Benzyladenine-Induced Increase in DNA Content per Cell, Chloroplast Size, and Chloroplast Number per Cell in Intact Bean Leaves

Primary leaves of intact bean plants (Phaseolus vulgaris L.)were treated with benzyladenine (BA) at different stages oftheir growth. BA induced a marked increase in DNA content percell in growing leaves where no cell division occurred. BA stimulatedthe chloroplast replication compared with untreated leaves.After the replication period, chloroplast size continued toincrease in BA- treated leaves, but not in untreated controls.     

Benzyladenine-Enhanced Cell Proliferation and -Suppressed Greening in Attached Young Bean Leaves

Changes in fresh weight, cell number, cell size and cell constituentswere studied during the growth of attached young bean (Phaseolusvulgaris L.) leaves after benzyladenine (BA) treatment. BA broughtabout the increase in DNA, RNA and protein contents and freshweight as well as the temporary inhibition of chlorophyll accumulationin growing leaves. After a lag period for about 12 hr, BA stimulatedcell division but not cell expansion. An increase in RNA contentoccurred during the lag period of BAinduced cell division growth. (Received November 5, 1980; Accepted March 10, 1981)     

Rapid Induction of Synthesis and Doubling of Nuclear DNA by Benzyladenine in Intact Bean Leaves

Primary leaves of young and old bean plants (Phaseolus vulgarisL.) were treated with benzyladenine (BA) after cell divisionhad been completed. Changes in the synthesis and amount of DNAin individual nuclei in mesophyll cells shortly after BA applicationwere studied. Cytofluorometric determination of nuclear DNAwith 4',6-diamidino-2-phenylindole (DAPI) showed that cellscontaining 4C nuclei had appeared in both young and old leavesby 24 h after BA application, while the nuclear DNA contentin control leaves remained at 2C. The number of 4C nuclei increaseduntil 48 h in young leaves, but not in old leaves. Autoradiographicanalysis showed that nuclei labelled with [6-³H]thymidine increasedover the control level 12 h after BA application. This effectpeaked at 24 h followed by a decline. There was no differencein the initial effect between young and old leaves, but theeffect diminished more rapidly in old leaves than in young ones.The results are discussed in relation to those obtained fromDNA measurements long after BA application in previous studies. ³ Present address: Bio-resources Technology Division, Forestryand Forest Products Research Institute, P. O. Box 16, TsukubaNorinkenkyu Danchi-nai, Ibaraki, 305 Japan (Received December 11, 1989; Accepted April 20, 1990)     

There is No Somatic Meiosis in Embryogenic Leaves of Cichorium

Several papers dealing with carrot cell cultures describe meiosis-likedivisions and haploid cells prior to somatic embryogenesis.We have studied the first division in embryogenic mesophyllcells of a diploidCichorium intybus L. and of a tetraploid hybridC.intybus L.xC. endivia L. which undergo direct somatic embryogenesisfrom single cells when leaf fragments are placed in a liquidagitated inductive medium (modified MS with 1x10^-7M NAA and2.5x10^-6M 2-iP), in darkness, at 35°C. MicrosporogenesisinC. intybus provided aspects of meiosis for comparison. Inleaves incubated in inductive conditions, DAPI staining of nucleishowed normal mitosis on days 3–6; about 0.6% cells inprophase had undergone spontaneous endoreduplication leadingto a tetraploid somatic embryo. Immunocytochemistry of tubulinrevealed the constant presence of a preprophase band, as ina normal mitosis. The first pluricellular somatic embryos becamevisible on day 5 of culture. Flow cytometric determination ofnuclear DNA on days 4, 5 and 6 did not show any peak correspondingto the 1C DNA level for the diploid plant or to the 2C DNA levelfor the tetraploid. Instead there was a weak but constant peakat the 4C and 8C levels. We conclude that inCichorium leaves,the first division of somatic embryogenesis is a normal mitosis,with a small shift to endoreduplication. In our opinion, somaticmeiosis is not a prerequisite during direct somatic embryogenesis. Cichorium ; chicory; somatic embryogenesis; cell division; flow cytometry; tubulin     

