Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.     

Floral morphogenesis in thin-layer tissue cultures of Nicotiana tabacum

The morphological changes in thin-layer tissues of Nicotiana labacum L. cv. Samsun, cultured on Murashige and Skoog medium with 1 μ M each of naphthalene acetic acid (NAA) and benzyladenine (BA), were studied during the first 8 days of culture with light and scanning electron microscopy. The first three days of culture arc characterized by enlargement of all cells and cell divisions starling in the cortical parenchyma cells adjacent to the medium. Between days 3 and 6, epidermal and/or subepidermal cells start to divide, resulting in division centers, which lead to flower bud formation. The hormones NAA and BA in different concentrations affect the formation and distribution of flower buds, bud morphology and callus formation. BA influences particularly bud formation and bud morphology, while NAA affects callus formation in particular. In addition, polarity may occur in the formation of both callus and flower buds, the degree of which depends upon the hormone concentrations.     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.     

Effect of inhibition of phenylalanine ammonia-lyase activity on growth of alfalfa cell suspension culture: Alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins and polyamines

The effect of inhibition of phenylpropanoid biosynthesis on the growth of Medicago sativa L. suspension culture was studied. 2-Aminoindan-2-phosphonic acid (AIP), a potent inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), caused a marked reduction in the amount of hydroxycinnamic acid derivatives in a few hours after cell inoculation into AIP medium. The treatment of alfalfa suspension culture with this inhibitor increased the extractable PAL activity and elevated ethylene production during the growth cycle. The addition of AIP (10 μ M ) stimulated cell division activity during the growth cycle, although the onset of cell division was slightly delayed. The maxima of cytokinin content as well as of the mitotic index were postponed in AIP-treated cells, however, the unchanged content of cytokinins did not correlate with increased mitotic activity of treated cells. The decreased level of hydroxycinnamic acid derivatives, which represent the phenolic conjugation partners of free polyamines (PAs), influenced the rate of PA conjugation. Consequently, the balance between free and conjugated PAs was shifted in favor of the free PA form. A potential role of the reduction of the pool of phenolic acids in the enhancement of cell division of alfalfa cell suspension culture is discussed.     

In vitro flower bud formation in tobacco: interaction of hormones

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate.     

In vitro studies on xylogenesis in citrus fruit vesicles. II. Effect of pH of the nutrient medium on the induction of cytodifferentiation

The initial pH of the nutrient medium influenced the type of cytodifferentiation occurring in cultured isolated fruit vesicles of Citrus limon (L) Burmann var. Assam lemon. Neither callus formation nor cytodifferentiation was found at pH values below 3.0. Three types of cytodifferentiation were found after 30 days culture in the presence of a liquid Murashige and Skoog (MS) basal medium supplemented with MS vitamins, indoleacetic acid (IAA) (10 mg/l), kinetin (0.2 mg/l), and sucrose (3% w/v): sclereids, xylem fibers and tracheary elements. The greatest numbers of sclereids and tracheary elements were found in callus grown on a medium with an initial pH 5.0–6.0, whereas pH 7.0 was optimal for the formation of xylem fibers.     

Changes in water potential and its components during shoot formation in tobacco callus

Addition of plant growth regulators (5 nM NAA and 5μM BAP) to a defined basal medium stimulated adventitious bud formation of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) cotyledon explants in culture. Cytoplasmic soluble proteins synthesized during early stages of adventitious bud formation were analyzed by electrophoresis of ³H- and ¹⁴C-leucine labeled proteins on SDS polyacrylamide gels. Increased synthesis of low molecular weight proteins (16,000 to 20,000 daltons) was detected after 2 days in culture and reached a maximal level at day 4. When cotyledon explants cultured on bud medium for 2 days were transferred to callus medium (which suppressed adventitious bud formation), suppression of the synthesis of low molecular weight proteins was also observed, suggesting that these proteins may be associated with early stages of adventitious bud formation.     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.Abbreviations TGase transglutaminase - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine     

Effect of partial oxygen pressure on survival, proliferation, and differentiation of mouse bone marrow mesenchymal stem cells

Effects of partial oxygen pressure in a gas mixture surrounding the culture medium on survival, proliferation and differentiation of mesenchymal stem cells (MSC) isolated from mouse bone marrow was studied. It was found that 3% oxygen increased the survival of cells seeded at low density; the rate and duration of the MSC proliferation were also elevated. Effect of oxygen concentration on replicative activity of MSC was manifested as early as the first few days after the onset of cell growth. Studies of colony formation revealed that preincubation of cells with 21% oxygen had a negative effect on cell growth under 3% oxygen, while preincubation with 3% oxygen stimulated MSC proliferation in the presence of 21% oxygen. Notwithstanding, this effect of oxygen cannot be interpreted as unequivocally deleterious. Low partial oxygen pressure inhibited osteogenic differentiation of MSC, but adipogenic differentiation was insensitive to oxygen concentration. It is concluded that proliferation and differentiation of MSC depend critically on oxygen content in the culture medium.     

