Investigations on Growth and Metabolism of Plant Cells: IV. Evidence on the Role of the Coconut-Milk Factor in Development

By the carrot-assay method it has been shown that the wateryendosperm of coconut contains the growth-promoting coconut-milkfactor at all stages of development. Some activity is shownby the parts of the immature embryo but not by the solid endosperm. Sources of analogous activity are in the endosperm of Zea maysin the milk stage, the gelatinous content of immature fruitsof Fuglans regia, and the young gametophyte of Ginkgo biloba.The data for other cases examined suggest that the materialdevelops best in nutritive tissues associated with delayed embryodevelopment.     

The Effect of the Female Gametophyte on the Growth of Cultured Douglas-fir Embryos

Several clones of Douglas-fir, Pseudotsuga menziesii (Mirb.)Franco, embryos grown in aseptic culture in the presence orabsence of the female gametophyte or a crushed aqueous extractof it, have shown striking differences in overall length, sizeand substance(s) responsible for this colour of the cotyledonsand in root length. The growth-promoting effect is diffusiblein agar, heat-stable and has detectable activity at a concentrationof about 0·1 to 1·0 per cent (v/v) in a basalnutrient medium. Evidence is provided that extracts of gametophytemay have synergistic effects with coconut water in promotingmorphogenesis of Douglas-fir cells in suspension culture. Pseudotsuga menziesii, Douglas-fir, female gametophyte, einbryo culture     

In vitro Growth of Vegetative Tomato Shoot Apices

Explants from the shoot apex of the tomato, comprising the apicaldome and youngest primordium together with small amounts ofsub-apical tissue were cultured for periods of 1 to 4 plastochrons.By the use of a simple parameter, the axillary distance, thegrowth-rate could be accurately monitored throughout each plastochron. Gibberellic acid, coconut milk, and kinetin, in addition tosucrose and inorganic salts, all promoted growth of the apex;a combination of gibberellic acid and coconut milk gave thefastest growth. Temperature had a large effect on the growth-ratewith an in vitro Q₁₀ of 2.1 contrasted with an in vivo Q₁₀ of1.2 over the range of 15 to 25 ?C. On gibberellic acid and coconutmilk at 15 ?C two-thirds of the in vivo growth--rate was sustainedin culture for two plastochrons after which the growth-rategradually declined; at 20 and 25 ?C growth-rates slightly higherthan in vivo rates were sustained for 1 plastochron before amore rapid decline. The anatomy of these in vitro apices wasnormal for 1? plastochrons after which there were small increasesin cell volume in the developing primordium. Reducing the amount of sub-apical tissue drastically reducedthe growth rate but had little effect on the responses to gibberellicacid and coconut milk. Explants are considered to be useful material for studying thechanges that take place in the apex during the course of 1 or2 plastochrons, but inadequate on the media tested for experimentsinvolving longer periods of growth. Explants also provide asensitive assay system for the effects of growth factors onthe rate of shoot apical growth.     

An Investigation of Factors which Influence the Production of Abnormal Terpenoids by Callus Cultures of Andrographis paniculata Nees

Callus and suspension cultures of Andrographis paniculata havebeen shown to produce three new sesquiterpene lactones whichhave been named paniculides. A, B, and C and not the andrographolides(diterpenes) produced by the intact plant. Cultures derived from leaves, stems, hypocotyls, roots, andembryos accumulated paniculides, but no andrographolides weredetected. Conversely andrographolides, but no paniculides weredetected in extracts from leaves, stems and roots of intactseedlings. Modifications of the medium including the use of different syntheticauxins and the replacement of coconut milk with kinetin andcasein hydrolysate did not influence paniculide production bythe hypocotyl cultures. In addition an established root isolatecultured for 9 months on a simple synthetic medium without auxin,meso-inositol or coconut milk still synthesized significantquantities of paniculides. Diurnal fluctuations of temperature and light did not influencethe pattern of compounds produced although cultures grown incomplete darkness produced the individual paniculides in differentproportions. Possible explanations for these findings are discussed.     