Increases in Nuclear DNA Content without Mitosis in Benzyladenine-treated Primary Leaves of Intact and Decapitated Bean Plants

Primary leaves of intact bean plants (Phaseolus vulgaris L.cv. Yamashiro-kurosando-saito) were treated with benzyladenine(BA) beginning on the seventh day after sowing when cell proliferationin the leaves had finished. Nuclear DNA contents were measuredby cytofluorometry combined with 4',6-diamidino-2-phenylindole(DAPI) staining. In the untreated controls, most mesophyll andabaxial epidermal cells contained a nucleus whose DNA contentwas 2C; whereas most adaxial epidermal cells contained a 4Cnucleus. Benzyladenine treatment induced 4C nuclec in mesophylland abaxial epidermal cells; but BA induced 8C nuclei in adaxialepidermal cells. To compare the effects of endogenous cytokininaccumulation, bean plants were decapitated above the primaryleaves on day 7 and continually disbudded thereafter. Changesin the nuclear DNA content in primary leaves attached to thedecapitated plants was similar to that for BA-treated primaryleaves. No multinucleate cells were formed and no mitotic figureswere present in the BA-treated leaves or in the primary leavesof decapitated plants. Our results indicate that both BA treatmentand decapitation induced one round of nuclear DNA synthesiswithout mitosis in a large number of mesophyll and epidermalcells.     

DNA Levels in Dividing and Developing Plastids in Expanding Primary Leaves of Avena sativa

The amounts of plastid DNA in the primary leaves of 4-d-oldlight- and dark-grown seedlings of Avena sativa were measuredby microspectrofluorometry using the DNA-fluorochrome DAPI (4',6-diamidino-2-phenylindole). In the light-grown primary leaves (40–45 mm long) therewas a marked increase in DNA level per plastid from 10.2 to18.5 ? 10^–15 g between 2.0 mm and 10 mm from the leafbase, resulting from the rate of plastid DNA synthesis beinghigher than the rate of plastid division. Beyond 30 mm the plastidDNA level was reduced to 14 ? 10^–15g due to chloroplastdivision rates being higher than the rate of plastid DNA synthesis,while from 20 mm plastid DNA levels were constant at 2.2 ? 10^–12g per cell, which corresponds to 16000 plastome copies per cell. Observations of dark-grown leaves establish that, in Avena,light is not necessary for plastid division and the dark-grownleaf cells accumulate higher amounts of plastid DNA than light-grownleaf cells. Plastid nucleoids showed a change of distribution after completionof plastid DNA synthesis in light-grown leaves. A change inthe distribution of plastid nucleoids was also observed duringthe greening of etioplasts of dark-grown leaves while plastidDNA level remained constant. Such changes in plastid nucleoiddistribution appear to be independent of plastid DNA synthesisand correlate with the formation of grana stacks. Key words: Avena sativa, microspectrofluorometry, plastid DNA     

Benzyladenine-induced increase in DNA content in the nucleus of bean leaf cells

Primary leaves of bean plants (Phaseolus vulgaris L.) were treated with benzyladenine (BA) from 7 days after sowing. Nuclei were isolated and the DNA content per nucleus was determined. BA increased the amount of DNA per nucleus of untreated controls by about 50% without nuclear division.     

Benzyladenine-induced increase in DNA content per chloroplast in intact bean leaves

Benzyladenine (BA) treatment was found to induce chloroplast DNA (ctDNA) synthesis after it had stopped in primary leaves of light-grown intact bean plants (Phaseolus vulgaris L.). The leaves were treated with BA from 7 days after sowing. Chloroplasts were isolated and the ctDNA content per chloroplast was determined. Chloroplast division occurred until 13 days after sowing in untreated leaves. BA stimulated the division keeping the level of ctDNA content per chloroplast the same as that in the untreated controls. After the division period, the ctDNA content per chloroplast increased in BA-treated leaves, but not in controls. Consequently, ctDNA per leaf (or per cell) increased immediately after the beginning of BA treatment, but remained constant in the control leaves.     

