Sugarbeet leaf disc culture: an improved procedure for inducing morphogenesis

In preparation for gene transfer experiments we investigated factors that might affect the production of shoots and somatic embryos from the wound callus of cultured sugarbeet leaf discs. A complex interaction was found between the leaf disc plating density, the disc culture medium, the source-shoot culture medium and the frequency of disc transfer to fresh medium. The most productive protocol utilized: source shoots maintained on MS medium containing 0.25 mg 1^-1 BA; multiple leaf discs (ten 4-mm discs/plate) plated onto an enriched modification of MS medium (RV) containing 1.0 mg 1^-1 BA and solidified with 0.3% Gelrite (not permitted to dry during hardening); and transfer of the discs to fresh medium every two weeks during the first month. This standard protocol produced more callus per plate and higher rates of morphogenesis per unit dry weight of callus than did the one-step method of Saunders and Doley. Water availability considerations were found to be critical to obtaining high morphogenic rates. Root induction frequency and quality was superior on shoots transplanted to MS medium containing 1 mg 1^-1 NAA as the sole growth regulator compared to IAA at the same concentration.Abbreviations BA N⁶-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Influence of agar on in vitro cultures: I. Physicochemical properties of agar and agar gelled media

Summary The success of in vitro culture is related to several factors. Beside factors associated with the plant material or the medium composition, the physicochemical characteristics of gelled media can play an important role. In this paper, the latter aspect has been considered and the nature of agar powders has been investigated. Moreover, the process of gel formation for three different media and the availability of water and minerals for the corresponding gels have been studied. Analysis of agar powders showed that they can contain different amounts of impurities and the dialysis of these powders suggested that the impurities might be available to the tissues. Thermal analysis on the hygroscopic properties of the agar brands suggest the importance of these data to obtain comparable and reproducible gelled media. The study on the process of formation of gelled media indicates that there is a critical temperature T_ss which can be used to control the gel processing. In fact, at this temperature, agar powders in water transform into a sol status through a rapid shift of electrical conductivity. Water potential of the medium, water loss from gels over the culture period, and the ease of releasing liquid from gels under pressure were shown to be different for different agar brands. A different availability of water and minerals in Murashige and Skoog medium was deduced from the gels prepared with three agar brands (Oxoid, Merck, and Roth).     

Leaf surface wetness in sorghum and resistance to shoot fly, Atherigona soccata: role of soil and plant water potentials

In experiments with potted plants, the relationships between soil matric potential, plant water potential and production of water droplets (leaf surface wetness) on the folded central whorl leaf of seedlings of sorghum genotypes that are either resistant or susceptible to shoot fly (Atherigona soccata) damage were investigated. Differences in soil matric potentials in the pots affected the plant water status, which in turn had profound effects on the production of water droplets on the central whorl leaf of the sorghum genotype susceptible to shoot fly. There was no consistent variation in the relationship between plant water potential and soil matric potential of resistant and susceptible sorghum genotypes. However, there was very little or practically no water droplets on the central whorl leaf of the resistant genotypes, indicating that the production of water droplets is not solely the result of internal water status of the plant. It is suggested that leaf surface wetness is genetically controlled and that an understanding of the mechanism by which water is transferred to the leaf surface will enhance breeding for resistance to shoot fly.     

The Level of Abutilon Mosaic Geminivirus in Leaf Discs and Wound Callus

The level of Abutilon mosaic geminivirus in leaf discs, wound callus and suspension culture was monitored by hybridization. Over 25 days the amount of AbMV DNA decreased in the leaf discs kept under various experimental conditions including different temperatures, light regimes and hormone concentrations. In primary callus or in suspension culture no viral DNA was detectable. When leaf discs were kept in liquid medium instead of on agar plates a shift from viral single-stranded DNA to replicative double-stranded DNA was observed when phytohormones were supplied. It is discussed here why under these conditions, rapidly dividing tissues seem to be virus-free.     

猕猴桃高频直接再生体系的建立

为了建立猕猴桃高频再生体系,以MS为基本培养基,猕猴桃(Actinidia deliciosaQinmei)茎及叶片为外植体,研究了2,4-D、6-BA和NAA在美味猕猴桃愈伤组织形成及分化过程中的作用。方差分析结果表明,6-BA能够显著促进愈伤组织形成,6-BA和NAA可以显著促进愈伤组织形成和分化,而2,4-D抑制愈伤组织形成。附加2.0 mg/L 6-BA、1.0 mg/L NAA和600 mg/L水解酪蛋白的MS培养基是茎段培养的最佳培养基,在该培养基上,以再生的无菌苗为起始材料,一个月时叶圆盘的直接再生频率达到100%,平均每个叶圆盘产生9.33个芽,其中23.21%芽高度超过0.5 cm。     

Development of morphogenic suspension cultures of garlic (Allium sativum L.)

