The Effect of Various Media on the Growth Responses of Different Clones of Carrot Explants

The induction of growth in otherwise quiescent tissue explantedfrom carrot root has been investigated with reference to theeffects of different kinds of growth-promoting substances addedas supplements to a basal medium, singly and in combination.The effects of these media upon different clones of carrot explantsare described. The idiosyncrasies of different clones of explantswere detected by their responses measured by the incidence ofcell division, the extent of cell enlargement, and by theirnucleic acid content. The basal medium which contains salts,sugar, and vitamins supported only a minimal amount of growth;the basal medium supplemented with casein hydrolysate and coconutmilk (10 per cent by volume) supported the highest level ofgrowth obtained in any of the treatments tried. The active componentsof the coconut milk (AF_cm) when refined required the furtherparticipation of either indole-3yl-acetic acid (IAA) or inositol,and were further stimulated by casein hydrolysate (CH). Thusthe over-all stimulus of the coconut milk comprised two parts—nowrecognized as growth-promoting systems I and II, respectively.The effects of System I were mediated by appropriate combinationsof inositol and the corresponding active growth-promoting factors(AF₁) which were, in turn, represented by a purified factorpreviously isolated from Aesculus (AF₂). System I induced bothcell division and cell enlargement in balance, whereas SystemII stimulated internal cell division more than cell enlargement.The effects of System II were mediated by appropriate combinationsof IAA and active growth-promoting factors (AF₂), which wererepresented by the substance zeatin. The maximum growth of anygiven clone of carrot explants isolated from a given carrotroot was only supported by exogenous requirements, over andabove a basal nutrient medium, which meet its specific endogenouslimitations. The paper shows how these limitations may be diagnosed,and discusses the over-all growth stimulus due to coconut milkin terms of the partial responses elicited by the known componentsof Systems I and II.     

Investigations on the growth and metabolism of cultured explants of Daucus carota

Summary Earlier papers of this series relate to different growth-promoting substances and systems which, singly and in combination, have interacted with trace elements (Mn and Mo) and Fe to induce growth and to affect the metabolism of aseptic cultures of carrot. The solutes of cultured carrot cells (K⁺, Na⁺, Cl^–, total solutes) are also affected. Two clones were grown in 9 combinations of growth factors and under 4 trace-element regimes (a complete complement including Fe, and this complement lacking either Mn or Mo, or both Mn and Mo), a total of 36 treatments under otherwise standardized experimental conditions. Under the treatments applied the number of cells varied over a 35fold range and their average size over a 7fold range; the concomitant effects on their solutes are expressed in terms of concentrations and of total content per cell. Both growth and the solutes accumulated were variously affected by carrot growth-promoting system I (mediated by inositol), by system II (mediated by IAA), and by coconut milk in the presence of Fe, with and without Mn, Mo, or Mn and Mo.The greatest concentrations of total solutes occurred in tissue cultured in nutrient solutions which lacked the stimuli to rapid cell multiplication and were also limited by the trace elements Mn and Mo. Moreover, specific regulatory effects of the trace elements on solute content, not solely attributable to their effects on cell growth, have been noted. An imbalanced growth-factor regime (zeatin acting alone, i.e. without IAA) shifted the normal preference for K⁺ over Na⁺ strongly toward Na⁺, a trend which could also be induced by certain trace elements and more balanced growth-factor regimes, e.g. in a basal coconut milk medium lacking only Mn.The data are interpreted in the context of views on the de-novo uptake of salts and solutes in cultured cells as they grow. These cells respond to a network, or matrix, of interacting factors by distinctive effects that are attributable to the component parts of the culture medium acting singly and in various combinations. These interactions (involving trace elements and exogenous growth factors) control growth (fresh weight, number and size of cells) and regulate the solutes (organic and inorganic; K⁺ vs. Na⁺; organic anions vs. Cl^–) which the cells acquire as they grow and develop. The intensity of the response of the cultures to balanced, or imbalanced, growth factors creates the internal spaces accessible to solutes; and the metabolism, as it is also affected by growth factors and trace elements, determines how these spaces are to be filled at a given osmotic value. The evidence shows the range of factors that affect the accumulation of solutes in cells as they grow and is to be contrasted with conventional observations on mature cells held in steady states under conditions that preclude all growth and when only a single ionic species is followed over a very short interval of time.     

