Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

Transformation and Characterization of Transgenic Plants of Solanum dulcamara L.--Incidence of Transgene Silencing

A transformation system is described for Solanum dulcamara usingthe supervirulentAgrobacterium tumefaciens strain 1065, carryingboth the ß-glucuronidase (gus) and neomycin phosphotransferaseII (npt II) genes adjacent to the right and left T-DNA borders,respectively. Leaf explants were more efficient for the productionof transformed plants compared to stem explants on medium containing50 mg l^-1of kanamycin sulphate. A 1:10 (v:v) dilution of anovernight culture ofAgrobacterium gave optimal transformationin terms of transgenic plant regeneration. From a total of 174kanamycin-resistant plants selected by their antibiotic resistance,16 failed to exhibit GUS activity. Southern analysis revealedthat these GUS-negative transformants originated from threeindependently transformed cell lines. Restriction enzyme analysesshowed that the GUS-negative plants had both the gus and nptII genes integrated into their genome (one plant had a singlecopy of each gene; the other two plants had multiple copies),with major rearrangement of the gus gene occurring in plantswith several copies of the transgene. GUS-negative plants showedleaf malformations, delayed flowering and a reduction in flower,fruit and seed production compared to GUS-positive and non-transformed(control) plants. Although gene silencing of the gus gene occurred,albeit at a low frequency (9.2%), the transformation systemdescribed generates large numbers of phenotypically normal,stably transformed plants. Copyright 2000 Annals of Botany Company Agrobacterium -mediated transformation, gene silencing, Solanum dulcamara L. (Bittersweet, Woody Nightshade), T-DNA truncation, transgene expression     

Biolistic Transformation of Rose (Rosa hybridaL.)

A reproducible method has been developed for the Biolistic transformationand regeneration of transgenic plants from embryogenic callusof rose (Rosa hybridaL.) cv. Glad Tidings. DNA delivery wasoptimized using the ß-glucuronidase (gus) gene. Thedistance between the stopping screen and target explants andsupplementation of pre-and post-bombardment culture media with0.25Mmyo-inositol influenced the transformation efficiency.Prior to culture on selection medium containing 250 mg l^-1kanamycinsulphate, embryogenic calli were bombarded, using optimizedgene delivery parameters, with a plasmid carrying the neomycinphosphotransferase (nptII) gene. Somatic embryo-derived kanamycin-resistantplants were regenerated and subsequently transferred to glasshouseconditions. Transformation was confirmed by kanamycin resistanceof calli and plants, NPT II ELISA assay and Southern analysis.All transgenic plants were morphologically normal (true-to-type).Copyright1998 Annals of Botany Company Biolistic; genetic engineering; rose;Rosa hybridaL.; transformation.     

Sulphate Transport into Plants and Excised Roots of Soybean (Glycine max L.)

The rates of sulphate transport into intact and excised rootsof soybean (Glycine max L.) were not significantly differentin the first hour and were maximal at pH 7. However, intactroots accumulated four times as much sulphate as excised rootsin 24 h, because of a marked reduction in the rate of transportby excised roots. The continued high rates of transport intointact roots were observed in plants kept in the light, andobserved in darkened plants growing in 1 per cent sucrose. Similarly,sulphate accumulation by excised roots was stimulated 2-foldby 1 per cent sucrose. The characteristics of sulphate accumulation by roots were notuseful in predicting sulphate translocation to the leaves. Transportto the leaves was maximal at pH 2–3, was almost totallylight-dependent and was not enhanced by growing plants in sucrose. Sulphate transport, Glycine max L., soybean, excised roots     

Reproductive Development and Nitrogen Fixation in Soybean (Glycine max [L.] Merril)

Gomes, M. A. F. and Sodek, L. 1987. Reproductive developmentand nitrogen fixation in soybean (Glycine max (L.) Merril).—J.exp. Bot. 38: 1982–1987. Nitrogenase activity (acetylene reduction) was measured duringthe growth cycle of soybean plants induced to flower at twodifferent ages. The decline in nitrogenase activity towardsthe end of the cycle was clearly associated with pod-fillingfor both flowering dates when plants were cultivated under lowerlight and temperature conditions (out of season). Under higherlight and temperature conditions (normal growing season) thedecline was independent of the flowering date. Furthermore,the timing of the decline was not altered when plants were maintainedunder long-day (vegetative) conditions nor when flowers wereremoved. It is suggested that under more favourable growth conditionsthe diversion of assimilates by the fruits is not the primarycause of the decline in nodule activity, but competition bythe fruits may be important when the production of photo-assimilatesis more limited. Key words: Glycine max, nitrogenase, source-sink     

Partitioning and Utilization of Photosynthate Produced at Different Growth Stages after Anthesis in Soybean (Glycine max L. Merr.): Analysis by Long-term 13C-Labelling Experiments