Variation in Concentration of Ribulose-1, 5-bisphosphate Carboxylase in Leaves of Barley, Wheat and Hybrids of Crested Wheatgrasses

Amounts of the enzyme ribulose-1,5-bisphosphate carboxylasewere estimated in seedling leaves of barley (Hordewn vulgareL.) and flag leaves of wheat (Triticum aestitum L.) by radialimmuno diffusion. A fourfold variation among barley varietiesfor amount of RuBPCase at the seedling stage was observed (c.3.5–15mg g^–1 fr. wt). Range in variation for amountof flag leaf RuBPCase among wheat varieties was 6-09-9.39 mgRuBPCase g^–1 fr. wt. F₁ hybrids from interspecific andintergeneric crosses of crested wheatgrasses (Agropyron andElymus spp.) and their amphidiploid analogues were comparedfor amount of RuBPCase in the most recent fully expanded leavesharvested before seed set. Amount of enzyme varied from 3.4to 77.6 mg g^–1 fr. wt among the hybrids. No effect chromosomenumber on enzyme concentration was observed among 13 hybridsand their amphidiploid counterparts. Key words: RuBPCase, wheatgrasses     

Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

The effect of benzyladenine (BA) on the diurnal changes in DNAand Chl contents per chloroplast and chloroplast replicationin primary leaves of bean plants (Phaseolus vulgaris L.) grownunder a 16 h light/8 h dark cycle was studied. Experiments weremade on primary leaves in the early expansion phase, where celldivision had been completed but chloroplasts were replicating.In untreated controls, chloroplast number, Chl content and freshweight per leaf showed daily periodic changes. Chl content perchloroplast increased in the light period every day, and freshweight per leaf increased most rapidly in the early dark period.Chloroplast number per leaf increased rapidly in the early darkperiod on day 9, though the increase began a little earlierand was less sharp on days 8 and 10. During these periods, DNAcontent per chloroplast was decreasing due to chloroplast divisionas chloroplast DNA (ctDNA) per leaf remained unchanged throughoutthe experimental period. BA induced increases in Chi contentper chloroplast, ctDNA content and fresh weight per leaf within6 h of its application, regardless of whether it was appliedat or 10 h after the beginning of the light period. Applicationof BA at 10 h in the light period shifted the start of chloroplastreplication by 6 h compared to that in untreated controls. However,when BA was applied at the beginning of illumination, the startof chloroplast replication showed the same relative change intime as above. 5-Fluorodeoxyuridine (5-FdU) promptly preventedBA-induced increase in Chl content and chloroplast number perleaf as well as ctDNA content per leaf.     

Photosynthetic Electron Transport in Plastids in Detached Cucumber Cotyledons Treated with 6-Benzylaminopurine and Potassium

Etiolated cotyledons of cucumber (Cucumis sativus L.) were excisedand incubated for 16 days on agar in the absence or in the presenceof 6-benzylaminopurine (BA) (3 µM) or KCl (10 mM) undercontinuous light. Treatment with BA or KCl increased growthand greening of cotyledons, KCl being particularly active duringthe last few days of the incubation. The rate of photosynthetic electron transport reached a maximumafter 3 days, as did the chlorophyll content, and then it declinedby approximately 95%. An uncoupling from phosphorylation wasobserved in older cotyledons. BA and KCl increased the rateof electron flow per cotyledon, and KCl delayed the onset ofthe decline in this rate. PS I and PS II activities per chlorophyllunit were lower in BA-treated cotyledons, but KCl-treated cotyledonsbehaved like the controls. Fluorescence analysis indicated that the level of active chlorophyllsinvolved in the transfer of energy in PS II decreased between3 and 7 days under all conditions tested. In BA-treated cotyledons,the proportion of active chlorophylls was consistently lowerthan that in the control. Thus, it appears that, in the absenceof exogenous K⁺, cytokinin has only a low antisenescence effecton photosynthetic activity, and that K⁺ may enhance the photosyntheticelectron flow between plastoquinone and plastocyanin via thecytochrome b₆-f complex. (Received August 2, 1989; Accepted January 23, 1990)     