Rapidly growing, regenerable suspension cultures were obtained from meristem-derived callus cultures of garlic (Allium sativum L.). The liquid culture medium consisted of MS salts, B5 vitamins, 3% sucrose, 1 mg l^–1 naphthalene-acetic acid (NAA) and 2 mg l^–1 6-benzyladenine (BA). The tissue in the suspension culture was yellow, smooth, organized, and proliferated as nodular clumps. Histological examination revealed that these morphogenic clumps had a well-defined epidermis. Following transfer of the morphogenic clumps to an agar-solidified medium, numerous meristems with green leaf primordia were produced.     

Genomic variability of sugar beet morphogenic and non-morphogenic callus

Cytogenetic characters and the level of DNA methylation were studied in sugarbeet callus cultures with different morphogenic potential which were obtained from the same leaf explant. It was shown that non-morphogenic callus in comparison with the morphogenic one was characterized by the higher level of cell ploidy and of the frequency of chromosome structural rearrangements even at the early stages of cultivation. Overspiralization of chromosomes and formation of condensed chromatin were observed in polyploid cells of non-morphogenic callus. At the same time cytosine hypermethylation occurs in site-specific CpCpGpG-sequences of DNA indicating the possible role of this DNA modification in the processes of genome rearrangements.     

Predator avoidance in phytophagous mites: response to present danger depends on alternative host quality

We studied whether volatiles released by putative host plants affect the antipredator response of an herbivorous mite, Tetranychus urticae, when the patch was invaded by Phytoseiulus persimilis. Tetranychus urticae laid a lower number of eggs on tomato leaves than on lima bean leaves, suggesting that lima bean is a preferred host food source for T. urticae. In addition, T. urticae preferred lima bean plant volatiles to tomato plant volatiles in a Y-tube olfactometer test. To investigate the antipredator response of T. urticae, we examined the migration of T. urticae from a lima bean leaf disc to a neighbouring leaf disc (either a tomato or lima bean leaf disc) when ten predators were introduced into the original lima bean disc. A Parafilm bridge allowed for migration between the leaf discs. No migrations occurred between leaf discs when there were no predators introduced to the original leaf disc. However, when predators were introduced migrations did occur. When the neighbouring leaf disc was upwind of the original disc, the migration rate of the mite from original lima bean leaf disc to a neighbouring tomato leaf disc was significantly lower than that to a neighbouring lima bean leaf disc. By contrast, when the neighbouring leaf disc was downwind of the original leaf disc, there was no difference in the migration rates between lima bean leaf discs and tomato leaf discs. The number of T. urticae killed by P. persimilis for each treatment was not different, and this clearly shows that the danger was the same in all treatments regardless of the decision made by T. urticae. From these results, we conclude that T. urticae change their antipredator response by evaluating the difference in host plant volatiles in the patch they inhabit.     

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Field and Vegetable Crops     

Protein variation accompanies leaf dedifferentiation in sugarcane (Sacchamm officinarum) and is influenced by genotype

Tissue culture lines (morphogenic and nonmorphogenic) were established in five genotypes of Saccharum officinarum L. Changes in protein expression after dedifferentiation of leaf tissue into callus were investigated by two-dimensional gel electrophoresis of cellular proteins. The findings demonstrated that protein expression was regulated both quantitatively and qualitatively in all five genotypes of sugarcane. Sixty-three dedifferention-proteins were identified, of which thirty-three were common to all genotypes. The expression of the remainder was dependent on the genotype, suggesting that the genotype within the same sugarcane species plays an important role in dedifferentiation. Three of the dedifferentiation-proteins were specific to morphogenic callus lines and one to nonmorphogenic callus lines. These proteins can be useful in characterizing the biochemical, molecular, and genetic properties of cultured cells in sugarcane, and in understanding the influence of genotype in the induction of dedifferentiation-proteins and their role in morphogenesis.     

Growth and Gas Exchange of Three Sorghum Cultivars Under Drought Stress

A field study was conducted to evaluate the drought tolerance of three sorghum [Sorghum bicolor (L.) Moench] cultivars, Gadambalia, Arous elRimal and Tabat, and quantify the physiological bases for differences in their drought tolerance. Water stress reduced shoot dry mass of Gadambalia, Arous elRimal and Tabat by 43, 46 and 58 %, respectively. The respective reduction in leaf area of the three cultivars was 28, 54 and 63 %. The reduction in net photosynthetic rate, stomatal conductance and transpiration rate due to water stress was lowest in Gadambalia and highest in Tabat. The leaf water potentials and relative water contents of Gadambalia under wet and dry treatments were similar, while those of Tabat were significantly reduced by water stress. The lowest and highest liquid water flow conductance was displayed by Tabat and Gadambalia, respectively. Drought tolerance in Gadambalia is associated with its smaller leaf area, higher liquid water flow conductance, and ability to maintain high leaf water potential, relative water content, stomatal conductance, transpiration rate and photosynthetic rate under drought stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

