Identification of novel low M(r) glutenin subunits in the high quality bread wheat cv Salmone and their effects on gluten quality

Southern-blot hybridization with a probe specific for genes encoding for low M_r glutenin subunits showed that the high quality bread wheat cv Salmone contains two DNA fragments designated as SF720 and SF750. These fragments were found to occur on the chromosome-1B satellite, and to be associated with the presence of two strongly staining low M_r glutenin subunits in the two-dimensional A-PAGE 2 SDS-PAGE pattern of cv Salmone. Comparison of 65 F₆ random lines derived from the cross between cv Salmone and the medium quality line FAP74809 revealed that the presence of fragments SF720 and SF750 had significant positive effects on several quality related parameters such as SDS sedimentation volume, Farinograph stability and Alveograph strength (W), tenacity (P) and elasticity (L). Additive effects of high M_r glutenin subunits 1 and 7+9 on gluten quality were found as well. Fragments SF720 and SF750 were suggested to occur at a locus other than Glu-B3, as indicated by their relatively high frequency of recombination with the Gli-B1 locus.     

Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

Metabolic Alterations Associated With Abscisic Acid-Induced Frost Hardiness in Bromegrass Suspension Culture Cells

Metabolic alterations associated with the induction of freezingtolerance by abscisic acid (ABA) were characterized by chemicalanalysis and by [U-^l4C]sucrose partitioning into cellular constituentsin bromegrass (Bromus inermis Leyss cv. Manchar) cell suspensioncultures. ABA caused a significant elevation in dry matter,particularly in the fraction insoluble in 85% ethanol, thatwas highly correlated with enhanced frost tolerance. Cell walls,the largest component of the insoluble fraction, increased significantlyas frost tolerance increased throughout the ABA treatment period.ABA stimulated total [¹⁴C]sucrose uptake by cells from 7% onday 1 to 97% on day 7 compared to control cells. Partitioningstudies detected a significant increase in ¹⁴CO₂ evolution at3, 5 and 7 days after ABA treatment and a significantly higherincorporation of [¹⁴C]sucrose into the ethanol insoluble fractionafter 5 and 7 days of treatment. Organic acid depletion in ABA-treatedcells was also highly correlated with the increase in hardiness.The concentration of total sugars was higher in ABA-treatedcells. The results indicate that most of the metabolic changesduring ABA-induced acclimation were similar to changes reportedfor cells acclimated in response to low temperature. ¹Oregon Agricultural Experiment Station Technical Paper No.9052 ²Present address: Department of Horticulture, University ofSaskatchewan, Saskatoon, Sask. Canada S7N 0W0 (Received November 1, 1989; Accepted March 13, 1990)     

Germination of synthetic seeds of pineapple (Ananas comosus L. Merr.)

. Tufts of multiple shoots were produced from dormant, axillary buds of pineapple in vitro. Tiny shoots (2-5 mm) isolated from the tuft of multiple shoots were encapsulated in 3% sodium alginate prepared using hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose. The encapsulated shoots represented synthetic seeds that germinated and formed roots in vitro after subculture onto one of the following media solidified with 0.8% agar: (1) hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose (Pin1), (2) Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol, 0.06 M sucrose, 9.67 µM 1-naphthalene acetic acid, 9.84 µM indole-3-butyric acid and 9.29 µM kinetin (Pin2), and (3) White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 0.54 µM 1-naphthalene acetic acid and 1.97 µM indole-3-butyric acid (Pin3). Pretreatment of shoots in either liquid Pin3 or Pin4 medium (White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 10.8 µM 1-naphthalene acetic acid and 39.4 µM indole-3-butryic acid) was required for development into plantlets with roots after culture on either Pin1, Pin2 or Pin3 media. One hundred percent germination of synthetic seeds to plantlets occurred after pretreatment of shoots in liquid Pin4 medium for 12 h followed by culture of synthetic seeds on Pin2 medium. Synthetic seeds stored at 4°C remained viable without sprouting for up to 45 days. Plantlets produced in vitro from synthetic seeds were successfully established in soil. The protocol provides an easy and novel propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Regeneration of Lonicera tatarica plants via adventitious organogenesis from cultured stem explants

We describe an efficient process for the regeneration of Lonicera tatarica plants from cultured stem sections. Induction of multiple shoots was achieved directly from cultured stem cuttings. The highest regeneration rate was achieved on Gamborg's B5 medium supplemented with 4% sucrose and 0.8% Difco bacto-agar in the absence of hormones. Differentiated shoots were elongated for 5-7 days on induction medium supplemented with 0.5 mg/l GA₃. Shoot induction and elongation experiments were carried out using original stem explants from either 2-, 6-, or 18-month-old donor plants. The age of the donor plant had no noticeable effect on either process. However, rooting of elongated shoots occurred only with shoots derived from 2-month-old donor plants. Rooting efficiency and proliferation were highest on half-strength WPM medium supplemented with 2 µM indole-3-butyric acid and 0.6% Keylis agar. The plants regenerated from stem explants were morphologically normal, and levels of loganin and secologanin were comparable to those detected in plants grown from seed and maintained through vegetative propagation.     

