BAP-solutions (10^-5M or more). In these cases the plants usually died during the first week. In some cases small amounts of BAP increased the growth of S. media, and high concentrations inhibited it causing characteristic morphological changes, such as creeping growth pattern, small, thick leaves and the turning up of the leaf margins. Benzylaminopurine also caused increased resistance to aging, especially in low concentrations (10^–7–10^–6M).     

Growth Cycles in Lemna gibba Cultures and Their Effects on Growth Rate and Ultrastructure

A long-established axenic culture of Lemna gibba G3 was maintained in exponential growth phase under controlled conditions. Weekly analyses for 2 years showed that the individual plants of the Lemna gibba clone fluctuated between two forms. One extreme consisted of plants light in weight, small in size, and with a high relative growth rate (RGR), the other of heavy, large, and more slowly growing plants. At intervals plants having intermediate characteristics dominated in the stock culture. Indication of an annual growth-cycle was also found. The magnitude of growth response (weight, RGR, area, and dry matter content) after treatment with abscisic acid (ABA), 6-benzylaminopurine (BAP), and a combination of the two was different for low-weight and heavy plants. The heavy plants were more sensitive to ABA and BAP treatment than the low-weight ones. The accumulation of starch was least in small untreated plants and greatest in ABA treated plants. Large electron transparent globules were found in the chloroplasts of the ABA treated plants and in heavy plants regardless of how they had been treated. The different physiological and ultrastructural characteristics of the two forms of Lemna plants probably reflect an ageing-rejuvenation cycle. Emphasis is placed on the importance of this cycle when Lemna is used as a model plant in physiological experiments.     

Stimulation of Pod and Ovule Growth of Soybean, Glycine max (L.) Merr. by 6-Benzylaminopurine

Flowers at distal nodes on soybean racemes usually fail to setpods and subsequently abscise. Physiological and histologicalstudies were performed to determine the influence of 6-benzylaminopurine(BAP) on distal pod development. The pedicels of fully openedflowers on terminal racemes of field-grown IX93-100 soybeanplants were treated three times with 200 mg kg^-1 BAP in lanolinover a 6-d period. Racemes were then excised and ³²P uptakewas recorded for each flower position within a raceme; histologicalfeatures of pedicels and ovules also were determined. Applicationof BAP increased pod and ovule length, width and weight at allfour distal nodes (D, D-1, D-2, D-3) relative to controls treatedwith lanolin. Length and width of parietal endosperm cells weresmaller in BAP-treated ovules at the most proximal node beingstudied (D-3), and greater numbers of parietal endosperm cellswere observed at D-1 and D-3 nodes when compared to lanolincontrols. Smaller amounts of starch were found in suspensorcells, endosperm, and integuments of lanolin-treated ovules,and starch depletion over time was observed within starch sheathsof pedicels from lanolin-treated pods when compared to BAP-treatedtissues. BAP-treated racemes had more ³²P uptake at the fourmost distal nodes. A higher rate of uptake (cpm mg^-1 f. wt)was evident in ovules than in ovary tissues. These results suggestthat for racemes otherwise destined to abscise, applicationof BAP promotes pod set and growth by stimulating ovule development.Copyright1993, 1999 Academic Press Pod, ovule soybean, abscission, 6-benzylaminopurine     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。     

Growth and carbohydrate changes in shoot cultures of Dioscorea species as influenced by photoperiod,exogenous sucrose and cytokinin concentrations

The yams (Dioscorea) are important as staple food and as products for synthesis of sexual and adrenocortical hormones. The vegetative propagation of commercial yams is by tuber-seeds. For medicinal uses, this system has several limitations, including long periods required for wild species to reach commercial production, the labour of cultivation and harvesting and the small number of tubers available. In the present investigation, two native species from Brazil, Dioscorea delicata R. Knuth. and D. olfersiana Kl., and the exotic species, D. bulbifera L. from Asia, were cultured in vitro under different daylength and in different concentrations of 6-benzylaminopurine and sucrose, and the leaves were analysed with respect to soluble carbohydrates, starch and proteins. The best explants for vegetative propagation were those with two or more nodal buds, although the culture conditions used did not stimulate bulbil production. Glucose and fructose were the major soluble carbohydrates present in the leaves and sucrose was found only in D. olfersiana. With increasing concentrations of 6-benzylaminopurine (0–22 M) and sucrose (1.5–8%), an accumulation of soluble carbohydrates and starch was observed, while the amount of soluble protein remained constant. The number of leaves produced and the age of cultures were positively correlated, despite the absence of significant differences among treatments.     