Yamagata, M., Kouchi, H. and Yoneyama, T. 1987. Partitioningand utilization of photosynthate produced at different growthstages after anthesis in soybean (Glycine max L. Merr.): Analysisby long term ¹³C-labelling experiments.—J. exp. Bot. 38:1247–1259. Soybean (Glycine max L. Merr. var. Akishirome) plants were allowedto assimilate ¹³CO₂ with a constant specific activity for 10h at different growth stages (a total of seven times at aboutone week intervals) after anthesis. The plants were harvestedperiodically until the time of full maturity and the partitioningof ¹³C into individual plant parts was investigated with anemphasis on the contribution of carbon assimilated at differentgrowth stages to the seed formation. Carbon assimilated at the middle to late seed-filling stagecontributed most to the seed production; one day contributionaccounted for 3–4% in total carbon of the seed at fullmaturity. Integrated contribution of carbon assimilated afteranthesis was estimated as 96% of the final seed carbon. An approximationbased on the temporal data of the incorporation of labelledcarbon into the seeds indicates that 77% of the final seed carboncame from direct transfer of current photosynthate from sourceleaves, which occurred within a few days after the photosyntheticfixation, while the rest originated from remobilization of carbonreserved mainly in leaves and stems plus petioles. In comparison with the total carbon accumulation in the seeds,protein carbon in the seeds was relatively more dependent onphotosynthate produced during the early period of reproductivegrowth stage, whereas lipid carbon was more dependent on photosynthateproduced during the later reproductive stage. Key words: Photosynthate partitioning, soybean (Glycine max L. Merr.), ¹³CO₂ assimilation, seed formation     

Source-sink Relationships, Seed Sucrose Levels and Seed Growth Rates in Soybean

Field experiments using two soybean (Glycine max L. Merrill)cultivars (‘Elgin 87’ and ‘Essex’) wereconducted for 2 years near Lexington, KY, USA to evaluate theeffect of source-sink alterations on seed carbohydrate statusand growth. Sucrose concentrations in developing cotyledonsof control plants were consistently low (<50 m M) early inseed development, but they increased to 100–150 m M byphysiological maturity. The concentrations increased in bothyears by 47 to 59% when 90% of the pods were removed from ‘Elgin87’, but the increase had no effect on individual seedgrowth rate (SGR). Shading (80%) reduced cotyledon sucrose levelsand SGR in both years. The critical cotyledon sucrose concentration(the concentration providing 80% of the maximum cotyledon growthrate) was estimated fromin vitro cotyledon growth at sucroseconcentrations of 0–200 m M. These critical concentrationsvaried from 72–124 m M;in planta control cotyledon sucroseconcentrations were below this critical level during the firsthalf of seed growth but exceeded it in the later stages of growthin all experiments. The estimated critical concentration wasconsistent with the failure of in planta SGR to respond to anincrease in assimilate supply and with the reduction in SGRassociated with a decrease in assimilate supply. The resultssuggest that soybean SGR is generally sink limited if photosynthesisincreases during seed filling, but source limited if photosynthesisis reduced. Copyright 2001 Annals of Botany Company Glycine max(L.) Merrill, soybean, source-sink ratios, sucrose, starch, depodding, shade, in vitro culture     

Herbicide-tolerant Transgenic Pineapple (Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple(Ananas comosus‘Phuket’) were produced followingmicroprojectile-mediated delivery of the plasmid AHC25, carryingthe ß-glucuronidase (gus) reporter gene and the bialaphosresistance (bar) gene for herbicide tolerance, into leaves ofmicropropagated shoots. Transformed plants were regeneratedfrom bombarded leaf bases on Murashige and Skoog-based mediumcontaining 0.5 mg l^-12,4-dichlorophenoxyacetic acid, 2.0 mgl^-16-benzylamino purine and 0.5 mg l^-1phosphinothricin. Integrationand expression of thebar gene in regenerated plants was confirmedby Southern analysis and RT-PCR, respectively. Regenerated plantswere assessed in vitro and under glasshouse conditions for theirtolerance to the commercial herbicide Basta^TM, containing glufosinateammonium as the active component. Plants sprayed with Basta^TMcontainingconcentrations of glufosinate ammonium up to 1400 mg l^-1remainedhealthy and retained their pigmentation. The generation of herbicide-tolerantpineapple will facilitate more efficient weed control in thiswidely cultivated tropical crop. Copyright 2001 Annals of BotanyCompany bar gene, Biolistics, herbicide tolerance, pineapple, phosphinothricin (PPT)     

Irreproducibility of the soybean pollen-tube pathway transformation procedure

The interest in developing tissue culture-independent genetic transformation methods for plants has been growth. The pollen-tube pathway transformation technique is one method; however, this method is controversial because it is difficult to duplicate and produces insufficient molecular evidence to confirm transformation. Our objective was to evaluate the robustness of the soybean pollen-tube pathway technique (Glycine max L. Merr.). Solutions of purified DNA constructs carrying abar marker gene and agus reporter gene or a gene of interest (npk1) were applied to severed styles of flowers 6–8 h after self-pollination. The experiment was repeated 3 summers in the field, in which 4 DNA constructs and 7 soybean genotypes were tested. A total of 4793 progeny seeds were harvested from 5590 individually treated soybean flowers. All seeds were germinated and screened for transformants with herbicide spray, histochemical GUS assay, and Southern blot analysis. Although 2% of progenies showed partial resistance to the herbicide, no positive plants were identified from GUS assay and Southern analysis. Our results indicate that soybean pollen-tube pathway transformation is not reproducible.     

