Isoperoxidases in Epidermal Layers of Tobacco and Changes during Organ Formation in vitro

The content and pattern of soluble isoperoxidases were determined in epidermal explants taken from different internodes of tobacco plants in the vegetative and floral states. There were qualitative and quantitative differences in the isoperoxidases, with a decrease in content and fewer bands being observed acropetally, i.e., in going from the base of the stem towards the apex. Epidermal explants from floral branches were grown in in vitro culture, with various media moditications, to form de novo floral or vegetative buds, roots or callus. Changes in soluble isoperoxidases were followed electrophoretically in relation to these varying morphogenetic pathways. In each of them, the number of bands increased on both the anodic and cathodic sides with time in culture. Compared to each other these four morphogenetic programmes were different in their peroxidase zymograms, mainly through varying kinetics in the development of activity of the isoenzymes. The changes observed during root and vegetative bud formation agree with previously published data, and the changes during floral bud formation agree with those observed in vivo.     

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.     

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.     

A HISTOCHEMICAL STUDY OF CALLUS INITIATION FROM CARROT TAPROOT PHLOEM CULTIVATED IN VITRO

Cylinders of carrot taproot secondary phloem were cultured on one of four media: 1) 2% sucrose + 1% agar (SA); 2) Heller's basal medium (NA); 3) NA + 10^-5 g/liter 2,4-D (H4); and 4) NA + H4 + 15% coconut milk (HW). Samples were taken from the cultured explants at 3-day intervals. A morphological study of the cultured explants revealed no differences between callus-initiating explants (cultured on HW medium) and noncallus-initiating explants (cultured on SA, NA, and H4 media) within the first 3 days of culture. All explants exhibited a typical wound response. Cell division ceased in the NA and SA explants after the sixth day in culture. Extensive cell division occurred in the subsurface layer of dividing cells in the HW explants and resulted in the formation of callus by the ninth day in culture. Histochemical staining revealed that the activity of NAD diaphorase, succinic dehydrogenase, and cytochrome oxidase were closely correlated with the wound response and with callus initiation in the cultured explants. The activity of these enzymes was high in the layer of dividing cells of all explants after 3 days of culture, but with longer periods of culture the activity of these enzymes was closely correlated with the extent of cell division. Acid phosphatase activity was associated with the dividing cell layers of all explants, but comparatively little acid phosphatase activity was observed in the NA, SA, and H4 explants as compared to the HW explants, and acid phosphatase was strongly correlated with callus initiation by the HW explants. Using the nitroso reaction, “catechol tannins” were found in the surface layers of the NA, SA, and H4 explants, while no nitroso-reaction-positive substances were detected in the HW explants during the period of callus initiation.     

Capacité caulogène de cellules de cal issues des couches cellulaires minces de type épidermique de ramifications florales de Nicotiana tabacum cv. Wise. 38 Par

Bud formation capacity of callus formed from thin epidermal cell loyers excised from floral branches of Nicotiana tabacum cv. Wise. 38. Subepidermal cells of thin tissue pieces with a few cell layers were capable of forming eitber buds, roots, (lowers or non-organ ogenetic callus. To determine wheiher this calltjs is able to dirferentiate into organs, we transferred it to media inducing eitber flowers, or buds, or roots. In this paper, we study ibe capacity of lbe callus to form buds. In 50% of the cases, the explants (being maintained for I day to 2 years in callus media) can still express the capacity to form buds. This percentage increased with increased agar concentration of the culture media. At the histological level, non-organogenetic callus is characterized by the absence of tracheid differentiation, whereas in the organogenetic callus, iracheids were induced after their transfer into a ‘Bud medium’ and indicate an organogenetic differentiation pattern.     

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.     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

Role of Ethylene in Abscisic Acid-induced Callus Formation in Citrus Bud Cultures

The induction of callus formation in cultured buds of Shamouti orange (Citrus sinensis [L.] Osbeck) by abscisic acid (ABA) is a multiphasic process. (Altman, and Goren 1974 Physiol Plant 32: 55.) A study of the mediation by ethylene on this effect of ABA was undertaken. It was found that: (a) ethylene and (2-chloroethyl) phosphonic acid, as well as ABA, induced callus formation; (b) callus induction is best attained when explants are exposed to ethylene during the 1st day after excision; and (c) ABA-induced callus formation is inhibited by rhizobitoxine analog, an inhibitor of ethylene biosynthesis. It is concluded that the effect of ABA on callus formation is mediated via ethylene.     