Effect of photoperiod and thermoperiod on microtuberization and carbohydrate levels in Cocoyam (Xanthosoma sagittifolium L. Schott)

Cocoyam (Xanthosoma sagittifolium L. Schott) is an important staple tuber crop for tropic populations and consists of three cultivars with different productivity. In vitro tuber induction of a broad range of genotypes could be a very useful way to propagate and increase valuable cocoyam. Shoot tips were cultured on a multiplication medium containing MS mineral salts, Morel and Wetmore (Ann J Bot 38:141–143, 1951) vitamins, 3% sucrose and 6% agar. Multiple shoots induced with 6-benzylaminopurine (BAP) were allowed to develop on the basal medium without plant growth regulators (PGRs). The subsequent explants obtained were used for the production of microtubers on MS-based PGRs free medium supplemented with 8% sucrose. Microtuberization was evaluated after 60 days of culture under different photo- and thermoperiod regimes. Changes in carbohydrates were also examined enzymatically during this process. Microtuberization response of three cocoyam cultivars (White, Red and Yellow) was influenced by photoperiod and thermoperiod regimes. A short photoperiod (8 h lighting), combined with an application of a day/night temperature regime 25/20°C for 10 days, followed by a continuous darkness at 20°C for 50 days, was the most effective treatment. The best response was observed with White followed by Red and Yellow cultivar. The sprouting frequency of microtubers varied significantly with cvs. after 15 days of culture (56% for White, 50% for Red and 40% for Yellow). Under tuber promoting condition, the onset of the process was characterized by an accumulation of starch and hexoses in leaves. The glucose:fructose ratio changed in favour of glucose earlier in White cultivar leaves where it doubled in 10 days. Moreover, in young developing tubers, the sucrose content increased concomitantly with starch.     

Plant regeneration in <Emphasis Type="Italic">Arachis stenosperma</Emphasis> Krapov. and W. C. Gregory from roots and calluses derived from leaflets of <Emphasis Type="Italic">in vitro</Emphasis> plants

Seed explants of A. stenosperma were cultured on MS medium supplemented with 6-benzylaminopurine with the aim of rescuing nonviable accessions stored in seed bank conditions. The regeneration potential of leaf explants from in vitro plants derived from embryonic axes was studied by using whole leaflets and leaflet segments. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine and naphthalene acetic acid. Indirect organogenesis was observed in response to 6-benzylaminopurine, either alone or in association with naphthalene acetic acid, in both explant types. Media supplemented with naphthalene acetic acid as the sole growth regulator induced rhizogenesis in whole leaflets and leaflet segments, with subsequent shoot production directly from the roots.     

Large‐scale screening and characterisation of Lemna aequinoctialis and Spirodela polyrhiza strains for starch production

• Duckweed is considered a promising feedstock for bioethanol production due to its high biomass and starch production. Selection of duckweed strains with high starch accumulation is essential for application of duckweeds to bioethanol production. Geographic differentiation had a large influence on genetic diversity of duckweeds.
• Biomass production, starch content and starch amount in geographically isolated strains of 20 Lemna aequinoctialis and Spirodela polyrhiza were calculated to evaluate their potential for bioethanol production. The influence of different collection time, culture medium and NaCl concentration on starch accumulation of the best strains were analysed.
• The results showed that biomass production, starch content and starch production of duckweeds demonstrated clonal dependency. The best strain was L. aequinoctialis 6000, with biomass production of 15.38 ± 1.47 g m^?2, starch content of 28.68 ± 1.10% and starch production of 4.39 ± 0.25 g m^?2. Furthermore, starch content of L. aequinoctialis 6000 was highest after 8 h of light, tap water was the best medium for starch induction, and NaCl did not induce starch accumulation.
• This study suggests duckweed biomass production and starch production demonstrate clonal dependency, indicating that extensive clonal comparisons will be required to identify the most suitable isolates for duckweed selective breeding for bioethanol.

