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.     

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

Summary The responses of carrot explants to various growth-promoting agents and to certain trace elements with which they interact have been investigated. A great range in the metabolic behavior of the tissue may be brought about in this way. The responses to the exogenously applied substances are described in terms of the growth of the carrot explants in fresh weight and number of cells and also in terms of their metabolism, as shown by the final content and composition of the non-protein N compounds, by the relations between protein and non-protein (alcohol-soluble) N and by the content of nucleic acid in the cultured tissue.The growth-promoting agents employed consisted of (1) the balanced complex of factors found in coconut milk, (2) an active isolate from Aesculus (AF_aesc), which is one of a class of growth factors (AF₁) that interact with inositol (AF₁+inositol) and which in this sense comprise growth-promoting System I, (3) the substance zeatin (Zeat) which is typical of a class of active factors (AF₂) that interact with indoleacetic acid (AF₂+IAA) and which, therefore, function as a growth promoting complex termed System II in the culture of carrot tissue.The carrot explants stimulated by coconut milk grew better than those stimulated by the other combinations of growth factors and they converted their soluble N more effectively to protein. The growth, whether it was induced by coconut milk or by System I or II, and other specific effects attributable to the growth factors employed were markedly affected also by the elements iron and molybdenum.The carrot explants that had responded to coconut milk emphasized alanine in their soluble, non-protein, nitrogenous pool, whereas those subjected to the active components of System I or of System II as clearly emphasized glutamine as the prominent non-protein, nitrogen-rich compound.The partial effects due to the component parts of System I (AF_aesc or inositol) and to the component parts of System II (Zeat. or IAA), as these interacted also with iron and molybdenum in an otherwise trace element free basal medium (B ^**), revealed a pattern of interlocking effects, due to trace elements and to growth factors, upon the metabolism (especially the nitrogen metabolism) of the aseptically cultured carrot explants. These effects show that the individual growth factors do not act alone and that their implications are far reaching. The interactions between growth promoting Systems I and II and their component parts, with each other, with various environmental factors, and especially with trace elements constitute a network, or a matrix, of parameters that will merit further investigation to reveal all that is required to control the growth and metabolism of carrot cells.This investigation was supported by PHS Research Grant No. GM 09609 to one of us (F.C.S.) from the National Institutes of Health.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.     

Coconut and Salmonella Infection

Raw, unprocessed coconut supports the growth of salmonellae as well as that of other enteric bacteria, salmonellae being particularly resistant to subsequent desiccation. Original contamination is not due to carriers or to polluted water supplies, but to contact with bacteria-containing soils followed by dispersion via infected coconut milk and shells. Pasteurization of raw coconut meat in a water bath at 80 C for 8 to 10 min effectively killed such bacteria, did not injure the product, and provided a prophylactic method now widely used by the coconut industry.     

Experimental Studies of Young Ginkgo Embryos—the Effect of Coconut Milk on the Embryos Cultured in Vitro

The present investigation deals with the effect of coconut milk upon the growth of in vitro culture of young Ginkgo embryos. The basic medium consists of White mineral salts in which Fe-citrate was used instead of Fe2(SO4)3 and vitamins (B1, 0.5 ppm, B6, 0.5 ppm, Ca-pantothenate, 0.5 ppm, niacin, 1 ppm and glycine, 2.5 ppm). 8% sucrose was used for younger embryos as the carbon source and 5% for the larger ones. The pH value of the medium was adjusted to about 6. 0.7% agar was used. The cultures were kept in an incubator (about 20–23 ℃) and no artificial light was used. The coconut milk, both filtered and autoclaved, was tested for its effect on the growth and structure of the young embryos. The important results obtained are summarized as follows: 1. The coconut milk, used both filtered and autoclaved, greatly promoted the growth and differentiation of the Ginkgo embryos cultured in vitro. 2. The optimum concentration of the coconut milk is 10%–20% for the younger embryos (900–1,600μ), while the higher concentrations (30% and over) may induce callus formation. 3. For the larger embryos the coconut milk was less effective, no callus formation occurred for the embryos over 2,800μ at isolation and for them 40% coconut milk was found more effective than 10% coconut milk. 4. There was no significant difference of the effect between the filtered and the autoclaved coconut milk. 5. From the experimental data obtained the authors conclude that the coconut milk at adequate concentration greatly promotes the rate of cell division and may initiate the meristematic regions around the shoot apex and over the whole surface of the cotyledons of the treated embryos.     

The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

Identification of Cell Division Inducing Compounds from Coconut Milk

The use of chromatographic techniques coupled with mass spectrometry, infra-red analysis and nuclear magnetic resonance revealed that the amino acid phenylalanine might be responsible for a large proportion of the cell division activity of coconut milk as determined by the soybean bioassay. The observation that zeatin can be removed very readily by partitioning against ethyl acetate at alkaline pH values, cautions against inclusion of this method as a purification step for extracting cytokinins from plant extracts. Mass spectrometry revealed the presence of an unidentified cell division substance with a molecular ion of 279 and which co-chromatographed with zeatinriboside on Sephadex LH-20.     

