Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses

Panax ginseng calluses were cultured for 5 weeks on solid MS medium supplemented with kinetin 0.46 mM (0.1 mg l^–1) and 2 mg l^–1 of 2,4-D (9.05 mM), IBA (9.98 mM) or NAA (10.74 mM). In the conditions studied, 2,4-D inhibited the organogenic capacity of the calluses, whereas IBA or NAA increased this capacity. IBA induced the formation of a high number of buds and roots, but the roots were thin and necrotized. Calluses grown with NAA produced fewer buds and roots than those grown in IBA medium, but the roots were thick and showed good growth. The highest ginsenoside content was found in root forming calluses grown in the presence of NAA.In calluses forming roots or buds, 2,4-D, NAA and especially IBA increased the Rb group of ginsenosides rather than that of the Rg group.     

The protein of rolB gene enhances shoot formation in tobacco leaf explants and thin cell layers from plants in different physiological states

The objective was to determine whether the protein of rolB affects shoot formation and whether this potential relationship depends on the developmental stages of the plant and/or on the culture conditions. Thin cell layers (TCL) and leaf explants were excised from tobacco plants in the vegetative and flowering stages and cultured under various hormonal conditions. In TCLs of vegetative-stage plants, the expression of rolB enhanced the formation of the shoot buds under hormone-free conditions and with specific concentrations of auxin and/or cytokinin. Histological examination showed that the induction of the shoot meristemoids was particularly enhanced by rolB protein and that meristemoid growth was accelerated. In leaf explants from vegetative-stage plants, the expression of rolB increased the formation of shoot buds in the presence of 1 M IAA plus 1 or 10 M cytokinin. With BA alone, at a 0.1 M concentration, shoot formation occurred in the transgenic explants only, whereas with concentrations ranging from 0.5 to 10 M, it was higher in these explants than in controls.RolB protein enhanced the formation of shoot buds in TCLs from flowering plants under all hormonal conditions. In the presence of 1 M IAA and kinetin, the protein also increased the flowering response. In leaf explants from flowering plants, the expression of rolB increased the number of shoot buds in the presence of 1 M IAA with 10 M BA.In conclusion, rolB protein promotes shoot formation; it seems to have a positive interaction with cytokinin and an effect on the induction of the meristematic condition.     

In Vitro Organogenetic Responses of Gloriosa superba

An efficient protocol was developed for regeneration of healthy plant derived from six categories of explants from both in vivo and in vitro raised plants, viz. roots, corm buds (dormant and nondormant), young leaves, stems, pedicels, and shoot tips from aerial shoots. MS medium supplemented with various concentrations and combinations of auxin, cytokinin, and organic acids was used. 98% of callus induction occurred in nondormant corm bud explants. The greatest number of multiple shoots (57) was observed in corm-derived calluses. Vigorous root formation occurred in all cases when multiple shoots were derived. Histomorphogenetic studies revealed that not only the origin of shoot and root buds in in vitro systems, but the morphology and structure of leaves resemble those of in vivo plants too.     

Cytokinin induces the developmentally restricted synthesis of an extracellular protein in Physcomitrella patens

Early development of the moss Physcomitrella patens follows a simple course leading to the formation of a filamentous protonema containing only two cell-types, chloronema and caulonema. The addition of the hormone cytokinin leads to the induction of multicellular buds from such protonema. The spectrum of extracellular proteins (ECPs) synthesized by P. patens has been investigated at defined stages of development and under defined hormone treatments. It is found that in contrast to the limited changes in intracellular protein synthesis detectable, in the extracellular environment major and specific changes in the patterns of proteins synthesized occur. For example, the presence of caulonema cells is characterized by the synthesis of a 25 kDa ECP whereas early chloronema differentiation is distinguished by the presence of a 38 kDa ECP. The analysis of the pattern of ECPs synthesized by developmental mutants altered in bud formation, and in response to cytokinin in tunicamycin treated protonema (in which bud induction is blocked) indicate that the synthesis of a 14 kDa ECP is specifically induced by cytokinin. This protein represents a novel cytokinin-induced ECP. These data show that the differentiation of particular cell types in plants is associated with the synthesis of particular ECPs, and suggest that hormones which induce specific morphogenic events may do so via the synthesis of specific ECPs.     

Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies

A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during post-germinative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattern typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue.     