A Method for the Removal of Mineral Salts from Coconut Milk and a Comparison of Some of the Growth-Promoting Properties of Whole and Desalted Coconut Milk in Carrot Root Tissue Cultures

Various methods of separating the growth-promoting activityof coconut milk from its mineral salts constituents are examined.It is shown that coconut milk can be freed from mineral saltselectrolytically without serious impairment of its ability tostimulate growth in carrot root tissue cultures. Fresh weightand dry weight data, cell counts, and anatomical observationsshow that the growth of cultures in electrolytically desaltedcoconut milk is substantially the same as in whole coconut milk. The anatomical heterogeneity of carrot tissue cultures grownin both desalted and whole coconut milk is demonstrated.     

Investigations on the growth and metabolism of cultured explants of Daucus carota

Summary Knowing that the element Fe essentially triggers the action of the coconut milk stimulus for the growth of carrot explants and thereafter interacts with added Mo and Mn, the effects of these trace elements on growth (fresh and dry weight) and metabolism of both nitrogenous and nitrogen-free metabolites have been determined. An outstanding effect of Fe is to determine the level of protein synthesized, and the combination of Mo with Fe increases both protein and the content of non-protein nitrogen compounds. The role of Mn seems to reside in the mobilization of the soluble (non-protein) nitrogen compounds but its effects, which tend to compete with Fe in combination with Mo, tend to divert these compounds from protein synthesis. The element Fe appears again as a key element which determines the linkage between the use of carbon from carbohydrate and its entry into nitrogen metabolism: this has been shown by the use of ¹⁴C-fructose as the source. Whereas Fe promotes the use of ¹⁴C from fructose and directs it into protein, neither Mo nor Mn could achieve this either separately or in combination. The paper presents both the individual effects due to the trace elements and their interactions when supplied in combination. Stress is laid upon the need to consider the effects which are due to the inorganic elements in combination with the componenents of such growth-promoting systems as those present in coconut milk. These interactions are illustrated by polygonal diagrams (Figs. 3, 4 and 5). The point is made that any of several single entities of such an interacting complex may be in a given case rendered limiting, and the consequences of this fact for the concept of kinins or cell division factors are outlined. Any concept that requires cell division to be mediated solely by a given kind of chemical substance, or cell-division factor, would on the evidence here given present problems.The collaboration with K. H. Neumann was made possible by a German-Cornell Exchange Scholarship tenable at Cornell University and awarded by the Deutsche Akademische Austauschdienst, Bad Godesberg. The collaboration with K. V. N. Rao was made possible by the award of a Fulbright Travel grant and a Smith-Mundt stipend. In both cases (K. H. N. and K. V. N. R.) work continued after their scholarships expired under arrangements made possible by a grant to one of us (F. C. S.) from the National Institutes of Health, Bethesda, Md. K. H. Neumann was associated with the work throughout both at Cornell University and subsequently at the Justus Liebig Universität at Giessen (Institute für Pflanzenernährung); K. V. N. Rao was concerned with the work in its later stages at Cornell.     

Investigations on the growth and metabolism of cultured explants of Daucus carota

