Characterization of Orthophosphate Absorption by Pea Root Protoplasts

Root protoplasts were isolated from 4 d old seedlings of Pisumsativum. Viability was verified by fluorescin diacetate, directblue and neutral red vital staining techniques. Phosphate influx of the protoplasts was similar to that of rootswith respect to phase 1 affinity for orthophosphate /{K_m = 99mmol m^–3) and sensitivity to pH and metabolic inhibitors.Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone decreaseduptake to 4% of the control and diethyl-stilboestrol inducedleakiness in the protoplasts. Influx was only slightly lessthan that in plasmolysed roots which in turn was 3.6 times lowerthan that of normal roots. Efflux from the protoplasts was highwith a value of approximately 2% of the cellular phosphate contentper hour. The elevated leakage indicated an essential differencebetween the effects of protoplast isolation on influx and effluxof phosphate. Key words: Root protoplasts, Phosphate absorption, Influx, Efflux, Metabolic inhibitors, Diethyl-stilboestrol, Subprotoplasts     

Cytokinins in Root Nodules of Phaseolus mungo

Four cytokinins have been separated from extracts of root nodulesof Phaseolus mungo by thin-layer chromatography. Their activitywas determined on the basis of their ability to induce betacyaninsynthesis in cotyledons of Amaranthus caudatus. Zeatin and itsriboside showed greater activity than N⁶ (²-(isopentenyl)) aminopurine and its riboside in the bioassay. Phaseolus mungo, mung bean, cytokinins, isopentenyl, amino-purine, zeatin, betacyanin synthesis, Amaranthus caudatus     

Cytokinin Production by Tomato Root: Occurrence of Cytokinins in Staled Medium of Root Culture

Cytokinins were released into the medium by cultures of excised tomato (Lycopersicon esculentum Mill.) root tips. A large portion was directly released from the proximal cut-end of the roots. The most active substance was found to co-chromatograph with zeatin riboside in both paper and Sephadex column chromatographies. The cytokinin activity after 7 days of growth was found about eight times higher in the medium as compared with that in the root tissues. When freshly excised root tips were subcultured throughout eight passages, the accumulation of cytokinin in the medium of each passage was approximately the same. These results show that the root tip is a main site of cytokinin synthesis.     

Enhanced shoot regeneration from Brassica campestris by silver nitrate

Summary The morphogenetic response of Brassica campestris genotype R500 to inhibitors of ethylene biosynthesis and action was investigated. A medium containing 1.0 mg.l^–1 NAA, 2.0 mg.l^–1 BAP, and 30 or 60 M AgNO₃ significantly enhanced both the percentage shoot regeneration and the number of shoots per cotyledon expiant. Although callus proliferation occurred on hypocotyl segments, no shoots were formed in response to AgNO₃ with expiants older than five days. Cotyledons older than six days formed shoots only with AgNO₃. Cobalt chloride at 20 and 30 M increased cotyledon shoot regeneration but was inferior to AgNO₃. Hypocotyl segments were unresponsive. Salicylic acid at 25 and 50 M prevented both shoot regeneration and callusing without any obvious toxic effects. Removal of expiants from AgNO₃ after 12 days did not alter the percentage of shoot regeneration but increased the number of shoots per expiant. This response was dependent on the level of BAP. Percentage shoot regeneration and number of shoots per cotyledon explant were not affected by removal of CoCl₂. These results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism.Abbreviations NAA Naphthalene Acetic Acid - BAP 6-Benzylamino Purine - MS Murashige and Skoog Medium     

Evaluation of a Bioassay for Cytokinins Using Soybean Hypocotyl Sections

The dose-response curves of several cytokinins were investigatedin a soybean hypocotyl bioassay. Zeatin riboside, zeatin-O-ß-D-glucoside,dihydrozeatin, and dihydrozeatin riboside produced linear responsesparallel to that for zeatin. The hypocotyl section assay wassuperior to the conventional soybean callus assay because theresponse (log₁₀ transformed data) was linear, exhibited lowvariability, and was more reproducible and more sensitive. Theassay was quicker to perform and required less cytokinin.     

Transgenic Rice Plants Produced by PEG-Mediated Plasmid Uptake into Protoplasts

Embryogenic cell suspension cultures were obtained from calli developed from mature rice seeds of a Japonica type Itahan cultivar, Roncarolo. Protoplasts were isolated and transformed by PEG-treated method with plasmid pHP23 carrying the NPT Ⅱ gene which encodes resistance to antibiotic G-418. Protoplast-derived colonies were selected in presence of the inhibitor. Plants were regenerated and transplanted into soil in the green house. The presence of foreign gene in the regenerated plants was verified by PCR and Southern analysis.     