Changes in Soybean Raceme and Petiole Anatomy Induced by 6-Benzylaminopurine

An enlargement of the peduncle and rachis of the terminal racemeand the petiole of the uppermost mainstem leaf was observedin soybean plants [Glycine max (L.) Merr.] treated with thecytokinin, 6-benzylaminopurine (BAP). Histological studies wereperformed to determine the timing and extent of anatomical changesaccompanying BAP treatment. Swelling of treated ‘Tracy-M’peduncles, rachises, and petioles was observed within 4–6d after treatment initiation. A significant increase in totalcross-sectional tissue area was observed at lower and intermediateinternodes of treated rachises after 11 d. Rachis enlargementwas due to increases in both cell size and cell number, particularlyof the vascular tissue. In treated petioles of IX93-100, procambialcells of vascular bundles were the first to respond to the BAPtreatment. These cells differentiated into a vascular cambiumwhich formed secondary xylem and phloem. Soybean, Glycine max (L.) Merr., anatomy, rachis, BAP.     

Plant Growth-promoting Rhizobacteria and Soybean [ Glycine max (L.) Merr.] Growth and Physiology at Suboptimal Root Zone Temperatures

Application of plant growth-promoting rhizobacteria (PGPR) hasbeen shown to increase legume growth and development under optimaltemperature conditions, and specifically to increase nodulationand nitrogen fixation of soybean [Glycine max (L.) Merr.] overa range of root zone temperatures (RZTs). Nine rhizobacteriaapplied into soybean rooting media were tested for their abilityto reduce the negative effects of low RZT on soybean growthand development by improving the physiological status of theplant. Three RZTs were tested: 25, 17.5, and 15 °C. At eachtemperature some PGPR strains increased plant growth and development,but the stimulatory strains varied with temperature. The strainsthat were most stimulatory at each temperatures were as follows:15 °C—Serratia proteamaculans 1–102; 17.5 °C—Aeromonashydrophila P73, and 25 °C—Serratia liquefaciens 2–68.Because enhancement of plant physiological activities were detectedbefore the onset of nitrogen fixation, these stimulatory effectscan be attributed to direct stimulation of the plant by thePGPR rather than stimulation of plant growth via improvementof the nitrogen fixation symbiosis. Legume; nitrogen fixation; nodulation; root zone temperature; PGPR     

Plant Growth Promoting Rhizobacteria and Soybean [ Glycine max (L.) Merr.] Nodulation and Nitrogen Fixation at Suboptimal Root Zone Temperatures

Co-inoculation of plant growth promoting rhizobacteria (PGPR)withBradyrhizobium has been shown to increase legume nodulationand nitrogen fixation at optimal soil temperatures. Nine rhizobacteriaco-inoculated withBradyrhizobium japonicum532C were tested fortheir ability to reduce the negative effects of low root zonetemperature (RZT) on soybean [Glycine max(L.) Merr.] nodulationand nitrogen fixation. Three RZTs were tested: 25 (optimal),17.5 (somewhat inhibitory), and 15°C (very inhibitory).At each temperature some PGPR strains increased the number ofnodules formed and the amount of fixed nitrogen when co-inoculatedwithB. japonicum,but the stimulatory strains varied with temperatures.The strains that were most stimulatory varied among temperaturesand were as follows: 15°C,Serratia proteamaculans 1-102;17.5°C,S. proteamaculans 1-102andAeromonas hydrophilaP73;25°C,Serratia liquefaciens2-68. Bradyrhizobium japonicum ; Glycine max; plant growth promoting rhizobacteria; suboptimal root zone temperatures     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenesmediated transformation of embryogenic calli

Genetically transformed roots and calli were induced from leafsegments of grapevine (Vitis vinifera L. cv. Koshusanjaku) afterco-cultivation with wild-type Agrobacterium rhizogenes strains,but plant regenera tion from them was not achieved. On the otherhand, transgenlc grapevine plants were obtained via somaticembryogenesis after co-cultivation of embryogenic calli withan engineered A. rhizogenes strain including both the neomycinphosphotransferase II (NPT II) and the ß-glucuronidase(GUS) genes, followed by selection of secondary embryos forkanamycin resistance. All these plants showed GUS gene expressionrevealed by histochemical assay. Southern blot analysis revealedthe stable integration of the GUS cording region in their genome.Transformants containing Ri T-DNA exhibited various phenotypes:most of them showed a typical Ri-transformed phenotype suchas wrinkled leaves, while the others looked normal. Key words: Agrobacterium rhizogenes, grapevine, transgenic plants, Vitis vinifera