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

The effects of auxins and cytokinin on callus formation, growth and regeneration of Gracilaria tenuistipitata Chang et Xia and G. perplexa Byrne et Zuccarello (Gracilariales, Rhodophyta) are reported. Plant growth regulators (PGR) in concentrations ranging from 0.1 to 100.0 μmol of indole‐3‐acetic acid, 2,4‐dichlorophenoxyacetic acid (2,4‐D), and kinetin (K) were added to the ASP 12‐NTA solid medium (0.7% agar), and apical and intercalary segments (5 mm long) were inoculated as initial explants. K stimulated growth rates of intercalary segments of G. tenuistipitata in a linear relation, and 2,4‐D (1.0 μmol) and K (10.0 μmol) stimulated growth rates of apical and intercalary segments of G. perplexa, respectively. The simultaneous formation of apical, basal, and intermediate calluses is reported for the first time in axenic tissue cultures of red algae. With intercalary segments of G. tenuistipitata, basal callus induction rates were higher than those of apical and intermediate calluses in the majority of treatments, and auxins had stimulatory effects on the formation of all callus types. In apical segments of G. perplexa, intermediate callus formation was stimulated only by treatment with 1.0 μmol of K, while apical callus formation was stimulated by indole‐3‐acetic acid (1.0–10.0 μmol), 2,4‐D (10.0–100.0 μmol), or K (0.1 μmol). Intercalary segments of G. perplexa developed only intermediate calluses, and the majority of treatments with PGR stimulated higher rates than those presented by apical segments. Potential for regeneration (development of adventitious plantlets originated from callus cells) was higher in apical calluses than in basal and intermediate calluses developed in intercalary segments of G. tenuistipitata. Moreover, auxins and cytokinin were essential to the induction of regeneration in intermediate calluses, while specific concentrations stimulated regeneration from basal and apical calluses. Plant regeneration in G. perplexa was observed only after transferring calluses from solid to liquid medium, and the majority of treatments with PGR had stimulatory effects. Regenerating plants of G. perplexa developed tetrasporangia, and released tetraspores giving rise to adult gametophytes. Our results indicate that auxins and cytokinin have a regulatory role in the growth and morphogenesis in G. tenuistipitata and G. perplexa, and diversity of responses presented by both species is related to specific developmental systems.     

The Effect of Cytokinins on Vitrification in Melon and Carnation

The influence of exogenous cytokinin on vitrification of melonand carnation buds grown in vitro was assessed. In melon, theanatomical traits of vitrification were found to be similarto those previously described for carnation grown on a highconcentration of auxin. In carnation, the plantlets treatedwith high cytokinin concentrations became bushy but the anatomicalstructure remained normal. The relation of cytokinin and vitrificationis discussed. Cucumis melo L., Dianthus caryophyllus L., vitrified plantlets, habituation, cytokinin     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

Factors affecting adventitious bud induction in Pinus elliottii (Engelm.) embryos cultured in vitro

Embryos of slash pine (Pinus elliottii Engelm.) were induced to form adventitious buds when placed in culture on nutrient media supplemented with cytokinin. Buds were induced on media containing Risser & White major salts. The high content in nitrogen of Murashige & Skoog formulation seems to be deleterious for this in vitro system, since morphogenic responses were only promoted when nitrogen concentration was drastically reduced in the macronutrient formulation. Factors such as concentration of cytokinin (6-benzyladenine) and time and method of exposure (liquid or solid induction medium) strongly influenced bud formation and development. The greatest number of buds and shoots were obtained from 22.0 M cytokinin, but these shoots showed less and slower development than those induced with low dosages of cytokinin. The presence of naphthaleneacetic acid in combination with cytokinin in the induction medium decreased the frequency of bud formation.Abbreviations (BA) 6-benzyladenine - (NAA) 1-naphthaleneacetic acid     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Axillary bud proliferation and organogenesis of <Emphasis Type="Italic">Euphorbia pulchurrima</Emphasis> winter rose

Summary Protocols for both axillary bud proliferation and shoot organogenesis of Euphorbia pulchurrima Winter Rose^TM were developed using terminal buds and leaf tissues. Greenhouse-grown terminal buds were placed on Murashige-Skoog (MS) basal medium supplemented with various concentrations of either benzlyaminopurine (BA) or thidiazuron (TDZ). Explants produced the greatest number of axillary buds on media containing between 2.2 and 8.8 μM BA. The number of explants that produced axillary buds increased with increasing BA concentration. TDZ at concentrations between 2.3 and 23.0 μM caused hyperhydricity of shoots and were not effective in promoting shoot proliferation. The most calluses and shoots were produced from leaf midvein sections from in vitro grown plants placed on the medium containing 8.8–13.3 μM BA and 17.1 μM indole-3-acetic acid (IAA) for 1 mo. before transferring to the medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Five-mo.-old shoots derived from shoot culture or organogenesis rooted readily in artificial soil with or without treatment with indolebutyric acid, and were acclimatized in the greenhouse.     

