Use of direct somatic organogenesis for Agrobacterium -mediated transformation of onion

Abstract

An Agrobacterium tumefaciens mediated DNA delivery is being developed for onion ( Allium cepa L.) using organogenic structures formed on the the ovaries as target tissue. An indirect transformation procedure using A. tumefaciens strains LBA4404 and EHA105 in combination with four plasmids was used for establishing an efficient transformation protocol. Studies were focused on different protocols for co-cultivation, selection and detection of optimal reporter and selection genes in onion. A histochemical GUS test and visual detection of the GFP protein were used for optimization of transformation treatments. Several pretreatment and co-cultivation protocols achieving transient expression were studied, and the optimal was a combination of sonication (10 s) and vacuum treatment (5 min, 35 mmHg). Among several selection media studied, optimal results were obtained using phosphinotricin (2.5 mg/l) as selection agent and timentin (150 mg/l) for good suppression of bacterial growth. The transgenic nature of individual regenerants after six months on selection media was confirmed by polymerase chain reaction using primers for detection of uidA and bar genes. Using this test, three regenerants were positive for both bar and uidA genes and three regenerants were positive for the bar gene only.

Keywords

tissue culture, genetic transformation, onion, Allium cepa L., direct somatic organogenesis, Agrobacterium , uidA , gfp , bar , PCR

Mycorrhizal potential of two forest research plots with respect to reduction of the emissions from the thermal power plant Šoštanj

Abstract

The mycorrhizal potential of two differently polluted forest research plots was determined in the emission area of the Thermal Power Plant Šoštanj. Zavodnje is the polluted, while Mislinja is the less polluted plot. Mycorrhizal potential of the soils from the two sites was estimated in a pot experiment. Types of ectomycorrhizae were identified in the soil cores and on short roots of Norway spruce seedlings. The fresh weight of needles and stems, number of short roots and the percentage of mycorrhizal short roots on seedlings from Zavodnje were significantly lower in comparison with Mislinja. The results indicate that the mycorrhizal potential of the more polluted site is lower. Mycorrhizal potential is discussed with respect to the results from our earlier studies.

Keywords

ectomycorrhizae, mycorrhizal potential, Norway spruce seedlings, forest research plots

Impact of air pollution on the mitotic activity in meristematic cells in shallot ( Allium cepa L. var. ascalonicum )

Abstract

Test shallot plants Allium cepa L. var ascalonicum were exposed to field conditions in research plots in the most polluted areas in Slovenia in the vegetation season in 1999. The intention of this research was to evaluate the influence of air pollution on mitotic activity in meristematic tissues of root tips of bioindication plants. At each sampling site the mitotic activity was determined under field conditions and in a pot experiment. The maturated bulbs were collected from the field and after winter dormancies cytogenetic analyses were run on them in a lab. Root tips were fixed in Clark´s fixative and afterwards stained with Schiff´s reagent.

Significant differences in the mitotic activity in different sampling plots in pot experiments were found. The results showed the presence of cytotoxic substances at chosen sampling sites which caused the decrease of mitotic cell division.

Keywords

Allium cepa L. var. ascalonicum , cytotoxicity, mitotic index, environmental pollution

Growth and root respiration of C4 plants under CO ₂ enrichment

Abstract

Respiratory measurements of apical root parts of several C4 plant species ( Echinochloa crus-galli var. crus-galli , Setaria pumila and Zea mays ‘DK 312’ (Dekalb, USA) subjected to an elevated CO ₂ regime during growth in climatic chambers or at natural CO ₂ springs were performed.

Biomass production, root respiratory potential and root respiration of Echinochloa was not significantly changed by high atmospheric CO ₂ treatment in the climatic chambers, compared to ambient CO2 treatment.

Root respiratory potential of C4 weeds ( Echinochloa crus-galli and Setaria pumila ) growing in natural CO ₂ spring area was not significantly affected by extremely high CO ₂ in the rhizosphere. Yet, respiratory potential of one and a half month old sown maize seedlings was significantly lower in the roots exposed to naturally elevated CO ₂ concentrations.

Keywords

root respiration, ETS activity, respiratory potential, C4 plants, Echinochloa crus-galli , Zea mays , Setaria pumila , elevated CO ₂ , natural CO ₂ springs, CO ₂ mofette

Influence of in vitro propagation on the economically important traits of strawberry cv. Marmolada

Abstract

In vitro propagation of strawberries is a promising alternative to traditional propagation, since it provides better sanitary status of plants. The possibility of somaclonal variation presents one of the drawbacks of this method, but this phenomena can be minimised by the choice of optimal tissue culture procedure.

To evaluate the influence of in vitro growing on economically important traits of strawberry cv. Marmolada, rooted plants were produced in vitro from a long term and from a newly established culture. These plants were used as mother plants for short multiplication in the field. Vegetative and generative traits of their runner plants were compared with conventionally produced cold storage plants and plants derived directly from tissue culture. Statistically significant differences were observed among different plant types in the number of runners per plant, number of flowers per plant, number of fruits per plant and yield per plant. The most pronounced differences were observed in the number of fruits per plant and in the yield per plant. Plants obtained directly from tissue culture were almost twice as productive as conventionally produced cold storage plants. Conventionally produced cold storage plants had statistically significantly higher yields and fruit number per plant than runner plants derived from micropropagated mother plants. High yields of plants obtained directly from in vitro were mainly the results of their significantly prolonged ripening.

Keywords

strawberry, micropropagation, yield, quality, somaclonal variation

In vitro plant regeneration from somatic tissue of strawberry Fragaria x ananassa Duch.

Abstract

Successful shoot regeneration in somatic tissue is the basic requirement for in vitro induction of genetic variability as the new tool in plant breeding.

Somatic tissue excised from in vitro multiplied strawberry plants were tested on ability for plant regeneration. Leaves, petiole and stipules were inoculated on initial medium with BA and 2,4-D, or on medium with BA only after 1 hour pulse treatment with 2,4-D. Callus was induced on all sliced surfaces of explants inoculated on initial medium with growth regulators BA and 2,4-D during first 7 days of culture. Explants inoculated on initial medium with BA, after pulse treatment with 2,4-D did not develop callus but abundantly produced fenolic compounds, and tured necrotic in the first 24 hours.

Spontaneous plant regeneration was noticed on leaves explants with less developed callus tissue on initial medium with growth regulators during second week of culture. High percentage of explants with regenerated shoots were obtained after transfer on hormone-free medium.

The highest plant regeneration ability was in leaf tissue, less in petiole and stipules.

Callus induced in leaf tissue showed ability for constant plant regeneration during three months of culture and careful 4-week interval transfer on basal MS medium with 4.4μM BA and 40 g/l sucrose.

Keywords

in vitro , somatic tissue, leaf explants, plant regeneration, strawberry, Fragaria x ananassa Duch., cv. Elsanta, cv. Marmolada