     

Effect of plant growth regulators on the cellular growth and levels of intracellular protein,starch and polyamines in embryogenic suspension cultures of Pinus taeda

Somatic embryogenesis is the most important in vitro culture system for conifer propagation. However, Pinus taeda has been considered recalcitrant to somatic embryogenesis in commercial scale-up. The study of biochemical and physiological aspects of cell growth could lead to a better understanding of somatic embryogenesis in this species. In the present work, we investigated the cell growth dynamics, intracellular levels of proteins, starch and polyamines in suspension cultures of Pinus taeda established in plant growth regulator-free medium (BM0) and in medium supplemented with 2 M 2,4-dichlorophenoxyacetic acid, 0.5 M 6-benzylaminopurine and 0.5 M Kinetin (BM2). Cell cultures growing in BM0 medium showed an increase in the sedimented cell volume from 3.77 to 17.73 ml after 24 days of culture. Those cultured in BM2 medium showed an increase in the sedimented cell volume from 4.23 to 25.17 ml after 20 days of culture. Intracellular proteins levels increased during the exponential growth phase and starch levels decreased until the exponential phase, followed by a synthesis up to the stationary phase, in both BM0 and BM2 media. Highest putrescine levels occurred in cultures growing in BM0 medium and this was associated with the low cellular growth.     

Effects of Fertilization and Irrigation on the Seasonal Changes of Carbohydrate Reserves in Different Age-Classes of Needle on 20-Year-Old Scots Pine Trees (Pinus silvestris)

The effects of fertilization, irrigation or both on the seasonal changes of starch and soluble carbohydrates (glucose, fructose, myo-inositol, pinitol and sucrose) in needles of 20-year-old Scots pine trees (Pinus silvestris L.) were studied during three consecutive years. The starch content of the mature needles increased during spring and early summer to about 25% of dry weight. Neither fertilization nor irrigation affected the general pattern of starch accumulation during the spring. The starch reserves were mobilized when the shoot started to grow. Starch content decreased more rapidly in needles from fertilized than in those from unfertilized trees. The current needles from the control trees accumulated starch while they were still growing. The current needles of the fertilized trees did so to a lesser extent. The amount of starch was closely correlated to the air temperature and to the growth rate. Large amounts were found at low temperatures and low growth rates. The concentrations of soluble carbohydrates showed the well-known seasonal variation, with the highest value during the winter. The levels of sugars were nearly similar, irrespective of fertilization. An exception was sucrose, which was found in small quantities in needles from fertilized plots. Small amounts of sucrose were also found in growing current needles. The results are discussed in relation to growth limitation by assimilate availability and indicate that the ‘sink demand’ is the limiting factor.     

木薯蔗糖合成、转运与块根淀粉积累关系研究

该研究以淀粉含量不同的两个木薯品种(辐选01和华南124)为材料,通过测定各品种不同生育期叶、茎和根的蔗糖含量及块根淀粉含量,分析了蔗糖合成、转运和块根淀粉积累过程的相关性.结果表明:与华南124相比,在整个生育期内辐选01叶、茎的蔗糖含量均较高,块根蔗糖含量在块根膨大初期以前高于华南124,块根膨大初期以后则相反.在木薯的整个生育期,与辐选01相比,华南124的淀粉合成量和淀粉合成速率均较低.叶和茎蔗糖含量的变化规律与淀粉合成速率的变化规律相反,即块根淀粉积累明显加快时叶和茎的蔗糖含量略呈下降趋势,而块根淀粉合成减慢时叶、茎的蔗糖含量又开始上升.随着生育期的延后,块根蔗糖含量越来越低.在块根形成初期,蔗糖含量最高的组织部位为块根,其次为茎秆,最低的是叶片;而在块根成熟期时则相反,即蔗糖含量最高的部位是叶片,其次为茎杆,块根的蔗糖含量最小.相关性分析结果表明,木薯叶片蔗糖含量与块根淀粉含量呈显著的正相关;茎秆蔗糖含量与块根淀粉积累量呈不显著的正相关;块根蔗糖含量与淀粉积累量呈显著的负相关.由此可见,木薯叶、茎和根蔗糖与块根淀粉积累过程密切相关,其中叶片合成蔗糖的能力与块根利用蔗糖的能力在淀粉的积累过程中发挥关键作用.该研究结果为木薯的生产选育与高效栽培提供了理论依据.     