Response of durum wheat cultivars to immature embryo culture,callus induction and in vitro salt stress

Response of twenty eight cultivars of durum wheat (Triticum turgidum var. durum) to immature embryo culture, callus production and in vitro salt tolerance was evaluated. For assessment of cultivars to salt tolerance, growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1% w/v) added to the culture medium during two subsequent subcultures (4 weeks each). Comparison of cultivars for callus induction from immature embryo was based on callus induction frequency and fresh weight growth of callus (FWG). While, for salt tolerance, the relative fresh weight growth (RFWG) and necrosis percent of callus were used. There were significant differences among cultivars for potential of regeneration from immature embryo, and ‘Shahivandi’ a native durum wheat cultivar originating from western Iran was superior among the cultivars tested. The FWG distinguished cultivars more than callus induction frequency did for callus induction evaluation. Hence, a range of FWG from 1.23 to 14.65 g was observed in ‘Mexical-75’ and ‘Omrabi-5’ cultivars, respectively. Growing calli derived from cultivars ‘PI 40100’ and ‘Dipper-6’ showed superiority for tolerating salinity under in vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of water stress and rewatering on leaf water relations of lemon plants

Potted two-year-old lemon plants (Citrus limon (L.) Burm. fil.) cv. Fino, growing under field conditions were subjected to drought by withholding irrigation for 13 d. After that, plants were re-irrigated and the recovery was studied for 5 d. Control plants were daily irrigated maintaining the soil matric potential at about -30 kPa. Young leaves of control plants presented higher leaf conductance (g1) and lower midday leaf water potential (Ψmd) than mature ones. Young leaves also showed higher leaf water potential at the turgor loss point (Ψtlp) than mature leaves. In both leaf types g1 decreased with increased vapour pressure deficit of the atmosphere. From day 1 of the withholding water, predawn and midday leaf water potentials (Ψpd and Ψmd) decreased, reaching in both cases minimum values of -5.5 MPa, with no significant differences between mature and young leaves. Water stress induced stomatal closure, leaf rolling and partial defoliation. No osmotic adjustment was found in response to water stress in either leaf type, but both were able to enhance the cell wall elasticity (elastic adjustment). After rewatering, leaf water potential recovered quickly (within 2 d) but g1 did not. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen. This revised version was published online in June 2006 with corrections to the Cover Date.     

A leaf disc method for measuring cuticular conductance

This paper describes a new method for the measurement of cuticularconductance (g_o;) using a leaf disc sealed in a specially-designedenvelope. Conductances for astomatous (adaxial) and stomatous(abaxial) surfaces of beech {Fagus sylvatica L.) were determinedfrom measurements of water flux. Leaf discs were punched outfrom attached leaves and placed inside individual envelopesthat provided a water supply. Water flux from an exposed epidermalsurface of the leaf discs was measured gravimetrically. Allmeasurements were made under darkness. Conductance of the adaxialsurface was referred to as g_c, whereas conductance of the abaxialsurface was considered as a minimum leaf surface conductance()- The main advantage of this method is that it enables measurement of g_c and from leaf samples with intact cuticles and a highrelative water content [RWC) for periods of up to 12 d. Conductancesof leaf discs in envelopes were compared with those of wholeleaves and leaf discs without envelopes. Data demonstratinga strong positive relationship between conductance and RWC ispresented. Key words: Cuticular conductance, leaf disc, relative water content     

Matric potential evaluations and measurements for gelled substrates

A new method for evaluating the matric potential of gelled media has been developed. The method allows the derivation of the matric potential as a limit of a series of measurements of water potential values from gelled media prepared without added components, from agar powders progressively cleaned of mineral impurities. Three commercial agar brands were tested, and for these the matric potential was found to contribute only between 1 and 2% of their total water potential. Thermodynamic features relating matric and osmotic potentials are described. New hypotheses for understanding the water flux mechanism from gel to tissue cultured explants are discussed. Movement of water along polymeric chains is postulated to be a facilitated step in comparison with bulk movement.     