Soyabean Stem In Vivo Nitrate Reductase Activity

Stem from three- and four-week-old Soyabean [Glycine max (L.)Merr. cv. Tracy] plants reduced from 0.3 to 0.7 µmol nitrateh^–l g^–l f. wt. Leaf activity was 4.7–7.6 µmolnitrate h^–l g^–l f. wt. Outer stem was two to fourtimes more active at reducing nitrate than was inner stem. Plantnitrate nutrition had a strong effect upon the ratio of activitypresent in stem and leaf. More nitrate increased the proportionpresent in leaves. Glycine max L., soyabean, nitrate assimilation, nitrogen metabolism, Rhizobium japonicum     

The Respiration of Mature Field Bean (Vicia faba L.) Leaves During Prolonged Darkness

Dark respiration in attached and detached mature leaves of thefield bean (Vicia faba L.) was studied whilst leaves experiencedup to 60 h of darkness. The results showed: (1) the initialrespiration rate to vary according to the irradiance duringthe previous photoperiod; (2) the dark respiration rate (perunit area) of attached leaves to be essentially constant duringa normal 12 h night although there was a rapid loss in leafd. wt during this time; (3) after 12 h, the respiration rateof attached leaves decayed to an asymptotic value at about 36h; (4) the respiration rate of leaves detached at the end ofthe photoperiod and maintained in the dark on deionised water,decayed only after 36 h of darkness; (5) there was no differencebetween the respiration rate of attached and detached leavesduring the normal 12 h night. It is concluded that the dark respiration of attached fieldbean leaves is intially related to the synthesis and translocationof sucrose in addition to maintenance. After about 36 h, whenthe rate of CO₂ efflux is more or less steady, the CO₂ effluxreflects the intensity of maintenance processes only. The maintenancerespiration rate (determined after 60 h in the dark) rangedfrom 062 to 151 mg CO₂ (g d. wt)^–1 h^–1 but wasrelatively unaffected by several applied treatments. Vicia faba L., field bean, respiration, maintenance, nitrate, non-structural carbohydrate, export     

The Effect of Benzyladenine on Changes in Nuclease and Protease Activities in Intact Bean Leaves during Ageing

Primary leaves of intact bean plants (Phaseolus vulgaris L.) were treated with benzyladenine (BA) at different stages during growth. Changes in DNase, RNase, and proteas activities in the leaves were followed. Unlike the case of various excised tissues, cytokinin raised the activities of these hydrolases in intact bean leaves. Because BA elevated the levels of DNA, RNA, and protein in intact leaves, it may stimulate both synthesis and decomposition of these cellular constituents. The hydrolase activities showed differential responses to BA according to the age at which the leaf received the hormone treatment.     

Effect of Benzyladenine on DNA, RNA, Protein, and Chlorophyll Contents in Intact Bean Leaves: Differential Responses to Benzyladenine According to Leaf Age

Benzyladenine (BA) was applied to intact bean (Phaseolus vulgaris) leaves at different stages of their growth. Changes in the amounts of cellular constituents resulting from the different treatments were followed and compared. RNA, protein, and chlorophyll contents, dry weight, fresh weight, and leaf area per single leaf continued increasing when leaves were treated with BA from an early stage, whereas in untreated leaves all these values levelled off or declined with advancing age. Besides these changes, BA treatment induced an increase in the DNA content. Changes in RNA content was more remarkable in response to application or deprival of BA treatment than the corresponding ones in protein and chlorophyll contents. The pattern of response to BA varied greatly according to the age at which the leaf received the treatment. As leaves aged, they lost the ability to increase their area and fresh weight in response to BA. However, continuous treatment with BA from an early stage kept the leaves young and able to respond.     