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.     

Purification and Partial Characterization of Polysaccharides From Coconut Milk

Ethanol-precipitated polysaccharides of the liquid endospermof coconut, Cocos nucifera L., were composed predominantly ofgalactose and arabinose with minor amounts of mannose and glucose.Gel filtration chromatography on Bio-Gel A-0.5 m revealed asingle major peak (Peak A) at the void volume and a minor peak(Peak B) partially included in the column volume. Peak A containedsome uronosyl residues, but was not susceptible to cleavageby endopolygalacturonase, indicating that it does not containsignificant amounts of polygalacturonic acid. Neutral glycosylresidue composition analysis of Peak A showed that it consistedof 72% galactose and 24% arabinose with minor amounts of glucoseand rhamnose. Coconut milk, Cocos nuciferaL, polysaccharides, glycosyl composition     

Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.     

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.     

Studies of the Utilization of Coconut Water Waste for the Production of the Food Yeast Saccharomyces fragilis

The accepted food yeast Saccharomyces fragilis was grown in batch and chemostat culture on coconut water and on a simulated coconut-water medium containing glucose, fructose, sucrose and sorbitol, to provide kinetic data for a feasibility study of microbial protein production. Analyses of growth on individual and mixed carbon substrates were made to determine sugar assimilation patterns in batch and chemostat cultures on coconut water. Growth on the polyol produced a much reduced specific growth rate, assimilation rate, growth yield and productivity compared to growth on the sugars. In mixed substrate fermentations a sequential utilization of the carbohydrates occurred. Both the monosaccharides repressed invertase synthesis and all three sugars repressed sorbitol assimilation. Complete carbon assimilation was only obtained by prolonged batch fermentation or in chemostat cultures at low dilution rates (<0.10 h^-1). Supplementation of coconut water with biotin and nicotinic acid increased biomass yields in chemostat cultures.     

GROWTH OF EXCISED PINE EMBRYOS AND THE ROLE OF THE COTYLEDONS DURING GERMINATION IN VITRO

A study was made of the role of the cotyledons, embryo orientation, surgical treatment, darkness, light, and autoclaved coconut milk on the growth of Pinus lambertiana Dougl. embryos in vitro. The embryos did not require an haustorial function of the cotyledons in vitro. Removal of the shoot meristem drastically altered the developmental physiology of the embryo and the function of the root meristem was severly inhibited. Positional effects on embryo growth were reversed by darkness. In the light vertical-inverted tube cultures displayed better growth than horizontal-inverted tube cultures, whereas in the dark the horizontal-inverted tube cultures displayed better growth than the ver ical-inverted tube cultures. Autoclaved coconut milk had no statistically demonstrable effect on embryo growth as measun d by the analysis of variance and Student-Newman-Keul's range test; however, the graphical analysis suggests that, in conjunction with the presence of the shoot meristem, there may be a slight beneficial response to autoclaved coconut milk.     

EFFECT OF INITIAL SIZE ON GROWTH OF PLANT TISSUE CULTURES

Caplin , Samuel M. (LOS Angeles State Coll., Los Angeles, Calif.) Effect of initial size on growth of plant tissue cultures . Amer. Jour. Bot. 50(1): 91–94. Illus. 1963.—Using different slice thicknesses and cannula diameters, cylindrical expiants of different size were obtained from tubers of Jerusalem artichoke. Slice thickness ranged from 0.5 to 2.5 mm and expiant diameter from 1.30 to 3.22 mm. Initial fresh weights ranged from 2.3 to 29.8 mg, a 13-fold range. After 17 days growth at 26 C in 10 ml basal medium containing 15% coconut milk, relative increase was inversely related to initial weight and ranged from 36-fold for 2.3-mg expiants to 8-fold for 29.8-mg expiants, a range of 4.5 × in relative increase between the largest and smallest cultures. Secondary phloem expiants of the same diameter from carrot root slices of different thickness, all removed at 1 mm from the cambium, also showed decreased relative growth with increasing size of explant; this was true for each of the different diameters used. Further investigation showed clearly that the growth of the large inocula was not limited by the amount of culture medium used during the period of culture. Initial weight has, therefore, a decided effect on the rate of relative growth.     

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.     

Activation of Dry Starter Cultures in Milk

The revitalization of mixed strain dried starter cultures at 22 and 32 C in sterile skim milk was materially accelerated when the substrate was fortified with 0.2% pancreas-extract solids. At 22 C, all cultures grew up satisfactorily in 18 hr, and in unfortified milk none of the cultures reached comparable growth in this period. When the cultures were grown at 32 C, the dried cultures developed adequately in 7.5 hr, but required 9 to 10 hr in plain milk. Culture growth was enhanced in milk containing pancreas extract to the extent that the amount of dried culture required to produce adequate acidity in normal incubation times could be markedly reduced. At 32 C, certain cultures could be reduced to 12.5% of recommended amounts, and at 22 C certain ones could be reduced by 50%. Revitalization of the dried cultures in milk containing pancreas extract did not affect the growth of subcultures in plain milk. Also, when dried cultures initiated growth in fortified milk at 32 C their subsequent growth at 22 C in milk alone was not affected. The faster rate of culture growth in milk containing pancreas extract should permit, with more certainty, the establishment of active mother and bulk starters. Furthermore, economy of dried cultures, as well as of time, could be realized by the use of fortified milk.     