The gradient between pre-dawn rhizoplane and bulk soil matric potentials, and its relation to the pre-dawn root and leaf water potentials of four species

Uptake of soil water by plants may result in significant gradients between bulk soil and soil in the vicinity of roots. Few experimental studies of water potential gradients in close proximity to roots, and no studies on the relationship of water potential gradients to the root and leaf water potentials, have been conducted. The occurrence and importance of pre-dawn gradients in the soil and their relation to the pre-dawn root and leaf water potentials were investigated with seedlings of four species. Pre-germinated seeds were grown without watering for 7 and lid in a silt loam soil with initial soil matric potentials of -0.02, -0.1 and -0.22 MPa. Significant gradients, independent of the species, were observed only at pre-dawn soil matric potentials lower than -0.25 MPa; the initial soil matric potentials were -0.1 MPa. At an initial bulk soil matric potential of -0.22 MPa, a steep gradient between bulk and rhizoplane soil was observed after 7 d for maize (Zea mays L. cv. Issa) and sunflower (Helianthus annuus L. cv. Nanus), in contrast to barley (Hordeum vulgare L. cv. Athos) and wheat (Triticum aestivum L. cv. Kolibri). Pre-dawn root water potentials were usually about the same as the bulk soil matric potential and were higher than the rhizoplane soil matric potential. Pre-dawn root and leaf water potentials tended to be much higher than rhizoplane soil matric potentials when the latter were lower than -0.5 MPa. It is concluded that plants tend to become equilibrated overnight with the wetter bulk soil or with wetter zones in the bulk soil. Plants can thus circumvent negative effects of localized steep pre-dawn soil matric potential gradients. This may be of considerable importance for water uptake and growth in drying soil.     

Involvement of ethylene on growth and plant regeneration in callus cultures of Heliconia psittacorum L.f.

The level of ethylene accumulated in morphogenic callus cultures of Heliconia psittacorum L.f. was only one quarter that of non-morphogenic cultures. The rate of ethylene production in the morphogenic callus cultures during early stages of differentiation of protocorm-like bodies leading to plantlet regeneration was 10-fold higher than that during callus proliferation. In cultures sealed with gastight serum caps, fresh weight gain was reduced 2-to 3-fold compared to those that were closed with Kaputs. Treatment with 1-aminocyclopropane-1-carboxylic acid ( 100 M) caused complete inhibition of plant regeneration from the morphogenic callus on subsequent culture under inductive conditions. Silver nitrate and aminoethoxyvinylglycine also reduced plant regeneration. These results indicate that while high levels of ethylene were inhibitory, a low level of endogenous ethylene production may be necessary during the plant regeneration phase in callus cultures of Heliconia.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AC activated charcoal - ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BM basal medium - CH casein hydrolysate - DM development medium - MM maintenance medium - PLB protocorm-like body     

A novel glass fiber disc culture system for testing of small amounts of compounds on growth and aflatoxin production byAspergillus flavus

A new method for growingAspergllius flavus for experimental studies is presented. The system consists of a humidified vial with a thick septum pierced by a pin on which a glass fiber disc is affixed. The disc contains the test solution and inoculum plus medium. The method has been used to assess the effect of variations in culture conditions on production of aflatoxin B₁ (AFB₁). The AFB₁ level was affected by the amount of medium placed on the disc and type of disc material. The results for different types of glass fiber and quartz discs were compared with AFB₁ produced by fungus grown in liquid medium or on paper discs. When compared to a liquid medium culture there was a 15 to 20-fold increase in AFB₁ for one type of disc. Incubations with less than 14 µl of medium gave satisfactory results. A crude phosphatidylcholine preparation at a concentration of 0.7% of the medium resulted in a 4-fold increase in AFB₁.Abbreviations AFB₁ aflatoxin B₁ - CV coefficient of variation - PC phosphatidylcholine - SD suspended disc     

Plant genotype and growth regulators interaction affecting in vitro morphogenesis of blackberry and raspberry

The morphogenic response of somatic (leaf and petiole) and de-differentiated tissue (callus) of two blackberry (Rubus fruticosus) and one raspberry (Rubus idaeus) cultivars have been studied in vitro. With the aim to induce regeneration the effect of two sets of plant growth regulator (PGR) combinations (high cytokinin/auxin ratios and high auxin/cytokinin ratios) in Murashige and Skoog basal medium, were analysed. The three cultivars were characterised by a qualitatively different morphogenic response to the PGR combinations. Raspberry adventitious shoot regeneration from somatic tissue was improved by the 6-benzylaminopurine (BAP)/indol-3-butyric acid (IBA) combinations. On the contrary, shoot regeneration of both blackberry cultivars was reduced by high concentrations of BAP and completely inhibited by BAP/IBA combination. Media supplemented with high auxin/cytokinin ratios promoted callus production and root differentiation according to genotype and type of auxin. All the genotypes responded to media supplemented with IBA. 2,4-dichlorophenoxyacetic acid induced good callus formation in blackberry, but was toxic to raspberry. Indirect shoot formation was observed only in callus of blackberry cultivar Hull Thornless cultivated on medium with 10 μM BAP, the same concentration able to trigger efficient direct shoot regeneration from leaf explants of the same cultivar. This revised version was published online in July 2006 with corrections to the Cover Date.