Action of cytokinins and anticytokinins on cotyledonary bud growth ofLycopersicon esculentum MILL

The action of two anticytokinins, 3-methyl-7-n-pentyl-aminopyrazolo[4,3-d] pyrimidine and 4-cyclopentylamino-2-methylthiopyrrolo [2,3-d] pyrimidine on the zeatin and 6-benzylaminopurine induced lateral bud growth ofLycopersicon esculentum Mill. cv. Fireball seedlings was studied. Bud growth stimulation by 0.1 mM zeatin was not overcome by application of anticytokinin at concentrations of 0.01 to 1.0 μ 24-h after zeatin application. However, application of 0.1 μM of anticytokinin 24-h before 0.1 mM zeatin caused a significant enhancement of bud growth. A simultaneous application of 1 mM anticytokinin and 6-benzylaminopurine or concurrent with young leaves excision significantly reduced bud growth.     

Calcium restriction induces cardenolide accumulation in cell suspension cultures of Digitalis thapsi L.

Summary The removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl₃ or ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.Abbreviations BA 6-benzylaminopurine - 2 4-D 2,4-dichlorophenoxyacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid - FW fresh weight - MS Murashige and Skoog (1962)     

Growth-Retarding Effect of 2-Aminoindan-2-phosphonic Acid on Spirodela punctata

2-Aminoindan-2-phosphonic acid (AIP) retarded the growth of duckweed, Spirodela punctata, and increased its dry mass. The accumulation of starch was observed at all concentrations of AIP at 8 days after treatment. The increase in starch was inversely proportional to the growth. The retarded growth of Spirodela by AIP was not limited only by excessive starch accumulation. Received December 8, 1997; accepted June 2, 1998     

Somatic embryogenesis in Nicotiana tabacum L.: induction by thidiazuron of direct embryo differentiation from cultured leaf discs

Summary Induction of somatic embryogenesis by different growth regulators was examined in leaf disc cultures of Nicotiana tabacum L. Direct differentiation of somatic embryos occurred on media supplemented with naphthaleneacetic acid (NAA) and N⁶-benzylaminopurine (BAP). Thidiazuron (N-phenyl-N-1, 2,3,-thiadiazol-5-ylurea; TDZ) not only substituted for the most effective NAA-BAP combination but also induced a higher frequency of somatic embryogenesis. Regenerated somatic embryos were capable of developing into plants.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - NAA Naphthaleneacetic acid - TDZ N-phenyl-N-1,2,3,- thiadiazol-5-ylurea     

Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: II. Heterotrophic conditions

The effect of the bacterium Azospirillum brasilense jointly immobilized with Chlorella vulgaris or C. sorokiniana in alginate beads on total carbohydrates and starch was studied under dark and heterotrophic conditions for 144h in synthetic growth medium supplemented with either d-glucose or Na-acetate as carbon sources. In all treatments, enhanced total carbohydrates and starch content per culture and per cell was obtained after 24h; only jointly immobilized C. vulgaris growing on d-glucose significantly increased total carbohydrates and starch content after 96h. Enhanced accumulation of carbohydrate and starch under jointly immobilized conditions was variable with time of sampling and substrate used. Similar results occurred when the microalgae was immobilized alone. In both microalgae growing on either carbon sources, the bacterium promoted accumulation of carbohydrates and starch; when the microalgae were immobilized alone, they used the carbon sources for cell multiplication. In jointly immobilized conditions with Chlorella spp., affinity to carbon source and volumetric productivity and yield were higher than when Chlorella spp. were immobilized alone; however, the growth rate was higher in microalgae immobilized alone. This study demonstrates that under heterotrophic conditions, A. brasilense promotes the accumulation of carbohydrates in two strains Chlorella spp. under certain time-substrate combinations, producing mainly starch. As such, this bacterium is a biological factor that can change the composition of compounds in microalgae in dark, heterotrophic conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and callogenesis in tissue culture of floral organs of the genus <Emphasis Type="Italic">Iris</Emphasis> (Iridaceae)