Effects of Auxin Concentration on the Dimensions and Patterns of Tracheary Elements Differentiating in Pith Explants

The optimal concentration of IAA (0.03 mM) for tracheary elementdifferentiation in lettuce pith explants was about ten timesgreater than the optimal concentration for callus proliferation.Related to this, the mean volume per tracheary element increasedwith increasing IAA concentration, 18-fold between 0.001 mMand 0.3 mM IAA. At the highest concentrations, some pith cellsappeared to differentiate directly into tracheary elements,without cell division, resulting in especially large trachearyelements. Tracheary strands developed at intermediate concentrationsof IAA, and led to a small increase in the mean length/breadthratio of tracheary elements. For tracheary elements differentiating from stem cambial derivatives,a reassessment of previous studies indicates that increase inauxin concentration brings greater tracheary element size atconcentrations up to the 0.03 mM optimum. Above this optimum,however, further increase in auxin concentration brings progressivelysmaller tracheary elements, as the high auxin curtails enlargementof the differentiating cells. This contrasts with the pith explants,in which tracheary element size increases with IAA concentrationmost markedly above the optimum concentration. The interpretationof these relations requires an understanding of the effectsof auxin concentration on interacting quantities such as initialsize of cells, rate of enlargement, and rate of differentiation. Lactuca sativa, lettuce, IAA concentration, pith explants, tracheary element dimensions     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

Hormonal Control of Cell Proliferation and Xylem Differentiation in Cultured Tissues of Glycine max var. Biloxi

The relationship between tracheary element differentiation, cell proliferation and growth hormones was examined in agar-grown soybean callus. The time course of cell division and tracheary element formation in tissues grown on a medium containing 5 x 10(-7)m kinetin and 10(-5)m NAA was determined by means of maceration technique. After a slight lag period, a logarithmic increase in cell number was observed through the twelfth day of the culture period. Cell numbers increased at a considerably slower rate after the twelfth day. The rate of tracheary element formation varied with the rate of cell proliferation. Tracheary elements increased logarithmically during the log phase of growth. As the rate of cell division decreased after the twelfth day of culture, the rate of tracheary element formation also decreased. In the presence of 10(-5)m NAA, cell number increased as the kinetin concentration was increased between 10(-9) and 10(-6)m. However, tracheary element formation was not initiated unless the kinetin concentration was 5 x 10(-8)m or above. When the Biloxi callus was subcultured repeatedly on media containing 10(-8)m kinetin, a tracheary element-free population of cells was obtained. This undifferentiated tissue produced tracheary elements upon transfer to a medium containing 5 x 10(-7)m kinetin. In the presence of 5 x 10(-7)m kinetin, NAA stimulated cell proliferation between 10(-7) and 10(-5)m, but no tracheary elements were formed without auxin, or with 10(-7)m NAA. Neither NAA nor kinetin at any concentration tested stimulated tracheary element formation in the absence of an effective level of the other hormone. However, 2,4-D at 10(-7) or 10(-6)m promoted both cell proliferation and tracheary element differentiation in the absence of an exogenous cytokinin.     

CELL DIVISION IN RELATION TO CYTODIFFERENTIATION IN CULTURED PEA ROOT SEGMENTS

One mm-thick segments cut 10–11 mm proximal to the root tip of germinating seeds of garden pea Pisum sativum were cultured in sterile nutrient medium containing auxin in the presence and absence of kinetin. In the absence of added cytokinin, pericyclic proliferation occurred, the cortical tissues showed no proliferation and were sloughed off, and a callus tissue of diploid cells was formed. In the presence of kinetin concentrations from 0.1–1.0 ppm cortical cells of the segments were induced to divide, beginning at the third day. From experiments with ³H-thymidine incorporation at different times of culture, from cytological squash preparations and from histological sections it was shown that the cortical cells stimulated to divide by cytokinin underwent DNA synthesis prior to division, were polyploid, and following cell division rapidly underwent cytodifferentiation at 5–7 days to form mature tracheary elements. At 10 days, when over 300,000 new cells had been formed per segment about 16% of these cells had formed tracheary elements. It was concluded that cytokinin, together with auxin, was essential for the initiation of DNA synthesis in the cortical cells, for their subsequent division, and finally for their specific cytodifferentiation.     