Summary The induction and maintenance of growth in small, standard explants of carrot root exposed to a trace-element-free basal nutrient medium (B ^**) have been investigated. The organic growth factors that induce the growth are complex as represented by a trace-element-limited coconut-milk preparation (CM^**) or the component parts of distinct growth promoting systems mediated by inositol or by 3-indoleacetic acid (IAA). In System I inositol interacts with growth factors from Aesculus, of which a known example is an IAA-rhamnoseglucose compound; in System II IAA interacts with adenyl compounds, of which zeatin is a known example. But the organic growth-promoting substances also interact with trace elements; therefore, the effects of the component parts of the growth promoting systems, separately and in combination, have been investigated with respect to their interactions with Mo, with Fe and also with these trace elements in combination. The growth so induced has been measured in terms of the fresh weight of the explants, the number and average size of their cells, as well as their total content of protein and nucleic acids. The metabolic responses of the cultured explants to the combined effects of growth factors and of trace elements are also described in terms of the principal soluble nitrogenous compounds. Each basis is informative, and suitable graphical devices are adopted so that the interactions of the multivariate factors that affect the behavior of the cultured tissue may be seen with respect to the various parameters selected. The study shows the range of complexity to be understood before the exogenous factors that determine cell growth and metabolism are both known and controllable; it also shows the limitations when attention is directed, simplistically, to one parameter or to the controlling influence of any single factor, or class, of growth factors.This investigation was supported by PHS Research Grant No. GM 09 609 to one of us (F. C. S.) from the National Institutes of Health, Bethesda, Maryland. The collaboration with Dr. K. V. N. Rao, later made possible by this grant, was first arranged under the terms of a Fulbright travel grant and the award of a Smith-Mundt stipend. Other assistance and facilities employed in the investigation depended upon research support made available through the Director of Research, New York State College of Agriculture.     

Investigations on the Growth and Metabolism of Plant Cells: V. Tumorous Growth in Relation to Growth Factors of the Type found in Coconut

1. It has been shown that aqueous extracts of plant tumours,induced by Agro-bacterium tumefaciens (Smith and Townsend) onBryophyllum and Kalanchoe, will act in place of coconut milkusing the tissue-culture procedures previously described inthis series of papers. 2. In a large number of experiments it has been shown that tumoursof this kind yield extracts which have a growth-promoting effectsimilar to that of coconut milk. This effect may be enhancedby, though it is distinguishable from the effects of, addedcasein- hydrolysate in the basal medium. The activity of thetumour extracts was consistently greater than the activity ofextracts of stems and leaves of the same plants and of normal,non-tumorous plants. 3. Partial fractionation of the tumour extracts has shown thatactivity was concentrated in the alcohol extracts, and to alesser extent in the water extracts. Activity was completelylacking in the ether extracts. 4. The effect of coconut milk, which is replaceable wholly orin part by tumour extracts, is primarily an effect on cell divisionin the carrot tissue. 5. The bearing of these results on tumorization in plants isdiscussed.     

The Localization in Cultured Carrot Cells of Factors that Induce their Growth

Various previously recognized parts of the complex of growthfactors present in the liquid endosperm of the coconut or inimmature fruits of Aesculus woerlitzensis were generally tritiated.The labeled growth factors were applied singly to culture mediawhich contained balanced requirements that had caused carrotexplants to proliferate and grow in accordance with combinationsof growth factors supplied. By the usc of electron microscopyand autoradiography, the radioactivity from each source wasdetected in the cells and its density and distribution, in theform of developed grains over different cellular compartmentsand organelles, was determined. The tabulated data relate tofour labeled sources as observed over seven cellular compartmentsunder six experimental treatments. Electron micrographs alsoshow how the radioactivity from the various sources relatedto organization of the cells. The distribution of radioactivity within the cells varied withthe source. Both ³H-myo-inositol and the tritiated growth factorsfrom Aesculus (³H-AF_1Aesc) with which it interacts (as in so-calledGrowth Promoting System I) contributed radioactivity, preferentially,to cell walls and sites of their formation in culturcd carrotcells. Both ³H-IAA and ³H-zatin (as in so-called Growth PromotingSystem II) contributed their radioactivity preferentially tothe nucleoli of the cultured cells. Some other conspicuous distributionsof radioactivity (e.g. from ³H-AF_1Aesc to plastids and from³H-IAA to the interstitial substance, i.e. middle lamella, whereenlarging cells separate) involved these tritiated moietieswithout regard to their counterparts in Growth Promoting SystemsI and II, respectively. The problems raised by such multiple effects due to differentgrowth factors acting singly and in combinations at differentcell sites are both recognized and discussed. growth factors, Aesculus woerlitzeensis, autoradiography, tritiation, cell sites, carrot, Daucus carota, coconut, electron microscopy     

MORPHOGENETIC STUDIES ON HAWORTHIA: ESTABLISHMENT OF TISSUE CULTURE AND CONTROL OF DIFFERENTIATION

Investigations were carried out on the in vitro morphogenetic responses of inflorescence segments and gynoecia of several species of Haworthia (Liliaceae). Morphogenetic responses of explants were not species specific. It was found that coconut milk was essential for the growth and differentiation of Haworthia tissue if White's basal medium was used. However, growth and differentiation could be supported by a modified Murashige and Skoog's medium, without any supplements. The investigations demonstrated’ the importance of inositol and ammonium nitrogen in the nutrition of Haworthia tissue cultures. A chemical control of callusing and shoot and root differentiation was obtained by providing appropriate amounts of auxin and cytokinin in the culture medium.     