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

A23187和EGTA对光周期诱导菊花成花及其过程中叶片Ca2+分布和碳水化合物的影响

以切花菊品种‘神马’为试材,研究光周期诱导菊花成花过程中Ca²⁺载体A23187和Ca²⁺螯合剂EGTA处理对花芽分化及其过程中叶片Ca²⁺分布和蔗糖、可溶性糖及淀粉含量变化的影响.结果表明：对照叶片Ca²⁺含量在花芽未分化期（Ⅰ）处于较低水平,而在花芽分化启动期（Ⅱ）迅速增加并达到高峰,之后下降;Ca²⁺亚细胞定位表明,在未分化期（Ⅰ）Ca²⁺沉淀主要分布在液泡、细胞壁和细胞间隙中,细胞质内较少,而在花芽分化启动期（Ⅱ）细胞质内积累大量的Ca²⁺沉淀.A23187处理的菊花花芽分化开始和结束时间比对照分别提前2 d和3 d,叶片Ca²⁺含量比对照显著增加;EGTA处理的叶片Ca²⁺含量比对照显著减少,花芽分化开始和结束时间分别比对照推迟4 d和8 d;A23187和EGTA处理的叶片Ca²⁺在花芽分化启动期（Ⅱ）均向细胞质流入并密集.A23187处理的蔗糖和可溶性糖含量在处理2 d时达到峰值,比对照达到峰值的时间提前2 d,与Ca²⁺达到峰值的时间一致,而EGTA处理的蔗糖和可溶性糖含量在处理2 d时没有明显变化,8 d时才迅速增加达到峰值,即所有处理的蔗糖、可溶性总糖含量在花芽分化启动期（Ⅱ）均增加并达到高峰,之后有所减少,但其在整个花芽分化过程均高于光周期诱导前的含量;对照和A23187处理的淀粉含量在处理2 d时开始减少,而EGTA则在处理8 d后开始减少,至花芽分化结束所有处理的淀粉含量均保持较低水平（低于诱导前）.表明Ca²⁺碳水化合物参与了光周期诱导的菊花成花过程.     

枣花芽分化过程中营养物质和内源激素含量及抗氧化酶活性变化研究

以新疆主栽品种灰枣和骏枣的花芽为材料,测定不同分化时期花芽的可溶性糖、还原糖、淀粉、可溶性蛋白含量,SOD、POD、PPO、CAT活性以及内源GA3、IAA、ABA、ZT水平的变化,并分析它们与花芽分化的关系,为枣花芽分化调控提供理论参考.结果表明:(1)灰枣和骏枣花芽可溶性糖、还原糖和淀粉含量在花芽分化过程的变化趋势...     

黄山药愈伤组织的诱导与分化

以黄山药的叶片、茎段和叶柄作外植体,以MS为基本培养基,试验了不同激素组合对愈伤组织诱导的影响,采用正交试验法研究了愈伤组织的分化效果。结果表明,以MS+2,4-D1~2mg/L+6-BA2mg/L培养基对叶片的愈伤组织诱导效果最好,接种12d后初见愈伤组织,20d后可形成大量的愈伤组织,而茎段和叶柄的诱导效果较差。分化试验结果表明,生根率最高可达85.3%,而出芽率最高仅达29.6%。     

Peroxidases and the metabolism of hydroxycinnamic acid amides in Poaceae

The arsenal of plants to fight off microorganisms and herbivores include hydroxycinnamic acid amides (HCAA) and their oxidation products. Hydroxycinnamic acid amides are widespread in the plant kingdom and in the recent years our knowledge of their biosynthesis and catabolism has increased substantially. Peroxidases are the primary candidates as the oxidative enzymes responsible for the turnover of hydroxycinnamic acid amide monomers. In barley, hydroxycinnamoylagmatine derivatives accumulate in young seedlings and in tissues infected with fungi. Hydroxycinnamoylagmatine is found as anti-fungal soluble dimers, called hordatines, and it is also a likely constituent of cell walls. Current evidence suggest that peroxidases are involved in the cross-linking of hydroxycinnamoylagmatine with cell wall components and possibly also in the synthesis of hordatines. Epidermal cell walls of barley respond to infection by the powdery mildew fungus with the deposition of polyphenolic material, that apparently contains hydroxycinnamic acid amides, at the site of attempted penetration. Accumulation of these compounds lowers the successful penetration by the fungus. The recent characterization of agmatine coumaroyl transferase (ACT), the N-hydroxycinnamoyltransferase responsible for the synthesis of hydroxycinnamoylagmatine in barley, has indicated that the production of these metabolites is widespread in the plant body and suggests multiple physiological functions for HCAA derivatives. The cloning of ACT has enabled the revelation of homologues genes in several monocots and the presence of a range of structurally diverse HCAAs in cereals suggests that their peroxidase-mediated metabolism is a common theme. The prospects for metabolic engineering of these pathways into other crops are discussed. Abbreviations: HCAA – hydroxycinnamic acid amide; HRPC – horseradish peroxidase C; ACT – agmatine coumaroyl transferase; THT – tyramine hydroxycinnamoyl transferase; HCBT – hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyl transferase; PHT – putrescine hydroxycinnamoyl transferase; SHT – spermidine/spermine hydroxycinnamoyl transferase; HHT – hydroxyanthranilate hydroxycinnamoyl transferase; p-CHA –p-coumaroyl hydroxyagmatine; p-CHDA –p-coumaroyl hydroxydehydroagmatine; PAL – phenylalanine ammonia lyase.     