Nitrate Reduction in the First Leaf and Roots of Barley Seedlings Grown in Sand and in Culture Solution

The in vivo method has been used to determine activity of nitratereductase in Hiproly and Proctor barley. Differences in activitybetween the cultivars were small and less than those due togrowing conditions. Activity in plants grown in culture solutionwas greater than that for sand-grown plants, especially in theroot The in vivo method gave values for nitrate reductase activitywhich are less than those found by the in vitro method, andevidence is presented to show that the in vivo method underestimatesthe rate of formation of organic nitrogen in barley seedlings.It is shown that significant nitrate reductase activity occursin roots but it is nevertheless concluded that the main siteof nitrate assimilation is in the leaves of this material.     

Flowering in Pisum: the Effect of Age on the Gene Sn and the Site of Action of Gene Hr

Late cultivars of peas behave as quantitative long day plants.The reason that they flower between nodes 20 and 35 under an8 h photoperiod is shown to be because the leaves and maturestem produce a more promotory ratio of the flowering hormonesas they age. Later formed leaves may also start with a slightlymore promotory ratio than the leaves produced at a lower node.The gene Sn controls the production of a flower inhibitor andit is suggested that the activity of this gene in a leaf isgradually reduced as the leaf ages. From grafting experiments,the site of action of the gene Hr is shown to be in the leavesor mature stem and not at the shoot apex. This supports a previoussuggestion that the gene Hr is a specific inhibitor of the ageingresponse of gene Sn. Gene Hr is shown to cause a substantial delay in the floweringnode of decotyledonized plants of genotype If e sn hr undershort day conditions, suggesting that Hr has little effect inthe cotyledons. It is argued that the gene sn is a leaky mutantand that gene Hr does not control a photoperiod response inits own right but has its effect through the Sn locus. From a comparison of intact plants and self-grafts of the lategenotype If e Sn hr it is shown that under the conditions usedphysiological age may be of more importance than chronologicalage in determining flowering in peas. Reasons for the smalleffect of defoliation treatments on flowering are discussedas well as possible reasons for the promotory effect of decotyledonizationon the flowering node of late lines. Pisum sativum L, flowering, ageing, genetic control     

Promotion and Inhibition of Inflorescence Growth by Long Days in Short-day Plants

Results of previous investigators have indicated that long periodsof light intercalated between inductive short-day cycles havean inhibitory effect on inflorescence growth in short-day plants.The present experiments show that such light periods can eitherpromote or inhibit inflorescence growth in Xanthium pemtsylvanicumand Chenopodium amaranticolor depending on their previous degreeof induction. Intercalated light exerts an inhibitory influence on the inductiveprocesses occurring during the dark period which follows itwhen unifoliate Xanthium plants have been previously exposedto not more than one short day and when fully foliated Chenopodiumplants have been previously exposed to not more than one ortwo short days. When plants are more strongly induced initially,an intercalated light period has a very marked promoting effecton the dark period succeeding it. In Xanthium this stimulatoryeffect increases with the duration of the light period up toan optimum of approximately 80 hours. It is suggested on the basis of available evidence that thepromotive effect of such intercalated light possibly affectsthe sensitivity of the apex to inductive stimuli and that itsinhibitory effect acts on the inductive processes occurringin the leaves.     

The Anther Smut of Sea Campion: A Study of the Role of Growth Regulators in the Dwarfing Symptom

The sympton of dwarfing in the sea campion, Silene vulgaris(Moench) Garcke sub sp. maritima(With.) A. & D. Löveinfected with the anther smut fungus Ustilago violacea (Pers.)Fuckel, a systemic, perennial parasite, has been investigated. Extracts of both healthy and diseased plants contain IAA andGA₃, but diseased plants contain less gibberellins than healthyplants. Neither IAA nor gibberellins were detected in significantquantities in the medium when U. violacea was grown in pureculture, but IAN was present. IAN was also found m extractsof diseased plants and it is tentatively suggested that it isformed by the fungus and may accumulate in the host owing tothe inability of the plant to convert IAN to IAA The exogenousapplication of IAN to healthy plants does not produce any diseasesymptoms. The dwarfing symptom of the diseased plant may be due to thelower levels of gibberellins which it contains compared withhealthy plants since the exogenous application of GA₃, restoresthe diseased plant to normal growth     