Ultrastructural and biochemical development of the photosynthetic apparatus during callus induction in carrot root explants

During the first 8–12 days of cultivation in a nutrient solution containing IAA, inositol and kinetin freshly isolated carrot root explants develop into a chlorophyllous and photosynthetically active tissue culture. Electron microscopy, low temperature pigment absorption spectra and fluorescence induction profile recording as well as the determination of the activity of some enzymes (ribulosebisphosphate carboxylase, phosphoenolpyruvate carboxylase) and CO₂-fixation experiments were carried out. Based on the results, a sequence of developmental stages of the photosynthetic system will be proposed. During the autotropic period from about the 20th day of culture onward the light reaction system of these tissue cultures is quite comparable to that of carrot leaves; however, some differences in the CO₂-fixation mechanism were observed.     

Rapid multiplication of Sapium sebiferum Roxb. by tissue culture

Multiple shoots formation and elongation was induced from stem explants of Sapium seedlings on media containing cytokinins. Leaf explants produced callus on a medium containing cytokinins, auxin, casein hydrolysate and coconut milk, which could be induced to form multiple shoots on transfer to a medium lacking casein hydrolysate, coconut milk and auxin. Rooting of isolated shoots by treatment with an auxin mixture (indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid) and transfer of the plantlets to field have also been successful.     

Investigations on Growth and Metabolism of Plant Cells: VII. Sources of Nitrogen for Tissue Cultures under Optimal Conditions for Their Growth

Tissue cultures from explants of carrot root and potato tuber,stimulated into rapid growth by the addition of coconut milkorAesculus liquid endosperm to the medium, become to a certaindegree heterotrophic for nitrogen. Maximum growth-rates areattained only when nitrate is supplemented with various reducednitrogen compounds. The effects of casein hydrolysate, amino-acidmixtures, ammonia, tryptophan, urea, and allantoin have beeninvestigated, and their possible biochemical roles are discussed.     

Adventive plants from ovules and nucelli in Citrus

Summary 1- to 8-week-old ovules and nucelli from three Citrus cultivars—Shamouti and Valencia (Citrus sinensis) oranges and Marsh Seedless (C. paradisi) grapefruit—were cultured in vitro. No embryo differentiation was observed in the explants prior to culture. The Shamouti ovules had degenerated and were apparently unfertilized. Embryoids formed on Murashige and Tucker nutrient medium supplemented with 500 mg/l malt extract. Whole plants developed on the same basal medium supplemented with kinetin and indole-3-acetic acid (IAA), coconut milk or gibberellic acid (GA₃). A higher kinetin/IAA ratio or the addition of coconut milk favoured stem elongation more than root formation while a lower kinetin/IAA ratio favoured root formation and inhibited stem elongation. The addition of GA₃ to the basal medium stimulated rooting and stem elongation. These results can be of aid in mutation research, allowing irradiation at stages prior to embryonic development.     

Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.)

A new culture medium, originally designed and shown to grow cell suspensions from a variety of loblolly pine (Pinus taeda L.) explants, was used to study growth and somatic embryogenesis of the wild carrot (Daucus carota L.) in cell suspensions. The new loblolly pine medium (LM) differed from the standard wild carrot medium (WCM) in having very low Ca²⁺, very high Mg²⁺, and enrichment with PO _inf4 ^sup3– and microelements. When WCM was altered to contain levels of Ca²⁺ or Ca²⁺ and Mg²⁺ equivalent to LM, it supported neither growth nor embryogenesis of the wild carrot. However, growth and embryogenesis in LM was superior to WCM. The phosphate level in WCM was found to be suboptimal.     