Soya Bean Seed Growth and Maturation In vitro without Pods

Immature Glycine max (L.) Merrill seeds, initially between 50and 450 mg f. wt, were grown and matured successfully in vitro.Excised seeds were floated in a liquid medium containing 5 percent sucrose, minerals and glutamine in flasks incubated at25 °C under 300 to 350 µE m^–2 s^–1 fluorescentlight. During 16 to 21 d in culture, seeds grew to a matured. wt of 100 to 600 mg per seed at an average rate of 5 to 25mg d. wt per seed d^–1 depending on initial size. Growthrates were maximal during the first 8 to 10 d in vitro but declinedwith loss of green colour in the cotyledons. Seed coats rupturedwith rapid cotyledon expansion during the first 2 d in culture.Embryos were tolerant to desiccation and 80 to 90 per cent germinatedif removed from culture before complete loss of green colour.The growth of excised seeds in vitro exceeded the growth ofseeds in detached pods, but when windows were cut in pods topermit direct exposure of seeds to the medium, seed growth wascomparable. Glycine max (L.) Merrill, soya bean, seed culture, seed growth, seed maturation, germination     

The Response of Phaseolus vulgaris L. to Root-zone Anaerobiosis, Waterlogging and High Sodium Chloride

Phaseolus vulgaris L. grown at a range of external concentrationsof NaCl (0 to 80 mM) responded differently to gaseous anaerobiosis(N₂ gas) in nutrient solution or stagnant waterlogging of theroot-zone. With similar patterns of distribution of Na⁺ andCl^- occurring in the plants with comparable NaCl treatments,and similar final concentrations of Na⁺ and Cl^- in plants grownunder both root-zone conditions, rates of uptake of Na⁺ andCl^- were much higher in plants with the stagnant waterloggedrootzones. After 72 h stagnant waterlogging, plant tops fromplants grown at 40 mM NaCl contained 1.42 per cent Na⁺ and 3.44per cent Cl^- (d. wt basis) while after 9 days exposure to NaClwith gaseous anaerobiosis, leaf tissue contained 1.49 per centNa⁺ and 4.28 per cen Cl^- (d. wt basis). Plants exposed to 40mM external NaCl were severely damaged within 72 h when grownwith stagnant waterlogged root-zones; those grown with N₂ anaerobiosiscontinued growth and development over the 9 d period. Plantsgrown in nutrient solution showed changes in distribution andconcentration of Na⁺ and Cl^- when oxygen concentration was reducedbelow 21 per cent O₂ (full aeration). Phaseolus vulgaris. L., bean, mineral salt distribution, anaerobiosis, salinity, waterlogging     

Response of Cultured Embryos of Phaseolus vulgaris and Hordeum vulgare to Farnesol

Excised embryos of Phaseolus vulgaris incubated in a mediumcontaining 10 mg dm^–3 farnesol showed enhanced root growthwhereas the leaves remained rudimentary At lower concentrationsof exogenous farnesol normal leaf development occurred and rootgrowth was comparable to untreated cultures. Enhanced root growthalso occurred when excised embryos of Hordeum vulgare were treatedwith farnesol but only at 10 mg dm^–3 and this treatmentdid not prevent leaf growth X-ray micro-probe analysis of leavesrevealed an increased phosphorus content in P vulgaris and adecreased sulphur content in H vulgare in comparison to untreatedplants. Hordeum vulgare L., barley, Phaseolus vulgaris, bean, embryo culture, farnesol, X-ray microprobe analysis, root growth