Transfer of resistance to Xanthomonas campestris pv campestris into Brassica oleracea L. by protoplast fusion

Black rot caused by the bacterium Xanthomonas campestris pv campestris is one of the most serious diseases of Brassica oleracea. Since sources of resistance to the disease within B. oleracea are insufficient and control means are limited, the development of resistant breeding lines is extremely desirable. Certain lines of B. napus contain very high resistance controlled by a dominant gene, but crossing the two species sexually is very difficult. Therefore, somatic hybrids were produced by protoplast fusion between rapid cycling B. oleracea and a B. napus line highly resistant to X. campestris pv campestris. Hybrid identity was confirmed by morphological studies, flow cytometric estimation of nuclear DNA content, and analysis of random amplified polymorphic DNA (RAPD). Inoculations with the pathogen identified four somatic hybrids with high resistance. The resistant hybrid plants were fertile and set seed when selfed or crossed reciprocally to the bridge line 15 (Quazi 1988). Direct crosses to B. oleracea were unsuccessful, but embryo rescue facilitated the production of a first-backcross generation. The BC₁ plants were resistant to the pathogen. Progeny from the crosses to line 15 were all susceptible. Embryo rescue techniques were not obligatory for the development of a second-backcross generation, and several resistant BC₂ plants were obtained.     

大白菜组织培养和快速繁殖(简报)

以大白菜无菌苗子叶为外植体,在添加AgNO3 2.0mg/L的MS 6-BA 2.0mg/L NAA 0.5mg/L培养基上,不定芽的诱导率达86.5%;依次转移到增殖、壮苗和生根培养基上培养,炼苗后移栽,成活率80%以上。     

Hydrogen Uptake and Exchange by Pea Root Nodules

Hydrogenase activity in pea root nodules was studied by followinggas exchanges of hydrogen and deuterium. It was found that thenodules did not evolve hydrogen but that hydrogen was takenup when it was provided in the gas mixture. When increasingpartial pressures of deuterium were supplied, hydrogen was evolvedat a rate which increased as the pressure of deuterium increased.Deuterium was taken up at the same time as this hydrogen wasevolved. Hydrogen evolution in the presence of deuterium wasinhibited by nitrogen, while the uptake of deuterium remainedunaffected. It was concluded that pea root nodules have at leasttwo separate hydrogenase system that are working in oppositedirections and must thus be situated in sites of different oxidation-reductionpotentials within the nodule.     

Polyethylene Glycol-induced Uptake of Bacteria into Yeast Protoplasts

Cells of the nitrogen-fixing bacterium Azotobacter vinelandii and the unicellular cyanobacterium Anacystis nidulans were introduced into protoplasts of Saccharomyces cerevisiae by the polyethylene glycol (PEG) method. Factors influencing the uptake frequency were examined, and experimental conditions were established for maximizing the uptake frequency. Under optimal conditions, each protoplast took-up a few bacterial cells. Electron-microscopic studies showed the localization of integrated bacterial cells in membrane-bound vesicles of the cytoplasm or large vacuoles. The protoplasts at the intermediate stages of uptake revealed two major mechanisms of uptake: (a) “endocytosis” by a single protoplast and (b) “cell fusion” between two or more protoplasts. Some bacterial cells disintegrated during the subsequent incubation period through a heterophagy-like process.     

Plant Regeneration from Petiole Protoplasts of Brassica napus

Two cultivars of Brassica napus, Altex and Canadian twins, were used as materials. Protoplasts isolated from petioles of plants grown in vitro were cultured in Nitsch medium supplemented with 0.5mg/L BA, 0.5mg/L NAA, lmg/L 2,4-D, 100mg/L serine, 800mg/L glutamine, 4% sucrose and 0.4mol/L mannitol. After 2 days of culture, the first division was observed. The division frequency estimated after 10 days of culture was 30-60%. One week after transferring onto MS medium containing 6mg/L GA3. and 3mg/L BA, protoplast-derived calli regenerated into shoots. The regeneration frequency of the two cultivars was 24% and 31% respectively. It was found that the protoplasts isolated from petioles could float on the surface of the 3% sucrose contained solution which was very favourable both to purification, and culture of the protoplasts.     

Regulation of monocarpic senescence of Brassica campestris by the developing pods

Senescence of Brassica campestris L. cv. B-9 was studied with regard to seed maturation and source-sink relationships. In normal control plants leaf senescence (as determined by the change in chlorophyll level) started and proceeded in a progressive manner from base to apex during the period of early pod setting. Complete yellowing of the leaves occurred well before the seed maturation and pod wall senescence. The pod wall always senesced before the attainment of final seed weight. In two different sets of acrocarpous plants containing 65 pods and 10 pods, respectively, leaf senescence was delayed during the pod filling period. It started non-sequentially after complete yellowing and senescence of the pod wall. The degree of leaf senescence at the post-pod filling stage was almost proportional to the number of pods present. When peduncles of the acrocarpous 10-podded plants were removed after the pod filling stage of the plant, leaf senescence was delayed compared to plants whose pedicels were removed, although the senescence pattern of the upper three leaves was nonsequential in both cases. Defruiting at an early stage of development delayed leaf senescence, although the pattern of such senescence remained unaltered (i.e. nonsequential). Defoliation hastened the seed-filling process and pod wall senescence. Plants containing fewer pods had higher average seed weight, although yield per plant was reduced.
These results suggest that the pod wall serves as a temporary as well as intermediary storage organ and that foliar senescence is not directly related to seed maturation. The possible cause of uncoupling between foliar senescence and seed development is discussed.     

