Dragan Vinterhalter

Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.

Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata

Agrobacterium rhizogenes A4M70GUS-mediated transformation of Savoy cabbage (Brassica oleracea L. var. sabauda) and two local lines of cabbage (B. oleracea L. var. capitata) was obtained using hypocotyl and cotyledon explants. The percentage of explants which formed roots was very high in all genotypes: 92.3 % in Savoy Gg-1, 64.4 % in cabbage P22I5, and 87.2 % in P34I5. Spontaneous shoot regeneration of excised root cultures grown on the hormone-free medium occurred in all three genotypes. In cabbage lines P22I5 and P34I5 shoot regeneration was higher (9.3 and 2.6 % respectively) than in Savoy cabbage Gg-1 (1.3 %). Transgenic nature of hairy root-derived plants was evaluated by GUS histological test and PCR analysis. All the tested cabbage shoots were GUS positive whilst in a Savoy cabbage GUS expression was registered only in 55 % of tested clones. PCR analysis demonstrated the presence of the GUS gene in regenerated shoot clones and in T1 progeny.

True-to-the type in vitro propagation of Aechmea fasciata Baker

Abstract In vitro propagation of Aechmea fasciata Baker was studied with the of decreasing the frequency of aberrant plant formation. Shoots differentiated from leaf explant callus were cultured on MS based agar solidified media. The frequency of aberrant plant formation was lower on kinetin (KIN)/indolyl-3-acetic acid (IAA) than on 6-benzyladenine/naphthyl acetic acid (NAA) supplemented media. A stable dark-green shoot culture clone was established by persistent use of media supplemented with 1.0 mg 1 −1 KIN and 1.0 mg 1 −1 IAA and by elimination of shoot clusters which contained shoots with pale green, light yellow and variegated leaves. The frequency of aberrant shoot formation in this clone was less than 1% prior to rooting and after potting and acclimatization aberrant plants could not be detected. On media supplemented with 0–2.0 mg 1 −1 NAA or indolyl-3-butyric acid adventitious roots were short. Their length significantly increased upon addition of 1% activated charcoal or if sucrose was replaced by equimolar glucose and fructose. The dark-green clone has been so far maintained for more than 40 subcultures throughout a period exceeding 5 years without significant changes in vigor or frequency of aberrant plant formation. Since summer 1992 it is in use for large scale commercial propagation.

Effect of Irradiance, Sugars and Nitrogen on Leaf Size of In Vitro Grown Ceratonia Siliqua L.

Carob (Ceratonia siliqua L.) has compound pinnate leaves consisting of 4 – 6 pairs of leaflets. However, in conditions of in vitro culture only one pair of leaflets develops. With increasing irradiance from 9.3 to 74.1 µmol m−2 s−1, leaf area increased 5-fold. Sucrose also significantly increased leaf area and the maxima were at concentration 147 mM at high irradiance and 233.6 mM at low irradiance. Sucrose was superior to fructose, glucose and combination of both in increasing leaf area. Decreasing concentration of KNO3 and NH4NO3 caused a 3-fold decline of leaf area.

Light-controlled root elongation in in vitro cultures of Dracaena fragrans Ker-Gawl

The effect of light on root elongation was studied in Dracaena fragrans Ker-Gawl shoot cultures grown in vitro. Root elongation depended on light conditions provided during rooting. In the irradiation range 1.0–17.0 W m-2 the rate of root elongation increased with increasing irradiance, while in darkness it was very low. Transfer of plants from darkness to light induced root elongation after a short lag period. The reverse transfer from light to darkness efficiently arrested root elongation but promoted appearance of lateral roots. Separate exposures of either shoots or developing roots to light or darkness revealed that the light effect on root elongation was exerted through the leaves. Root elongation was not dependent on sucrose and it occurred even on sucrose-free medium. Far-red light had the same effect as darkness, i.e. it did not support root elongation, while rooting in red and blue light was irradiance-dependent. The results presented in this paper show that the growth of D. fragrans root system can be manipulated by light.

In vitro propagation of Gentiana dinarica Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l−1 BA and 0.1 mg l−1 NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l−1 IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.

Effect of inorganic nutrition on the formation of lateral roots in Dracaena fragrans Ker-Gawl cultured in vitro

The effect of inorganic nutrition on the formation of lateral roots has been studied in Dracaena fragrans Ker-Gawl cultured in vitro. MS macronutrient salts imposed inhibition of lateral root formation in ≃95% of primary adventitious roots which could be overcome by:decrease of concentration of all 5 macronutrient salts.significant decrease or omission of MgSO4, ordecrease or omission of both KNO3 and NH4NO3. Inhibitory effect has been further confirmed to appear as the result of inadequate balance of SO42- with NO3- and NH4+ ions in the MS formulation of inorganic salts.

In vitro propagation of green-foliaged Dracaena fragrans Ker.

Callus tissue was induced in young stem segments cultured on MS based media supplemented with 0.25–0.5 mg l-1 2, 4-D. Shoots were differentiated on media containing 0.5–1.0 mg l-1 BA and 0.5–2.0 mg l-1 IBA or 0.1–0.2 mg l-1 NAA. The same media were suitable for shoot multiplication. Shoot elongation and rooting were strongly inhibited by BA and stimulated by auxins IBA and NAA. Medium containing 0.5 mg l-1 IBA was optimal for rooting. Root elongation was stimulated by light and inhibited in darkness. Transfer of rooted plantlets to outdoor conditions was feasible and special hardening procedures were not required. Among more than 5000 plants produced by this procedure only 9 off-type plants with variegated leaves were found.

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm−3 6-benzyladenine and 0.5 mg dm−3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm−3 phosphinothricin (L-PPT) supplemented medium were confirmed by GUS assay and PCR analysis. The transformation rate was 58 % with AGL1/ pDM805 and 25 % with LB5-1. Rooted plantlets were acclimated and then again screened for Basta®-resistance by spraying with 15 – 60 mg dm−3 L-PPT. Surviving plants were selfed and Basta®-resistance was demonstrated in T1 progeny.

Secondary somatic embryogenesis versus caulogenesis from somatic embryos of Aesculus carnea Hayne.: developmental stage impact

Somatic embryos of red horse chestnut, derived from cultures maintained through repetitive somatic embryogenesis for a few years, were subjected to induction of secondary regeneration. The embryos were divided in four classes on the basis of their size (I-1, II-5, III-10 and IV-30 mm), and sub-cultured on MS media containing 0, 1, 5 or 10 μM kinetin (Kin) or benzyladenine (BA). The pathway of secondary regeneration, somatic embryogenesis or caulogenesis, depended on the primary somatic embryo (PSE) stage of development. The embryogenic capacity declined and bud-forming capacity increased with the degree of PSE maturity. The PSE of the Classes I and II produced only secondary somatic embryos (SSE), the Class III PSE formed both SSE and adventitious buds, whereas the Class IV PSE developed almost solely adventitious buds. The process of secondary somatic embryogenesis was most effective in the Class II PSE at 5 μM BA, and the process of adventive organogenesis was most effective in the Class IV PSE at 10 μM BA. On plant growth regulator (PGR)-free medium, PSE of A. carnea followed the same pattern of adventive regeneration, as those cultured on cytokinin containing media. The cytokinins only amplified the response, in a certain range of concentrations. BA promoted bud induction at a much higher rate than Kin, while their embryogenic effect was similar.