The Effect of (2-chloroethyl)-trimethylammonium Chloride on the Growth of Barley

Barley (Hordeum vulgare L. C.I.666) was shown to be susceptibleto the growth retardant (2-chloroethyl)-trimethylammonium chloride(CCC). The estimation of cell number in the dwarfed third leafblade indicated that a decrease in mitotic activity had occurredin treated plants. There was also a decrease in cell size intreated plants. The dwarfing action of CCC was reversed by exogenousgibberellic acid (GA₃) but this was shown to be the result ofincreased cell elongation only. GA₃ did not promote cell divisionin healthy or CCC-treated plants. Assay of endogenous gibberellinsshowed a significant reduction in the level of a substance correspondingto GA₃ in CCC-treated plants. It is suggested that CCC-induceddwarfing of barley is largely the result of a reduction in meristematicactivity. This may be related to an effect on gibberellin biosynthesisbut is not reversed by the application of exogenous GA ₃.     

Correlative Inhibition of Lateral Bud Growth in Pisum sativum L. and Phaseolus vulgaris L.: Studies of the Role of Abscisic Acid

The possibility has been investigated that abscisic acid (ABA)might act as a correlative inhibitor of lateral bud growth inPisum sativum and Phaseolus vulgaris. Application of ABA insmall quantities (2µg) to axillary buds on decapitatedplants of P. sativum caused appreciable inhibition of theirgrowth, and induced a compensatory growth of the bud on an adjacentnode. Application of this same quantity of ABA to axillary budson decapitated plants of Phaseolus vulgaris was without effect,but a high concentration in lanolin (1 mg g^–1) did substantiallyreduce bud outgrowth. Endogenous ABA-like substances in Phaseolusvulgaris, detected by bioassay and electron capture g.l.c.,were present in similar concentrations in shoot tips, lateralbuds on intact plants and lateral buds on plants decapitated24 h earlier. The effects of applied ABA suggested that it might be involvedin the mechanism of correlative inhibition in Pisum sativum,but it was not possible to test this hypothesis by determiningendogenous ABA levels in axillary buds because of their smallsize. The evidence presented here suggests that ABA is not acorrelative inhibitor in Phaseolus vulgaris even though at highconcentration it can inhibit the growth of axillary buds.     

Growth Substances in Relation the the Rooting of Salix fragilis Cuttings

An attempt has been made to correlate seasonal variations inthe rooting responses of Salix fragilis cuttings with theircontent of growth-promoting substances. Although they were bothlow in early dormancy and higher in late dormancy and in summerthere was not an exact correlation in all months. It was found that extracts of S. fragilis cuttings taken indormancy strongly inhibited the action of IAA on the growthof Avena coleoptile segments. This inhibitory influence wasless in extracts of cuttings taken at the end of dormancy andin summer. Extracts of cuttings which had been left in waterto root showed a striking decrease in their inhibitory actionon IAA.     

In Vitro Induction of Haploid, Diploid, and Triploid Androgenic Embryoids and Plantlets in Datura metal L.

Pollen plantlets of Datura metel L. have been successfully rearedin vitro through anther culture on a nutrient medium supplementedwith coconut milk (15 per cent v/v) The early segmentation patternof the proembryos and their subsequent differentiation intoheart, torpedo, and cotyledonary stages were traced. More than100 plantlets were grown to maturity on soil. They grew normallyand produced flowers. Root-tip and leaf-squash preparationsshowed variable numbers of chromosomes in their cells, and thisindicated the occurrence of ploidy plants among the population.Frequency determinations showed the predominant types to bediploid (70 per cent), triploid (24 per cent), and haploid (6per cent). Differences in the morphology of mature plants inregard to floral size, meiotic irregularities in PMCs, formationof giant pollen grains, differential fruitset, somatic mosaicismin respect of chromosome number, and aneuploidy of cells, wererecorded. The origin of diploid and triploid plants is discussed.     