A structured growth model for Cynara cardunculus cell suspension

In this study a model was developed to describe the growth of Cynara cardunculus L. suspended cells as a function of the availability of two substrates, sucrose as the carbon and energy source and phosphate. It was assumed that the maintenance energy need was fulfilled by the consumption of extracellular carbohydrates, in non-limiting conditions, or by the consumption of structural biomass when sucrose is depleted. A production of secondary metabolites was also assumed. This model was developed based on a structured model previously described by Van Gulik et al. (1993). The model was applied to the experimental results of C. cardunculus suspended cells grown in a Gamborg B₅ medium supplemented with 2% sucrose, using a non-linear regression program.     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

In vitro flowering of bitter melon

Flowers were formed from shoot tips of bitter melon (Momordica charantia L.) cultured on Murashige and Skoog medium supplemented with 90 mM sucrose, 0.05 mM Fe²⁺ and 4 µM N⁶-benzyladenine (BA). The addition of 0.05 mM Fe²⁺ to the medium prevented chlorosis of the explant and promoted normal flowering. Increasing the ratio of carbon to nitrogen promoted male flower formation but intensively inhibited vegetative growth. The influence of cytokinin on the morphogenesis of the explant was highly notable. Flowers could be formed after a 15- to 20-day exposure to kinetin (Kin) or BA. Kin and BA had opposite effects with regard to the development of the explant. Kin promoted flower formation, especially female, but inhibited branch bud formation. Conversely, BA promoted branch bud formation and also promoted male flower formation when present at a concentration of 1-2 µM, but completely inhibited flower formation at 4-8 µM. Fluorescein diacetate staining and in vitro germination showed that in vitro pollen were of a fairly high viability.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Candidate genes and QTLs for sugar and organic acid content in peach [ Prunus persica (L.) Batsch

The identification of genes involved in variation of peach fruit quality would assist breeders in creating new cultivars with improved fruit quality. Major genes and quantitative trait loci (QTLs) for physical and chemical components of fruit quality have already been detected, based on the peach [Prunus persica (L.) Batsch] cv. Ferjalou Jalousia^® (low-acid peach) 2 cv. Fantasia (normally-acid nectarine) F₂ intraspecific cross. Our aim was to associate these QTLs to structural genes using a candidate gene/QTL approach. Eighteen cDNAs encoding key proteins in soluble sugar and organic acid metabolic pathways as well as in cell expansion were isolated from peach fruit. A single-strand conformation polymorphism strategy based on specific cDNA-based primers was used to map the corresponding genes. Since no polymorphism could be detected in the Ferjalou Jalousia^® 2 Fantasia population, gene mapping was performed on the almond [Prunus amygdalus (P. dulcis)] cv. Texas 2 peach cv. Earlygold F₂ interspecific cross from which a saturated map was available. Twelve candidate genes were assigned to four linkage groups of the peach genome. In a second step, the previous QTL detection was enhanced by integrating anchor loci between the Ferjalou Jalousia^® 2 Fantasia and Texas 2 Earlygold maps and data from a third year of trait assessment on the Ferjalou Jalousia^® 2 Fantasia population. Comparative mapping allowed us to detect a candidate gene/QTL co-location. It involved a cDNA encoding a vacuolar H⁺-pyrophosphatase (PRUpe;Vp2) that energises solute accumulation, and QTLs for sucrose and soluble solid content. This preliminary result may be the first step in the future development of marker-assisted selection for peach fruit sucrose and soluble solid content.     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Effects of Sucrose Concentration on the Photosynthetic Ability of Rose Shoots In Vitro

Reducing the concentration of sucrose in the culture mediumover successive subcultures has been tested as a method forincreasing the ability of rose shoots grown in vitro (Rosa cvsIceberg and Peace) to take up CO₂. Shoots maintained on ‘constant’10, 20 and 40 g I^–1 sucrose showed decreased levels ofCO² uptake at higher sucrose concentrations, although cv. Peacegrew least at 10 g l^–1 and showed correspondingly lowamounts of CO₂ uptake compared with 20 and 40 g l^–1. Bothcultivars died when sucrose was omitted from the medium. Assucrose concentration was reduced in the medium, so CO₂ uptakeof shoots initially cultured on 20 and 40 g l^–1 sucrosewas found to increase, although a concentration of 10 gl ^–1sucrose seemed to be limiting, below which the growth and chlorophylllevels of shoots declined. Rosa hybrid, rose, shoot culture in vitro, photosynthetic ability, sucrose, infra-red gas analysis     