Chromosome pairing in haploids of Brassica campestris

Summary The maximum chromosome pairing observed in haploids of Brassica campestris was two bivalents plus one trivalent but differences were observed in the chromosome pairing frequencies of the four haploids studied. This pairing supports the theorem that the species is hexasomic for one chromosome, tetrasomic for two and disomic for three others but it is emphasized that some of the observed pairing might be explained by a phenomenon other than homology.     

甘蓝下胚轴原生质体再生植株

经纯化后,甘蓝下胚轴原生质体的产量为1．5×10⁶g^-1(Fw),采用液体浅层培养的方法进行培养。2～3d后,发生第一次分裂,第10天,统计分裂频率为6l％,5周内形成大量的细胞团和小愈伤组织,统计植板率为1．1％,把小愈伤组织转到与原生质体培养基相同激素的MS固体培养基上增殖。当愈伤组织长到3～5mm大小时,接到分化培养基上,芽分化率为46.7％．分化出来的芽长到3～4cm长时,从基部切下,插入生根培养基,两星期左右即可长成完整植株。     

The Selective Uptake of Alkali Cations by Red Beet Root Tissue

The selective uptake of alkali cations by red beet root tissuefrom solutions of chlorides has been investigated. It is shownthat when disks are transferred from distilled water to a solutionof salts, there is a rapid initial uptake of cations which isneither particularly selective, nor directly related to metabolism.On the other hand, the prolonged active accumulation of cationsexhibits strong selectivity, Na being preferred to other ions. Evidence is presented to show that the alkali cations competewith one another for the same absorption mechanism. In thisrespect the material apparently differs from that investigatedby Epstein and Hagen, in which the operation of distinct mechanismsfor some of these ions was visualized. The validity of Epsteinand Hagen's conclusion is discussed in relation to the resultspresented here.     

Plant Eegeneration from Protoplasts of Brassica carinata Braun

Protoplasts of Brassica carinata Braun. (accession No. 84A165) were enzymatically isolated from hypocotyls and cotyledons of 3–5 day-old test-tube seedlings or first true leaves taken from greenhouse grown plants at a three-leaf stage. The protoplasts were suspended in a P-B liquid medium solidified with 0.15%–0.3% low melting agarose which formed a thin layer floating on the surface of the liquid medium. The optimum protoplast density was ranging from 5× l03 to 1 × 104/ml. As for the hypocotyl protoplasts, the first division was observed after 48 h in the culture. The division frequency reached 21% and 34% at day 3 and 6 respectively. The initiation of cell division in the case of cotyledon and mesophyll protoplast culture was late, usually at day 5, and the division frequency was also somewhat lower. One week after culture, the cultures were transferred to fluorescent light condition with an intensity of about 1000 lx. A dilution medium DPDK3 was then added and the dilution procedure was repeated at one week interval thereafter. One month after culture, microcalli with 300–500 μm in size were formed. It was also found that in some cases globular embryoid structure protruded on the callus surface. Totally, a 2%–3% plating efficiency was achieved. Shoot regeneration occurred when cotyledon and mesophyll protoplast-derived calli were transferred onto a modified MS medium supplemented with NAA 0.1, BA 3 mg/l. Individual shoots were rooted on a rooting medium supplemented with 0.2 mg/l of IAA. Intact plants with normal morphology were eventually produced.     

Differential Rates of Uptake of Glucose, Fructose and Sucrose by Pea Stem Protoplasts

Protoplasts from growing regions of etiolated pea stems takeup glucose more rapidly than fructose when supplied for briefperiods at low concentrations. The uptake of the two hexosesis differentially inhibited by galactoac and by reagents thatcurtail ATP synthesis, and uptake of one hexose is not preventedby the other, even at a 100-fold excess. Sucrose uptake is muchslower than that of either hexose and is correlated with theappearance of invertase activity in the medium. Label from [¹⁴C-glc]-sucroseis taken up more rapidly than from [¹⁴C-fru]-sucrose. It isconcluded that these cells take up supplied sucrose only afterhydrolysis to hexoses, which are then absorbed by differentcarrier-mediated processes. Key words: Glucose, fructose, invertase, pea, protoplast, sucrose