Callus growth and somatic embryogenesis in thalamus tissue ofRanunculus asiaticus L. CultivatedIn vitro: Cytokinin effect and phenol metabolism

Summary The effect of cytokinin on growth and plant regeneration of thalamus-derived calluses ofRanunculus asiaticus L. has been investigated with various concentrations of 6-benzyladenine and 6-furfurylaminopurine (kinetin), in a medium containing 2,4-dichlorophenoxyacetic acid levels, which was decreased to 0 over three subcultures. Cytokinins, although not essential, for initiating callus production, improved subsequent callus growth and plant regeneration. No somatic embryogenesis was observed on calluses grown on media lacking cytokinins or containing only kinetin. Calluses manifested embryogenesis on media containing 6-benzyladenie plus kinetin or only 6-benzyladenine. Nondifferentiating callus was characterized by a high content of phenolic polymers and an elevated peroxidase and polyphenol oxidase activity in comparison with differentiating callus. Differences in simple phenol concentrations were observed in the two kinds of callus.     

Morphogenetic Effects of Indole-3-acetic Acid and Benz(a)anthracene on Tobacco Callus

Callus obtained from haploid plants of Nicotiana tabacum was inoculated on media containing indole-3-acetic acid (IAA) and benz(a)anthracene (BaA) in various combinations. No cytokinin was included in the medium. Vegetative buds and roots differentiated on this callus. Higher concentrations of BaA favored vegetative bud initiation while higher IAA concentrations promoted root formation. However, these treatments proved ineffective on the callus derived from diploid tobacco plants.     

CERTAIN EMBRYOLOGICAL AND BIOCHEMICAL ASPECTS OF CYTOKININ SD 8339 IN CONVERTING SEX OF A MALE VITIS VINIFERA (SYLVESTRIS)

Certain embryological and biochemical effects of the cytokinin (SD 8339) in converting flower sex from male to hermaphrodite were studied in a clone of Vitis vinifera L. (sylvestris). The cytokinin accelerated the meiotic division of the megaspore mother cell, mitotic divisions of the megaspore and cells of pistillate tissue, and increased the rate of protein synthesis in flower buds. Two working hypotheses for the possible mode of action of the cytokinin in sex conversion are presented.     

Change in the spectra of peroxidases in regenerants of Stachys sieboldii (Miq.) as a result of hormonal and mutagenic effects

Nodular segments of the stem and stolons of aseptic Stachys sieboldii (Miq.) plants were cultured in Gamborg's B5 medium at a low (1-2 mg/l) or high (10 mg/l) concentration of cytokinins (6-BAP, kinetin) and produced many adventitious buds and shoots without callus formation. Several plants were regenerated from nodular segments incubated at various concentrations of 5-bromodeoxiuridine (5-BdU) for three weeks. All regenerants were morphologically normal. Peroxidase electrophoretic patterns were analyzed to determine whether somaclonal variation is associated with S. sieboldii micropropagation. The frequency of changed forms was higher with the high 6-BAP concentration and with mutagenic treatment. Changed forms were not detected in plants grown at a low cytokinin concentration, which can be used in S. sieboldii micropropagation.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

Plantlet regeneration from petiole explants of the african horned cucumber,Cucumis metuliferus

Plantlet regeneration in Cucumis metuliferus from several explant sources, including cotyledons, leaves, hypocotyls and petioles, was evaluated on Murashige and Skoog's medium containing various combinations of auxin (IAA, NAA, 2,4-d) and cytokinin (BA, kinetin, zeatin), Callus development was obtained within 4 to 5 weeks on all growth regulator combinations which were tested at concentrations ranging from 1.0 M to 4.0 M of each. The response was similar when the tissues were incubated under light or in continuous darkness. Differentiation of callus to form adventitious buds or shoot primordia occurred only with petiole explants on medium containing NAA/BA or 2,4-d/BA at 2.0/1.0 M; none of these calluses, however, differentiated further to form shoots. When the differentiated calluses derived from petiole explants which had been initiated on 2,4-d/BA at 2.0/1.0 M were transferred onto medium with 2.0 M zeatin, formation of shoots occurred within 2 to 3 weeks. The frequency of shoot formation was 14.6%. Subculture of these shoots onto MS medium without growth regulators gave rise to plantlets of normal appearance. Regeneration in C. metuliferus requires callus initiation on an appropriate growth regulator regime followed by transfer to a medium containing the cytokinin, zeatin, and can be achieved within 10–12 weeks.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid     

金桔组织培养中愈伤组织和芽形成的组织细胞学观察

用金桔茎段为外植体,培养在附加1.0毫克/升BA和0.l毫克/升IBA的MS培养基上,诱导愈伤组织和芽形成。观察了愈伤组织和芽形成过程中的组织细胞学变化。培养一周后,在茎组织切口两端开始膨大,细胞增大和开始分裂。培养两周后,开始形成瘤状愈伤组织。在愈伤组织中有形成层状分生组织、维管组织结节和分生细胞团。培养四周后,表层的分生细胞团分化形成大量芽原基,同时愈伤组织深层也出现分生细胞团。带节茎段可从切口两端的愈伤组织分化形成芽,亦可从叶腋的潜伏芽直接形成芽。     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.     

Controlled Cytokinin Production in Transgenic Tobacco Using a Copper-Inducible Promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu²⁺ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.