Temperature influence on stable T-DNA integration in plant cells

Four co-cultivation temperatures (15°C, 19°C, 25°C, and 32°C) were evaluated to determine their effects on T-DNA transfer and stable integration. Tobacco leaf explants were co-cultivated with Agrobacterium tumefaciens LBA4404 containing plasmids encoding resistance to the herbicide phosphinothricin, and Bt for insect resistance. Transgenic plants were evaluated for insect and herbicide resistance as well as at the molecular level for foreign gene integration. Even though 19°C has been reported as the optimal temperature for Agrobacterium-mediated gene transfer, co-culture at 25°C led to the highest number of stable transformed plants. Although 19°C may be the best temperature for the Agrobacterium transfer machinery, co-culture at 25°C appears beneficial for plant cell susceptibility to infection and for stable T-DNA insertion into the plant chromosomes.     

An effective and reproducible transformation protocol for the model resurrection plant Craterostigma plantagineum Hochst.

Procedures previously established for plant regeneration and Agrobacterium tumefaciens-mediated genetic transformation of the desiccation-tolerant plant, Craterostigma plantagineum, have been further developed. A highly effective tissue culture system was established based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. The undesirable hyperhydricity of Craterostigma tissue in tissue culture was also circumvented by these modifications, which serve as an alternative to the previously described procedures. The high efficiency of plant regeneration from the callus phase provided the basis for optimising genetic transformation in Craterostigma. For gene delivery, both a standard (method A) and a modified protocol (method B), the latter having previously resulted in successful Agrobacterium-mediated transformation of monocot cereals, were applied. Physical and biochemical key variables in transformation were evaluated, such as gene gun-mediated microwounding of plant explants, infiltration of Agrobacterium suspension cultures into target tissues and the influence of in vitro pre-induction of vir genes. While the physical enhancement of Agrobacterium infection (microwounding, infiltration) had no positive effect, the in vitro pre-induction of vir genes (biochemical enhancement) resulted in a twofold increase in the transformation frequency as compared to the conventional protocol (method A).     

Allometry in the giant tropical ant, Paraponera clavata

Summary: Workers of the ponerine ant, Paraponera clavata, grow allometrically, with the head disproportionately larger in larger workers. The anterior (clypeus)-posterior (occiput) dimension of the head capsule is allometric with the lateral (interocular) dimension, so that larger workers have elongate heads. The degree of head capsule allometry varies among colonies; this could result either from differences in colony maturity or from variation in developmental patterns among colonies. These data resolve a previously controversial issue concerning caste in Paraponera clavata.     

Efficient recovery of transgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker gene expression

Our previous studies have shown that tCUP, a cryptic promoter from tobacco, functions in all living plant cell types in a wide range of plant species. This led us to investigate if an enhanced derivative, EntCUP(, could be used to drive the neomycin phosphotransferase II (nptII) gene and select for kanamycin resistance in crop species that regenerate by organogenesis or embryogenesis. Tobacco (leaves), cauliflower (hypocotyls) and alfalfa (leaves, petioles, stems) explants were co-cultivated with Agrobacterium containing either EntCUP(-nptII-nos or 35S-nptII-nos to compare the efficiency of selection for kanamycin resistance. The infected alfalfa explants were placed in somatic embryo induction media, whereas tobacco and cauliflower explants were placed in shoot induction media with kanamycin at concentrations that normally inhibit regeneration. Transgenic plants were recovered from all of the explants with both selectable marker gene constructs. The transformation efficiencies using tCUP(-nptII-nos were comparable to or higher than those using 35S-nptII-nos in all three species tested. This study demonstrated that promoters which are not associated with expressed plant genes can be used as alternatives for the expression of selectable marker genes in a broad range of tissues and species for the generation of transgenic plants.     

Interactions between extrafloral nectaries, aphids and ants: are there competition effects between plant and homopteran sugar sources?