Culture treatments for enhancing post-thaw recovery of cryopreserved suspension cells of potato cv. Desiree

An efficient and reproducible protocol has been developed for the cryopreservation of cell suspension cultures of the potato (Solanum tuberosum L.) cv. Desiree. An evaluation was made of the effectiveness of different pre-culture and post-thaw treatments on cell growth, as measured by changes in biomass. Cell suspensions were cultured in UM medium supplemented with 0.25, 0.5, 0.625, 0.75 or 1.0 M sucrose prior to cryopreservation. Sucrose-treated cells were harvested from suspension and 0.75 ml packed cell volumes placed in 2 ml capacity polypropylene vials with 0.5 ml of chilled cryoprotectant (glycerol 46.0 g 1(-1), dimethylsulphoxide 39.0 g 1(-1), sucrose 342.0 g 1(-1) proline 5.0 g 1(-1); pH 5.8). Cells were frozen at -0.5 degrees C min(-1) from 0 to -35 degrees C, held at -35 degrees C for 35 min and stored, for 10 days, in liquid nitrogen (-196 degrees C). The most effective pre-treatment, in terms of subsequent post-thaw cell viability as assessed by fluorescein diacetate uptake or triphenyltetrazolium chloride reduction, was culture with 0.75 M sucrose. For this treatment, the mean absorbance (490 nm) following triphenyltetrazolium chloride reduction was 88% greater (p < 0.05) than control and 59% greater (p < 0.05) for thawed cells also cultured on supporting filter paper discs.     

Cryopreservation of Panax ginseng cells

The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution, cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%) occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%); the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after several subcultures. Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000     

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones. Received: 28 October 1996 / Revision received: 28 January 1997 / Accepted: 15 March 1997     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Regeneration and molecular characterization of intergeneric somatic hybrids between Citrus reticulata and Poncirus trifoliata

Citrus exocortis viroid (CEV) is widespread in citrus production areas where trifoliate orange [Poncirus trifoliata (L.) Raf.] is used as rootstock. Citrus reticulata Blanco cv. Red tangerine, a different rootstock, is tolerant to CEV. Embryogenic protoplasts of C. reticulata cv. Red tangerine were electrically fused with mesophyll protoplasts from P. trifoliata, and five embryoids were regenerated after 40 days of culture. The embryoids were cut into several pieces and subcultured on shoot induction medium. After 5 months and several subcultures, shoots initially regenerated. The plants grew vigorously with well-developed root systems and exhibited the trifoliate leaf character of P. trifoliata. Chromosome counts on four randomly selected root tips revealed them to be tetraploids (2n=4x=36). RAPD analysis of four randomly selected plants verified their hybridity. This hybridity was further confirmed by AFLP analysis using four primer pairs, from which a total of 65 specific bands were detected. Cytoplasmic genome analysis using universal primers revealed that their chloroplast DNA banding pattern was identical to that of trifoliate orange, while the banding pattern of mitochondrial DNA was identical to that of Red tangerine. The potential of this somatic hybrid as a means to control tree size and provide multi-resistance is discussed.     

Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos

A protocol for the production of transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] was developed via Agrobacterium-mediated genetic transformation of somatic embryos. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the nptII gene and gus-intron were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While pre-culture, wounding and acetosyringone treatment were inhibitory, the bacterial growth phase (optical density; OD₆₀₀ = 0.6), cell density (10⁹/ml), co-cultivation period (5 days) and pH of the co-cultivation medium (5.6) had positive effects on transformation. Following co-cultivation, globular somatic embryos were placed on multiplication medium and stressed with kanamycin (50 µg/ml). Further selection occurred in the maturation and germination medium at an elevated kanamycin level (75 µg/ml). An average of 40% transient expression was evident based on the GUS histochemical assay. Kanamycin-resistant, GUS-positive embryos were germinated, and the resulting microshoots were multiplied in vitro. Integration of the transgenes into the tea nuclear genome was confirmed by PCR analysis using nptII- and gus-specific primers and by Southern hybridization using an nptII-specific probe. The transgenic shoots were micrografted onto seed-grown rootstocks of cv. Kangra Jat and eventually hardened in a walk-in polyhouse. This is the first report on the production of transgenic tea.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998