Branka 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.

Propagation and xanthone content of Gentianella austriaca shoot cultures

Shoot cultures of Gentianella austriaca (A. & J. Kerner) Dostal established from seedling epicotyls were maintained on MS medium supplemented with 2.22xa0μM BA and 0.54xa0μM NAA. A characteristic feature of these cultures was precocious flowering, which appeared in all rapidly elongating shoots. Flower development arrested shoot elongation and multiplication of shoot cultures. Continuous shoot propagation was possible only by use of small axillary or adventitious buds as explants for subculturing. Flowering could not be suppressed by GA3 addition or by cultivation in short-day conditions. The highest rooting percentage (47.3% with 7.83 roots per explant) was achieved on media with 4.92xa0μM IBA. Shoot cultures contained the same types of secondary metabolites as plants from nature. Xanthones were the major constituents, with DMB (demethylbellidifolin), DGL (demethylbellidifolin-8-O-glucoside) and BGL (bellidifolin-8-O-glucoside) present at roughly two times lower concentrations than in samples from nature. Secondary metabolite production was strongly affected by the presence of BA in the medium.

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of Alyssum murale Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92xa0μM kinetin. Optimal shoot multiplication was at 0.46xa0μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A.xa0rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30xa0days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46xa0μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700xa0μgxa0g−1 dry weight.

Phytodecta fornicata Bruggemann resistance mediated by oryzacystatin II proteinase inhibitor transgene

Phytodecta fornicata Brüggemann is a serious pest of alfalfa (Medicagosativa L.) that causes significant crop loss in the Balkan peninsula of Europe. We introduced a wound-inducible oryzacystatin II (OCII) gene to alfalfa to evaluate its effect on survival of P.xa0fornicata larvae. Feeding bioassays with second, third and fourth instars were carried out using transgenic plants that were shown to express OCII at 24 and 48xa0h after wounding. Second and third instars were the most sensitive to the ingestion of OCII, whereas no effects were observed with fourth instars. About 80% of the second and third instars died after 2xa0days of feeding on the transgenic plants as compared to 0–40% on the controls. This is the first report that demonstrates significant increase in mortality of P.xa0fornicata on transgenic plants that express a cysteine proteinase inhibitor gene, and this knowledge should lead to the development of effective management strategies for this devastating pest of alfalfa.

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.

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1xa0mgxa0l−1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8xa0mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5xa0%, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10xa0months (ten cycles). A total of 20xa0% of the mature SSEs from cabbage and 55xa0% from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1xa0mgxa0l−1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56xa0% in cabbage and 79xa0% in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.

The effects of IAA and tetcyclacis on tuberization in potato (Solanum tuberosum L.) shoot cultures in vitro

Potato (Solanum tuberosum cv. Désirée) shoots grown inxa0vitro in continuous darkness or in long days (LDs), were used to investigate indole-3-acetic acid (IAA) effects on stolon initiation and tuber formation, combining IAA with increased or decreased gibberellin levels. An increased gibberellin (GA) level was achieved by the applying 1xa0μM GA3, while decreased gibberellin level was presumably realized by the adding 3xa0μM tetcyclacis (Tc). About 15% of potato shoots developed stolons both in LDs and in darkness. Stolon initiation was stimulated by GA3 in darkness and by Tc in LDs. Tuber formation was strongly inhibited in LDs and by GA3 both in light and darkness, but stimulated in darkness at low GA level. Exceptionally, tuber formation occurred in LDs at the highest Tc concentrations, in about 25% of explants. Indole-3-acetic acid alone stimulated stolon formation in LDs, both in the presence or absence of GA3. IAA alone also stimulated tuber formation in dark-grown shoots, but could not overcome the inhibitory effect of LDs. Indications that, depending on their concentration ratio, IAA may interact with GA3 in different tuberization phases, have been discussed.

Somatic embryogenesis and plant regeneration from root sections of Allium schoenoprasum L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5xa0μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10xa0μM. The highest frequencies of callus induction were achieved on media with 5xa0μM 2,4-D in combination with 5xa0μM TDZ or 10xa0μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1xa0μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5xa0μM 2,4-D in combination with 10xa0μM BA or 10xa0μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5xa0μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.

In vitro shoot organogenesis and comparative analysis of endogenous phytohormones in kohlrabi (Brassica oleracea var. gongylodes): effects of genotype, explant type and applied cytokinins

Kohlrabi (Brassica oleracea var. gongylodes) cultivars Vienna Purple (VP) and Vienna White (VW) were tested for their ability of de novo organogenesis in vitro. Root, cotyledon, hypocotyl explants and intact seedlings were cultivated on Murashige and Skoog (MS) media supplemented with different cytokinins: benzyladenine (BA), thidiazuron (TDZ), trans- or cis-zeatin. All tested cytokinins, including cis-zeatin, induced shoot regeneration from hypocotyl explants and intact seedlings, with seedlings being most successful for regeneration efficiency and viability of regenerated shoots in both cultivars. The highest frequency of shoot regeneration was achieved on MS with BA (60xa0%) or TDZ (50xa0%) for VP; and with BA (50xa0%), TDZ (47.5xa0%) or transZ (37.5xa0%) for VW. Measurements of the endogenous cytokinin and indole-3-acetic acid (IAA) contents in both hypocotyl explants and seedlings with regenerated shoots (HRSs and SRSs) suggested that the observed differences in organogenic response between these two types of explants were related to their cytokinin and IAA contents. HRSs generally exhibited elevated amounts of total cytokinins, while SRSs displayed a higher IAA/bioactive cytokinins ratio. Shoots regenerated from seedlings were further successfully multiplicated on a medium supplemented with BA (0.5xa0mgxa0L−1). The rooting potential of multiplicated shoots was tested on media supplemented with 2 or 4xa0mgxa0L−1 indole-3-butyric acid (IBA), with the higher concentration of IBA leading to more efficient rooting. Rooted plantlets were successfully planted into soil and flow cytometric analysis did not reveal ploidy variations, indicating that the described protocol is fast and efficient for kohlrabi regeneration.