Histological Study of Adventttious bud Formation on Lactuca Sativa Cotyledons Cultured In Vitro

Abstract

Cyto-histological changes accompanying the formation of adventitious buds in excised cotyledons of Lactuca sativa were studied during the first 12 days after planting in vitro. Prospective proliferating cells can first be recognized, already on the first day after planting, by a marked increase in nuclear and nucleolar volumes, followed on the second day by a burst of cell divisions involving particularly mesophyll cells. Then lignified elements develop together with meristematic center, forming a callus-like tissue in the inner part of the cotyledons. At the third day of culture, the epidermal cells start to divide with a periclinal wall followed by an anticlinal division. In the following days of culture the epidermal cells, which divide mainly with periclinal walls, form layers of cells below the surface, gradually filling up the intercellular spaces. From the 8^th day on, the buds protude above the surface and develops into shoots. These results are discussed in relation to DNA content of nuclei of Lactuca sativa cotyledons and to the time course of cell division and tracheary element formation. The very regular sequence of changes associated with the initiation and development of the bud makes the in vitro culture of Lactuca cotyledons an appropriate System for histochemical and biochemical studies.     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Interaction of auxin, cytokinin, and gibberellin on cell division and xylem differentiation in cultured explants of Jerusalem artichoke.

Dark-cultured explants of parenchymatous cells isolated fromJerusalem artichoke tubers were induced to divide and differentiateas tracheary elements on Murashige and Skoog medium containingdifferent combinations of plant growth-hormones such as auxin(IAA), cytokinin (zeatin), and gibberellin (GA₃). Addition ofauxin to the growth-medium induced after a short lag period,very rapid cell division which was followed by differentiationof some of the divided cells as tracheary elements. At the optimallevel of IAA (5.0 mg/liter), the percentage of tracheids differentiatedwith respect to the total number of cell population was 13.54.When the explants were cultured in the presence of both auxin(IAA 5.0 mg/liter) and one cytokinin (zeatin 0.1 mg/liter),not only a strong interaction on cell division and trachearyelement formation was observed but also an increase in the percentageof tracheids differentiated in relation to the total cell population.Auxin-gibberellin and auxin-gibberellin-cytokinin treatmentsalso produced interaction on cell division and cytodifferentiation.In explants treated with the three growth-hormones about 20%of the total cell population differentiated as tracheary elements.Further, all the hormonal treatments gave different patternsof cytodifferentiation which reflected meristematic patterns. ¹ This research was supported by a grant from C. N. R. Italy. (Received April 18, 1973; )     

Structural physiological, and biochemical gradients in tobacco pith tissue

Explants of tobacco pith taken at various distances from the apex of a mature stem show a sharp gradient in growth potential in vitro; growth is highest in the extreme apical and basal explants, and is minimal in explants removed ca. 75 cm from the apex. Calluses produced by the vigorously growing basal explants are harder and more compact than those produced from more apical explants. The gradient in growth potential is directly correlated with gradients in RNA, protein of cell sap and soluble N per unit fresh weight, but is inversely correlated with peroxidase activity. Cell size increases from apex to base of plants.

The peroxidase activity of pith explants is electrophoretically resolvable into 2 isoperoxidases, moving anodically at pH 9.0. During in vitro culture, this activity rises, due to the formation of several new isozymes moving toward the cathode. The appearance of these isozymes occurs most rapidly in apical and extreme basal explants.