Growth Hormone Changes in Chrysanthemum morifolium: Effects of Environmental Factors Controlling Flowering

Simultaneous quantitative analyses have been made of the endogenouslevels of auxin- and gibberellin like substances, growth inhibitors,and auxin-oxidizing enzyme activity in the cold-requiring Chrysanthemummorifolium cv. Sunbeam subjected to different daylength, lightintensity and temperature regimes known to affect flowering.While little hormone or enzyme activity was found in extractsfrom unvernalized plants, a striking rise in auxin-oxidizingenzyme activity occurred rapidly after the end of cold treatment.Increased auxin activity was also recorded shortly after vernalization.At 28 °C both enzyme and auxin activity declined over aperiod of 3–4 weeks; at 20 °C this response was delayed.Gibberellin activity at 28 °C rose steeply about 2 weeksfrom vernalization and declined several weeks later; at 20 °Ca similar response was less marked. Low light intensity treatment,which may have increased endogenous auxin levels, or exogenousauxin application reduced gibberellin-like substance levelsand cause d devernalization.Phosphon D treatment also loweredgibberellin levels and prevented flowering. An extract fromvernalized plants containing gibberellin-like substances intensifiedthe flowering of partially vernalized test plants. Persistenceof high auxin activity in vernalized plants on long days wasassociated with failure to form normal flower buds. Stem elongationrates correlated in general with levels of endogenous auxin-and gibberellin-like substances. Significant amounts of an abscisin-likeinhibitor were found in extracts of flower buds. The mechanismof natural devernalization is discussed in relation to theseobservations.     

In Vitro Culture of Orobanche aegyptiaca

Calli derived from seedlings of the parasitic plant Orobancheaegyptiaca were grown on enriched nutrient agar. Growth wasenhanced by the addition of 0.1 mg 1^–1 NAA and 5 % coconutmilk. The calli developed two kinds of elongated protrusions:thin ones which resemble the young root radicle and thick oneswhich resemble the adventitious roots of Orobanche plants. Bothformed pad-like structures when touching the substrate. In liquidculture, some of the calli were observed to surround isolatedcultured tomato roots. Orobanche aegyptiaca Pers., in vitro culture, germination, coconut milk, root parasite     

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.     

Kinetics Studies on the Photoinduction of Xanthium strumarium L. and Isolation of a Flower Primordia Growth-Regulating Fractionl

The kinetics of flower primordia formation have been re-examinedin photoinduced Xanthium strumarium L. to determine the numberof short-day treatments that promote optimum primordia initiationand development. Plants given 4 short-day cycles showed thehighest rate of primordia development. Attempts were made toisolate flower-promoting substance(s) from the young leavesof such optimally induced plants by methanol extraction followedby ether fractionation. A Xanthium bioassay was developed fortesting the flower-promoting activity of aqueous concentratesobtained from the acidic, neutral-basic and total ether extracts.No flower-primordia inducing activity was found in any of thefractions, but growth-accelerating activity on young primordiawas shown by substance(s) present in the combined neutral-basicfraction isolated from photoinduced leaves. ¹C.R.D.A. Contribution No. 170. (Received June 14, 1989; Accepted September 20, 1989)     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

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.     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Studies on the Properties of Carboxylating Enzymes in the Epidermis of Commelina communis

A spectrophotometric assay has been used to measure the activityof PEP carboxylase and RuBP carboxylase in the epidermal andmesophyll tissue of Commelina communis. On both a chlorophylland protein basis the PEP carboxylase activity was always greaterin the epidermis than in the mesophyll, whereas RuBP carboxylaseactivity was always highest in the mesophyll. PEP carboxylaseactivity in epidermal extracts was lost very slowly and itspH optimum was a broad one in the range 7·5–8·0.The K_m values for PEP carboxylase in the epidermis and mesophyllobtained from light- and dark-treated plants were not very differentalthough its V_max was much lower in dark-treated tissue. Thesedata are discussed in relation to the possible role of PEP carboxylasein guard cell metabolism.