Broad bean (Vicia faba), an annual plant bearing extrafloral nectaries (EFN) at the base of the upper leaves, is regularly infested by two aphid species, Aphis fabae and Acyrthosiphon pisum. EFN and A. fabae are commonly attended by the ant, Lasius niger, while Ac. pisum usually remains uninfested. Sugar concentration and sugar composition of extrafloral nectar did not change significantly after aphid infestation. The sugar concentration was significantly higher in EFN (c. 271 µg µl^-1) than in the honeydew of A. fabae (37.5 µg µl^-1). The presence of small A. fabae colonies had no significant effect on ant attendance of EFN, which remained at the same level as that on plants without A. fabae. Obviously, there was no significant competitive effect between the two sugar sources. We suggest that the high sugar concentration in the extrafloral nectar may outweigh the higher quality (due to the presence of melezitose) and quantity of aphid honeydew. Ants and the presence of EFN influenced aphid colony growth. While A. fabae colonies generally grew better in the presence of ants, Ac. pisum colonies declined on plants with EFN or A. fabae colonies. We conclude that EFN may provide some degree of protection for V. faba against those sucking herbivores that are not able to attract ants.     

Isolation and molecular characterization of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> from coffee plants in Costa Rica

Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica’s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as “crespera” disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 μm in width and 1.5 to 3.0 μm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.     

How does colony growth influence communication in ants?

Summary. As Lasius niger societies grow from incipient nests to mature colonies, their foraging strategies shift from the individual exploitation of food sources to mass recruitment. Colony size instead of age is the key factor that shapes the exploratory and foraging responses of Lasius niger: a drastic reduction (or increase) of the population elicits an activity profile similar to that observed in younger (or older) societies of the same size. As a colony grows, the proportion of patrollers significantly decreases while the proportion of conveyors remains rather constant. As regards the energetic return, it increases with incipient nest size due to the replacement of minims by ordinary workers of larger crop capacity. We also demonstrate that minims of incipient nests modulate their trail-laying behaviour according to the social context, in this case the colony size. During their ontogenesis, L. niger colonies exhibit a progressive integration of individual foragers into a network of communication, the adaptive significance of which is discussed.     

Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene

Ponkan (Citrus reticulata Blanco), one of the most important commercial cultivars of mandarin, is very seedy. In this study, the chimeric ribonuclease gene (barnase) driven by an anther tapetum-specific promoter (pTA29) was introduced into embryogenic callus of Ponkan by Agrobacterium-mediated transformation using the bar gene as a selectable marker. In contrast to previous reports, embryogenic calluses were used as the explant for Agrobacterium infection and transgenic plant regeneration. Selection of transformed callus was accomplished using basta. After 3 days of co-culture, calluses were transferred to MT medium with 50 mg/l basta and 400 mg/l cefotaxime. Resistant calluses were recovered and proliferated after three to four subcultures and then regenerated plantlets. A total of 52 resistant plants were recovered, of which 43 were verified to be transformants by polymerase chain reaction amplification of a fragment of the transgene. Southern hybridization of seven randomly selected transformed plants further confirmed their transgenic nature. The potential of this strategy for breeding citrus seedless types is discussed.     

Desiccation of plant tissues post-<Emphasis Type="Italic">Agrobacterium</Emphasis> infection enhances T-DNA delivery and increases stable transformation efficiency in wheat

Summary Factors influencing the Agrobacterium-mediated transformation of both monocotyledonous and dicotyledonous plant species have been widely investigated. These factors include manipulating Agrobacterium strains and plasmids, growth conditions for vir gene induction, plant genotype, inoculation and co-culture conditions, and the selection agents and their application regime. We report here a novel physical parameter during co-culture, desiccation of plant cells or tissues post-Agrobacterium infection, which greatly enhances transfer DNA (T-DNA) delivery and increases stable transformation efficiency in wheat. Desiccation during co-culture dramatically suppressed Agrobacterium growth, which is one of the factors known to favor plant cell recovery. Osmotic and abscisic acid treatments and desiccation prior to inoculation did not have the same enhancement effect as desiccation during co-culture on T-DNA delivery in wheat. An efficient transformation protocol has been developed based on desiccation and is suitable for both paromomycin and glyphosate selection. Southern analysis showed approximately 67% of transgenic wheat plants received a single copy of the transgene.     

The effects of interspecific interactions on resource use and behavior in a desert ant

We examined how interspecific competition in ants affects resource use and behavior. To test how neighboring Myrmecocystus colonies influence the desert ant Aphaenogaster cockerelli, we placed temporary enclosures around Myrmecocystus spp. colonies and recorded the resources collected by A. cockerelli and the numbers of A. cockerelli ants engaged in various tasks outside the nest. When neighbors were enclosed, A. cockerelli colonies collected a significantly higher proportion of termites and significantly less plant matter than when neighbors were active. The numbers of A. cockerelli ants engaged in foraging behavior and nest maintenance work increased when Myrmecocystus colonies were enclosed. Interspecific interactions thus can affect the behavior and resource use of A. cockerelli colonies and may influence colony fitness.     