Coconut products alleviate hyperglycaemic,hyperlipidimic and nephropathy indices in streptozotocin-induced diabetic wistar rats

Type 2 diabetes mellitus (T2DM) is a chronic and one of the most common metabolic diseases affecting large proportion of world population. Diabetes-induced changes in lipid and renal parameters are major risk factors contributing to diabetic complications such as diabetic nephropathy and cardiovascular diseases. Due to adverse effects associated with pharmacological intervention in the T2DM treatment, there is an increased interest in the research focussing on identifying novel plant based therapeutic agents. Here we report the effects of various coconut products on diabetic, lipid and renal parameters in streptozotocin (STZ)-induced diabetic rat model. Diabetic rats demonstrated a significant increase in serum glucose, and glycated haemoglobin levels (HbA1c). Lipid parameters including triglycerides, total cholesterol, low density lipoprotein cholesterol (LDL-cholesterol) and very low density lipoprotein cholesterol (VLDL-cholesterol) were found to be significantly increased, while high density lipoprotein cholesterol (HDL-cholesterol) was significantly declined in diabetic rats. Diabetic rats also displayed increased serum and kidney creatinine, urea, and total protein levels and increased urine glucose, urea, albumin and creatinine levels. Contrastingly, treatment with virgin and filtered coconut oils, coconut water and coconut milk resulted in a significant reversal in the levels of above studied parameters in diabetic rats. Further, these coconut products markedly prevented diabetes induced histopathological changes in kidney tissue. Collectively, the data demonstrate the antidiabetic, hypolipidemic and renal protective properties of various coconut products and underscore the importance of regular consumption of plant based medicinal products in the treatment of T2DM and its complications.     

Coconut as a Medium for the Experimental Production of Aflatoxin

Fresh, grated coconut has been found to be an excellent medium for aflatoxin production by Aspergillus flavus. Under optimal conditions, yields of 8 mg of total aflatoxin per g of substrate were obtained. Continuous agitation of the growth medium under moist conditions at 24 C produced highest yields. Aflatoxin was assayed both biologically and chromatographically. The aflatoxin content of cultures varied biphasically with the duration of incubation. It is suggested that this pattern could result from the sequential operation of factors promoting aflatoxin formation on the one hand and a detoxifying mechanism on the other.     

Thermal Resistance of Staphylococcus aureus in Milk, Whey, and Phosphate Buffer

The thermal resistance of four strains of coagulase-positive Staphylococcus aureus was determined in phosphate buffer, whole milk, skim milk, and Cheddar cheese whey. The logarithmic order of death prevailed until about 99.99 to 99.999% of the organisms were destroyed, after which there was a decline in the rate of destruction. The organisms were more resistant in skim milk and Cheddar cheese whey than in phosphate buffer and whole milk. Thermal resistance varied among strains of S. aureus but was consistent with individual strains. As the age of cultures of strain B-120 increased from 12 to 228 hr, the D(55) values increased from 0.95 to 3.0. The thermal resistance of cultures obtained from survivors to partial thermal destruction was similar to that of the parent cultures.     

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.     

Milk intake and bone mineral acquisition in adolescent girls: randomised,controlled intervention trial.

OBJECTIVES: To investigate the effect of milk supplementation on total body bone mineral acquisition in adolescent girls. DESIGN: 18 month, open randomised intervention trial. SUBJECTS: 82 white girls aged 12.2 (SD 0.3) years, recruited from four secondary schools in Sheffield. INTERVENTION: 568 ml (one pint) of whole or reduced fat milk per day for 18 months. MAIN OUTCOME MEASURES: Total body bone mineral content and bone mineral density measured by dual energy x ray absorptiometry. Outcome measures to evaluate mechanism included biochemical markers of bone turnover (osteocalcin, bone alkaline phosphatase, deoxypyridinoline, N-telopeptide of type I collagen), and hormones important to skeletal growth (parathyroid hormone, oestradiol, insulin-like growth factor I). RESULTS: 80 subjects completed the trial. Daily milk intake at baseline averaged 150 ml in both groups. The intervention group consumed, on average, an additional 300 ml a day throughout the trial. Compared with the control group, the intervention group had greater increases of bone mineral density (9.6% v 8.5%, P = 0.017; repeated measures analysis of variance) and bone mineral content (27.0% v 24.1%, P = 0.009). No significant differences in increments in height, weight, lean body mass, and fat mass were observed between the groups. Bone turnover was not affected by milk supplementation. Serum concentrations of insulin-like growth factor I increased in the milk group compared with the control group (35% v 25%, P = 0.02). CONCLUSION: Increased milk consumption significantly enhances bone mineral acquisition in adolescent girls and could favourably modify attainment of peak bone mass.