We tested the differentiation and morphogenetic capacity of floral organs of Iris ensata, I. setosa, and I. sanguinea cultured in vitro. Organogenesis through direct formation of shoots from explants, callogenesis, and floral organogenesis were demonstrated in I. ensata callus culture in vitro. These processes depended on the plant species and on the content of phytohormones in the medium. Adventitious shoots proved to develop on the basal part of the perianth tube and on the apical part of the ovary, while roots were not formed. Direct organogenesis was induced by the following phytohormones: -naphthylacetic acid and 6-benzylaminopurine for I. ensata and 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine for I. setosa and I. sanguinea; while callogenesis was induced by 2,4-dichlorophenoxyacetic acid. The obtained data indicate that development of adventitious structures from iris floral organs requires the presence of 6-benzylaminopurine in the growth medium.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 174–179.Original Russian Text Copyright © 2005 by Boltenkov, Zarembo.     

Accumulation of non-utilizable starch in laticifers of Euphorbia heterophylla and E. myrsinites

Starch biosynthesis and degradation was studied in seedlings and mature plants of Euphorbia heterophylla L. and E. myrsinites L. Mature embryos, which lack starch grains in the non-articulated laticifers, develop into seedlings that accumulate starch rapidly when grown either in the light or the dark. Starch accumulation in laticifers of dark-grown seedlings was ca. 47 and 43% of total starch in light-grown controls in E. heterophylla and E. myrsinites, respectively. In light-grown seedlings, starch was present in laticifers as well as parenchyma of stems and leaves, whereas in dark-grown seedlings starch synthesis was almost exclusively limited to laticifers. In 7-month-old plants placed into total darkness, the starch in chyma was depleted within 6 d, whereas starch in laticifers was not mobilized. The starch content of latex in plants during development of floral primordia, flowering, and subsequent fruit formation remained rather constant. The results indicate that laticifers in seedlings divert embryonal storage reserves to synthesize starch even under stress conditions (darkness) in contrast to other cells, and that starch accumulated in laticifers does not serve as a metabolic reserve. The laticifer in Euphorbia functions in the accumulation and storage of secondary metabolites yet retains the capacity to produce, but not utilize starch, a primary metabolite.     

Developmental changes in enzymes involved in the conversion of hexose phosphate and its subsequent metabolites during early tuberization of potato

A highly synchronized in vitro tuberization system, based on single-node cuttings containing an axillary bud, was used to investigate the activity patterns of enzymes involved in the conversion of hexose phosphates and related products during stolon-to-tuber transition of potato (Solanum tuberosum L.). At tuberization the activity of enzymes involved in glycolysis and the oxidative pentose phosphate pathway (OPPP) showed a small but clear increase. This increase reflects a higher capacity of respiratory(-related) metabolism, presumably due to the onset of rapid cell division in the apical part of the tuberizing stolon. During the phase of successive tuber growth these enzymes decreased in activity, suggesting that the concomitant massive starch accumulation is not accompanied by a large increase in respiration. A high degree of positive correlation between the activities of these enzymes could be observed, implying that the level of respiratory metabolism-related enzymes is co-ordinately regulated by the same mechanism of coarse control. The activity pattern of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) showed no developmental change and does not resemble the activity pattern of the enzymes participating in respiratory(-related) metabolism. Instead, its level of activity is very likely the result of metabolic regulation. The level of the content of the metabolites UDP-glucose (UDPGlc) and glucose-6-phosphate (Glc6P) decreased after the onset of tuberization. This decline indicates that tuber induction is not accompanied by an appreciable increase in the level of the cytosolic hexose phosphate (hexose-P) content but that it rather remains on a low level, which might be a prerequisite in order to maintain a high net rate of sucrose degradation during tuber development. In contrast to UDPGlc and Glc6P, the content of fructose-1,6-bisphosphate (Fru1,6bisP) showed an increase after tuber induction. The overall activities of ADP-glucose pyrophosphorylase (AGPase) and starch phosphorylase (STP) both showed a large increase after tuber initiation, which is consistent with their presumed role in the process of starch synthesis and accumulation during rapid tuber growth.