Theobroma cacao L.: an axillary bud in vitro propagation procedure

An axillary bud derived in vitro propagation procedure for Theobroma cacao is described. The method allows bud expansion and elongation, including leaf development, as early as 13 days after placing explants on establishment medium. Shoots have been maintained in culture vessels for six months with little evidence of decline. Propagules have been induced to form roots, hardened off, and moved to the greenhouse within six weeks of initial explant establishment. Some plants have been in the greenhouse for twelve months and are growing in an apparently normal manner. No major differences in response from UF-667 or EQX-100 derived seed-grown trees were found suggesting that the method may not be limited by genotype. Long-term growth without added plant growth regulators has been seen in over 2400 explants.Abbreviations NAA naphthaleneacetic acid - IBA indolebutyric acid - BAP benzylaminopurine - WPM Lloyd-McCown woody plant medium Published as paper No. 8148, Journal Series, Pennsylvania Agricultural Experiment Station     

Polyamines, hydroxycinnamoylputrescines, and root formation in leaf explants of tobacco cultivated in vitro: effects of the suicide inhibitors of putrescine synthesis

In vitro formation of roots is obtained directly, without intermediate growth of callus, from foliar explants of a tobacco (Nicotiana tabacum) plant cultured on Murashige and Skoog medium containing IAA. Auxin-induced root formation was accompanied by significant changes in hydroxycinnamoylputrescine levels. Increasing levels were found in leaf explants during the first 14 days in culture; this was followed by a sharp decline after 20 days. Early changes in putrescine conjugates were detected in leaf explants before the visible appearance of roots. An early and transitory accumulation of hydroxycinnamoylputrescines was observed in the roots. Free polyamines (putrescine, spermidine, and spermine) in leaf explants and roots were always at a low level and only small changes in their concentrations were observed, α-dl-difluoromethylarginine and α-dl-difluoromethylornithine, specific, irreversible inhibitors of arginine decarboxylase and ornithine decarboxylase, respectively, inhibited putrescine accumulation and root initiation and reduced the fresh and dry weights of leaf explants. These effects were reversed by free putrescine or hydroxycinnamoylputrescines. The results reported here suggest that hydroxycinnamoylputrescines are associated with root formation. The relationship among free polyamines, hydroxycinnamoylputrescines, cell division, and root formation is discussed.     

Changes of Endogenous Hormone Contents During Floral Bud and Vegetative Bud Differentiation in Thin Cell Layer Culture of Cichorium intybus L. Explant

The sectioned thin cell layers (TCL) of flower stalk of Cichorium intybus L. were cultured in MS medium supplemented with NAA and BA or IAA and BA where floral and vegetative buds were developed from the explant. Endogenous IAA, DHZ+DHZR, iPA increased significantly during the floral bud formation, while Z+ZR remained changed. The levels of cytokinins, DHZ +DHZR, iPA, and Z-f-ZR all increased significantly during the vegetative bud formation, however IAA level was reduced during the first 7 days of culture and increased to two-thirds of initial values on the day when the bud primordia were formed. The results suggested that the initiation of floral buds was associated with a high IAA/CTK ratio, whereas the induction of vegetative bud differentiation was related to a low IAA/CTK ratio.     

Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.

Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development.     

苦瓜离体培养过程中内源激素含量的变化

针对苦瓜(Momordica charantia)离体培养中外植体易于产生愈伤组织而难以再生不定芽的问题,本文采用酶联免疫吸附法, 研究了苦瓜组织培养过程中不同发育阶段各外植体内源激素含量的变化, 以探讨不定芽分化与激素水平变化之间的关系, 以及苦瓜难以再生不定芽的内在制约因素。结果表明: (1)苦瓜外植体中IAA含量较高, 而iPAs含量过低, 是苦瓜易于产生愈伤组织而不定芽再生困难的主要原因;(2)不定芽的分化与IAA/iPAs的变化有密切的关系; (3)在离体培养过程中, 保持外源细胞分裂素类物质(如ZT)的适当浓度并及时继代, 有利于苦瓜不定芽的分化。