     

Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxydase (DAO) free amine and conjugated amine titers were estimated in leaf explants of Chrysanthemum morifolium Ramat. var. Spinder cultivated in vitro in relation to hormone treatment. Addition of benzyladenine (BA) to a basal medium caused the formation of buds on the explants. BA plus 2,4 dichlorophenoxyacetic acid (2,4 D) caused callus formation and proliferation. Formation of roots was obtained by addition of indolylacetic acid (IAA). Arginine decarboxylase (ADC) ornithine decarboxylase (ODC) and diamine oxidase (DAO) activities increased during the first days of culture when cell multiplication was rapid, followed by a sharp decline as the rate of cell division decreased and differentiation took place. DAO activities increased rapidly in proliferating and growing organs and decreased during maturity. This increase was concomitant with ADC and ODC activities and polyamine content (free and conjugated polyamines). The biosynthesis and oxidation of polyamines which occurred simultaneously in physiological states of intense metabolism such as cell division or organ formation were directly correlated. In callus cultures DAO activity was blocked throughout development and regulated neither the cellular levels of polyamines nor polyamine conjugates. Levels of polyamine conjugates were high in callus cultures throughout development. In foliar explants cultivated on a medium promoting callus, inhibition of ODC activity by DFMO (-DL-difluoromethylornithine, a specific enzyme-activated ODC inhibitor) resulting in an amide deficiency facilated the expression of differentiated cell function; substantial activation of DAO was observed until the emergence of the buds. On a medium promoting bud formation, -OH ethylhydrazine (DAO inhibitor) promoted callus formation without differentiation. In this system DAO activity was blocked and there were high levels of polyamines, especially polyamine conjugates, throughout the culture period. The relationship among free and conjugated polyamines related biosynthetic enzyme activities, DAO activities, cell division and organ formation is discussed.Abbreviations ADC = arginine decarboxylase - ODC = ornithine decarboxylase - DOA = diamine oxidase - DFMA = -DL-difluoromethylarginine - DFMO = -DL-difluoromethylornithine - Put = putrescine     

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     

K-Nutrition, Growth Bud Formation, and Amine and Hydroxycinnamic Acid Amide Contents in Leaf Explants of Nicotiana tabacum Variety Xanthi n.c. Cultivated in Vitro

The effects of K-nutrition on growth (increase of fresh weight), bud formation (time of emergence, number of buds), and amine and hydroxycinnamic acid amide contents in foliar explants of Nicotiana tabacum cv Xanthi n.c. cultivated in vitro were examined. In K-deficient medium and in high K medium growth and bud formation were markedly inhibited. Marked changes of amine content (a diamine, putrescine; a phenolic amine, phenethylamine) were observed after a few days of culture. No apparent relationship was found between these amines and growth or bud differentiation. In contrast, changes in hydroxycinnamic acid levels were shown to correlate well with growth and bud formation. The greatest stimulation of budding and growth was correlated with the greatest accumulation of these amides. The highest contents of hydroxycinnamic acid amides were found during the first 15 days in culture when intensive cell division took place. Then they declined sharply after 26 days in culture as the rate of cell division decreased and differentiation occurred.     

The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis

The higher plant tumors are convenient models for studying the genetic control mechanism of plant cell division. There are two types of tumors: induced by the pathogenic factor and genetically determined. The development of both tumor types was related to the changes in cytokinin metabolism and/or signal transduction. In this work, the effect of synthetic cytokinins on the in vitro morphogenesis of cotyledon explants and isolated apices of radish seedlings was studied in several inbred radish lines (Raphanus sativus var. radicula Pers.) that differed in their in vivo tumorigenic properties. It was noted that root formation was stronger affected by kinetin while the treatment with thidiazuron tended to induce active callus formation in cotyledon explants of all inbred lines, except IIa. Growing with benzyladenine produced an intermediate effect as regards all morphogenetic responses. Cytokinin treatment of tumorigenic lines enhanced necrotic development in cotyledon explants. Culturing isolated apices of regenerated plants produced tumors anatomically and morphologically similar to those developing in vivo. Some of the lines nontumorigenic in vivo with enhanced formation of calli on cotyledon explants also developed tumors on apical explants in vitro when treated with cytokinins. These data suggest that different mechanisms for tumor formation operate in various radish lines. The radish lines are classified into three types: (1) necrotic lines with tumor formation putatively related to endogenous cytokinin level, (2) callus-forming lines with cell division enhanced in response to cytokinins, and (3) necrosis-and callus-forming lines with both mechanisms of tumor formation involved.