VARIATIONS WITHIN SINGLE CELL CLONES OF TOBACCO TISSUE CULTURES

Comparative studies were made utilizing two series of secondary clones (single cell clones derived from single cell clones H 196 and H 241) of hybrid tobacco (Nicotiana tabacum ♂ × Nicotiana glutinosa ♀ ) tissue grown in vitro. Secondary clones derived from a single parent varied in color, consistency, the ability to grow, and rate of growth with various carbohydrates and growth-promoting substances. The growth of the secondary clones generally resembled that of the parent clone from which derived. Many of the 23 secondary clones of H 196 grew satisfactorily on media supplemented with sucrose, dextrose, levulose, or maltose; lactose, galactose, and xylose were unsatisfactory supplements. Similarly, the series of 30 secondary clones isolated from H 241 grew well on some media but poorly on others. Growth generally decreased when α-napthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was omitted from the basal coconut milk medium. Growth decreased considerably when coconut milk was omitted from the basal medium. The optimum sugar concentration was 1/2 to 1 per cent.     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

Morphogenesis in Stem-callus Cultures of Convolvulus arvensis L

Convolvulus arvensis stem explants form callus when inoculatedon to 0.5 mgm/1 2, 4-D, 15 per cent coconut milk, and on to1 mgm/1 kinetin, 15 per cent coconut milk. In the latter casenumerous shoots are formed on the callus. Callus formed on explantson 0.05 mgm/1 2, 4-D, 15 per cent coconut milk formed shootswith in-creasing vigour when transferred to a medium (1) inwhich the 2, 4–D concentration is lowered by one-tenth,(2) which contains no auxin but kinetin at 1 mgm/1, (3) whichcontains kinetin at 1 mgm/1 and 15 per cent coconut milk. OnNAA/ kinetin media shoots may form in which the intenode developmentis suppressed, giving compressed shoots. The capacity of callusto form shoots may be retained or lost through repeated sub-culture.Roots are formed erratically. The results are discussed, particularlywith regard to the loss of morphogenetic potential and whetherthis is reversible.     

Embryoids derived from isolated protoplasts of carrot

Summary Protoplasts isolated enzymatically from carrot root tissues developed into cell clusters in a liquid medium containing coconut milk and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Cells of the resulting calluses differentiated into embryoids on an agar medium containing coconut milk or kinetin.     

Partial purification of a legume nodulation factor present in coconut water

The nodulation of adventitious roots growing from segments of bean hypocotyl tissue was used as a bioassay for the material present in coconut water which stimulated nodulation. The active material in coconut water is acidic, but it was not possible to extract it from an acid solution with organic solvents. A purification of approximately 70-fold (on a dry wt basis) was obtained using activated charcoal, but at least 10 different compounds were present in the active fractions. A purified fraction of coconut water, which is stimulatory to the growth of carrot root explants, was active in the nodulation assay at a concentration of 2 μg/ml. This represents a 4000-fold purification of the diffusible fraction of coconut water. The charcoal fractionation procedure can be applied to the active material present in extracts of bean leaves.     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Callus Formation, Plant Regeneration and Clonal Propagation in Vitro of Gynura aurantiaca (Blume) DC.

Methods for culture in vitro of Gynura aurantiaca (Blume) DC.are described. Callus formation from gynura explants was initiallydifficult in spite of the multiple combinations of plant growthregulators assayed. In order to induce high growth rate, incorporationof several organic addenda to a basal medium was necessary,the best of these being coconut milk. Unorganized cell linescould be obtained by subculturing on C medium supplemented with10% coconut milk, and transfers had to be done at short intervals. A morphogenetic cell line without apparent loss in its organogenicpotential through more than 8 months of subculturing was establishedfollowing a sequence of culture media. Methods for rapid and efficient clonal propagation in vitroof this plant species are also reported. The possible utilization of these tissue culture techniquesdeveloped for gynura, a suitable indicator host for Citrus ExocortisViroid, in further studies concerning pathogenesis and replicationof this viroid is discussed. (Received April 22, 1985; Accepted October 11, 1985)