Reproductive schedule and factors affecting soldier production in the eusocial bamboo aphid Pseudoregma bambucicola (Homoptera, Aphididae)

Summary: The reproductive characteristics of the soldier-producing aphid Pseudoregma bambucicola were studied in Kagoshima, Southern Japan, to know the factors affecting soldier production of eusocial aphids. The soldier proportion in aphid colonies was highest from October to November. In some large colonies, soldiers were observed in all seasons except in July when colony size was relatively small. Multiple regression analysis showed that the colony size was a principal factor affecting soldier proportion throughout a year. Other social or environmental factors such as aphid composition, host plant conditions and predator abundance were not always significant. Rearing experiments revealed that large colonies (̿,000 individuals) produced soldiers in almost all seasons while small colonies (<1,000) never produced any soldiers. The caste-production schedule of adult females was examined in the field. When solitary females produced both castes, they usually produced normal nymphs first and then soldiers. Females from large colonies tended to produce more soldiers in the earlier period of their lifetime, whereas females from newly established small colonies produced no or only a few soldiers at later times. The average number of soldiers and normal nymphs produced consecutively by a single female was >10 and >20, respectively. Because they have a small number of ovarioles (<15 on average), females should alter caste production within the same ovarioles according to changes in environmental conditions. Artificial removal or introduction of predators and reduction of colony size did not affect soldier production over two successive generations, revealing maternal effects on soldier production. Females cannot shift caste production quickly in response to changes in predator abundance and colony size. This is probably due to early developmental determination of castes within the mother's body.     

Prevalence of virus-like particles within a staghorn scleractinian coral (<Emphasis Type="Italic">Acropora muricata</Emphasis>) from the Great Barrier Reef

Transmission electron microscopy (TEM) was used to determine whether Acropora muricata coral colonies from the Great Barrier Reef (GBR), Australia, harboured virus-like particles (VLPs). VLPs were present in all coral colonies sampled at Heron Island (southern GBR) and in tagged coral colonies sampled in at least two of the three sampling periods at Lizard Island (northern GBR). VLPs were observed within gastrodermal and epidermal tissues, and on rarer occasions, within the mesoglea. These VLPs had similar morphologies to known prokaryotic and eukaryotic viruses in other systems. Icosahedral VLPs were observed most frequently, however, filamentous VLPs (FVLPs) and phage were also noted. There were no clear differences in VLP size, morphology or location within the tissues with respect to sample date, coral health status or site. The most common VLP morphotype exhibited icosahedral symmetry, 120–150 nm in diameter, with an electron-dense core and an electronlucent membrane. Larger VLPs of similar morphology were also common. VLPs occurred as single entities, in groups, or in dense clusters, either as free particles within coral tissues, or within membrane-bound vacuoles. VLPs were commonly observed within the perinuclear region, with mitochondria, golgi apparatus and crescent-shaped particles frequently observed within close proximity. The host(s) of these observed VLPs was not clear; however, the different sizes and morphologies of VLPs observed within A. muricata tissues suggest that viruses are infecting either the coral animal, zooxanthellae, intracellular bacteria and/or other coral-associated microbiota, or that the one host is susceptible to infection from more than one type of virus. These results add to the limited but emerging body of evidence that viruses represent another potentially important component of the coral holobiont.     

Transient expression of ipt gene enhances regeneration and transformation rates of sunflower shoot apices (Helianthus annuus L.)

The limiting step in the transformation protocol for sunflower (Helianthus annuus L.) is the induction of adventitious shoots from embryonic axes. This specific element of an established protocol was addressed in the present study. The explants were bombarded with tungsten particles coated with a construct harbouring the ipt gene of Agrobacterium vitis, involved in the de novo synthesis of cytokinins, in addition to the usual co-culture with an Agrobacterium strain harbouring the gene of interest, i.e. the uidA gene. The proportion of GUS-expressing shoots was increased in the bombarded samples. The expression of the ipt gene was transient and the construct was not incorporated in the regenerated shoots. After introduction of this additional step in the transformation protocol, the rate of recovery of transgenic shoots after Agrobacterium-mediated transformation was 6.2%, corresponding to approximately 3 times the control value obtained in experiments without the additional bombardment step or where explants were bombarded with naked particles.     

Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were inoculated with 1 ml of diluted (OD_600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin^?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin^?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration. Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997     

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.     

Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression

Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of microwounds into the target plant tissue. Using immature cotyledons of soybean as explants, we evaluated the effects of the following parameters on transient β-glucuronidase (GUS) activity: cultivars, binary vectors, optical density of Agrobacterium during infection, duration of sonication treatment, co-culture conditions, length of explant preculture and addition of acetosyringone during co-culture. The extent of tissue disruption caused by sonication was also determined. The highest GUS expression was obtained when immature cotyledons were sonicated for 2 s in the presence of Agrobacterium (0.11 OD_600nm) followed by co-cultivation with the abaxial side of the explant in contact with the culture medium for 3 days at 27°C. The addition of acetosyringone to the co-culture medium enhanced transient expression. No differences were observed when different cultivars or binary vectors were used. Cotyledons sonicated for 2 s had moderate tissue disruption, while the longer treatments resulted in more extensive damage. Received: 1 October 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998     

Colony formation by chicken hematopoietic cells and virus-induced myeloblasts

Leukemic myeloblasts and cells derived from normal chick hematopoietic tissue produced colonies in soft agar. Colonies produced by leukemic myeloblasts differed from normal chick tissue in their morphological characteristics, in the greater initial number of cells required for colony formation and in their decreased dependence on conditioned medium for development. The colony forming cells for both types were enriched when allowed to grow for several days in liquid growth medium. In soft agar, myeloblasts differentiated into more mature granulocytic cells and macrophages. These differentiated cells accumulated between one and two weeks after seeding. When tested for release of avian myeloblastosis virus (AMV), 6 out of 18 colonies were releasing AMV at one week whereas 3 out of 39 were releasing AMV at two weeks. Five two week old colonies which were negative for AMV were producing myeloblastosis associated viruses (MAVs). Normal colony forming cells were present in leukemic buffy coat and although colonies made by these cells contained MAVs, no AMV could be detected. The data obtained with normal avian tissues were similar to those obtained by others with mammalian hematopoietic tissue. Colony formation by normal hematopoietic tissues was strictly dependent on factors present in conditioned medium. Tissues producing colonies included bone marrow, yolk sac, spleen and peripheral leukocytes. Colonies were not obtained from thymus and bursa. Furthermore, the colony origin did not appear to be erythroid in nature.     

The biology of Brevineura froggatti and phylogenetic conservatism in Australian allodapine bees (Apidae,Allodapini)

Summary: We present the first data on the social biology of the allodapine bee, Brevineura froggatti. Colony sizes are small, and only 12.5% of nests contained more than two females. Brood rearing occurs throughout the year, including winter, as it does in the other species of Brevineura studied to date. In both Brevineura species, per capita brood production is much higher in multi-female nests than single female nests, raising the question of why more colonies aren't multifemale. The occurrence of small colony sizes, despite large benefits to group living, differs strongly from species of the sister clade Exoneura. These findings, combined with previous allodapine studies, indicate conservatism in voltinism, brood phenology and colony size within, but not between, Australian allodapine genera.     

Applying dimorphic yeasts as model organisms to study mycelial growth: Part 1. Experimental investigation of the spatio-temporal development of filamentous yeast colonies

Colony development of the dimorphic yeasts Yarrowia lipolytica and Candida boidinii on solid agar substrates under glucose limitation served as a model system for mycelial development of higher filamentous fungi. Strong differences were observed in the behaviour of both yeasts: C. boidinii colonies reached a final colony extension which was small compared to the size of the growth field. They formed cell-density profiles which steeply declined along the colony radius and no biomass decay processes could be detected. The stop of colony extension coincided with the depletion of glucose from the growth substrate. These findings supported the hypothesis that glucose-limited C. boidinii colonies can be regarded as populations of single cells which grow according to a diffusion-limited growth mechanism. Y. lipolytica colonies continued to extend after the depletion of the primary nutrient resource, glucose, until the populations covered the entire growth field which was accomplished by utilization of mycelial biomass.