Silvia Schiff

Callus cultures of Artemisia absinthium L.: initiation, growth optimization and organogenesis

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 μM 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 μM BA and 2.69 μM NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.

Response of in vitro cultures of Nicotiana tabacum L. to copper stress and selection of plants from Cu-tolerant callus

In Nicotiana tabacum L. var. BEL W3 copper (Cu) at concentrations higher than 50 μM significantly inhibited callus growth and shoot regeneration. After 5–6 months of culture only a few morphogenic callus lines survived in the presence of 100 μM Cu. These calluses showed the capacity to grow and regenerate shoots through successive subcultures on medium containing 100 μM Cu. The 100 μM Cu-tolerant shoots, in comparison to regenerated control shoots, formed roots only when cultured in the presence of 100 μM Cu. From five independent Cu-tolerant callus lines in a culture period of 4–5 months more than 50 plants (defined ‘tolerant’) able to grow in presence of 100 μM Cu were obtained. These plants showed normal xylem tissue formation while control regenerated plants growing in normal Cu MS content (0.1 μM) had few xylem elements in the central cylinder. No difference as far chlorophyll content and chloroplast structure was found among Cu-tolerant and control plants. In Cu-tolerant plants, dry matter production was higher than in controls, particularly in roots.

Effects of manganese on Nicotiana species cultivated in vitro and characterization of regenerated Mn-tolerant tobacco plants

Abstract Increasing manganese (Mn) concentrations to 5, 10, 15 and 20 mM caused a significant decrease in callus formation and shoot regeneration in comparison to 0.1 (control medium) and 2 mM in Nicotiana tabacum var. BEL W3 grown on Murashige and Skoog’s medium (MS) with 1 mg l−1 α-naphtaleneacetic acid and 1 mg l−1 kinetin. Concentrations of Mn higher than 5 mM also reduced callus growth in Nicotiana bigelovii bigelovii grown on MS solid medium. Callus cultures of N. tabacum with enhanced tolerance to Mn were selected by progressively elevating the level of MnSO4·4H2O in the culture medium. Selection of Mn-tolerant plants occurred only at 2 and 5 mM, because higher concentrations inhibited callus and shoot growth. Differences in mitotic phase indices in plants grown at 2 and 5 mM Mn were found in comparison to the control. In these plants, several mitotic alterations as well as damaged chloroplasts and a reduction in chloroplast number were also observed, especially for those treated with 5 mM Mn. Mn was also shown to affect the number of xylem elements and the degree of lignification, which differed between plants grown at 2 or 5 mM compared to the control.

In vitro culture of species and varieties of four Amaranthus L. species

SummaryCallus induction, callus growth and organogenetic processes were investigated in hypocotyl and stem cultures of four species of Amaranthus each of which comprised several varieties. Callus formation occurred in almost all the varieties in 100% of explants. The combinations of naphtaleneacetic acid plus benzylaminopurine or 2,4-dichlorophenoxyacetic acid plus Kinetin were very effective in causing callus formation; the results for callus growth in presence of the same growth regulator combinations revealed in some cases significant differences among the varieties.As far organogenesis-based on a few varieties-only A. caudatus and A. hypochondriacus responded well forming shoots from callus when cultured in presence of indoleacetic acid plus Kinetin and/or indoleacetic acid plus benzylaminopurine. Root regeneration was also observed in several varieties. Complete plantlets were obtained from rooted shoots.The results are discussed in relation to hormonal effect and the genotype importance.

Studies on callus growth and morphogenesis in several species and lines of t Amaranthus

A study has been carried out on several Amaranthus L species and breeding lines (A. caudatus, A. cruentus, A. hybridus, A. hypochondriacus), some of which have previously shown poor or no plant regeneration from callusing primary explants. Callus formation occurred from explants from almost all the lines tested. Significant differences in callus growth were observed among the lines depending on the growth regulator combinations used. Cultural conditions such as type and growth regulator combination, as well as age-dependent competence of the explant tissues, also influenced the frequency of shoot (and/or root) regeneration in Amaranthus species that had been shown difficult to regenerate in the past. The results obtained demonstrate that in Amaranthus the genotype, growth regulator dose and combination, the type and the physiological stage of explant are factors of great importance for in vitro plant regeneration.

In vitro culture of Foeniculum vulgare: callus characteristics in relation to morphogenesis

Callus induction and morphogenic response of several fennel populations were determined by genotype and hormonal treatment. 100% callus formation occurred only in the Francia Pernod population under the action of 2, 4-dichlorophenoxyacetic acid or α-naphthaleneacetic acid and kinetin. Only this last hormonal treatment induced shoot regeneration. Plant regeneration was observed especially in Francia Pernod population. Ths calluses grown in presence of 2,4-dichlorophenoxyacetic acid or α-naphthaleneacetic acid plus kinetin, showed considerable differences at cytological and histological level which were correlated to their different morphogenic capability.

Manganese toxicity to different growth processes in vitro in Nicotiana

Manganese toxicity to germination, callus induction and shoot regeneration was studied on three cultivars of Nicotiana tabacum: BEL W3, Burley 21, Bright NC 944. All materials were cultured on MS solid medium containing 0.1 (control), 2, 5, 10, 15 and 20 mM Mn, to which 5 µM NAA and 5 µM kinetin were added for callus induction and shoot regeneration. Mn toxicity to callus growth was tested using habituated callus of Nicotiana bigelovii var. bigelovii grown on MS medium without growth regulators. Mn concentrations higher than 2 mM were toxic for germination, and concentrations higher than 5 mM were toxic for callus induction, shoot regeneration and callus growth. Among the cultivars examined, Bright tobacco appeared more tolerant to high Mn concentrations during callus formation and shoot regeneration. However, many regenerated plants capable of growing in vitro in the presence of 2 and 5 mM Mn were obtained.

Morphogenic effect of colchicine in Cichorium intybus L. root explants cultured in vitro

Abstract A simple and efficient protocol for in vitro shoot regeneration from storage root expiants of witloof chicory (Cichorium intybus L.) has been used to study the effect of colchicine at different concentrations, and colchicine in combination with kinetin or 2,4-D or with 2,4-D and kinetin, in comparison with kinetin and control (no treatment), upon this type of morphogenic event. The results demonstrated a strong stimulatory effect of colchicine at low concentration on shoot regeneration frequency when compared to the number of shoots formed in the control. This effect was comparable to that of kinetin, a growth regulator that induces shoot differentiation. By contrast, the presence of 2,4-D completely inhibited shoot regeneration. Colchicine at high concentration did not induce shoots, and caused the complete death of the expiants. A carefully histological analysis of the expiants during the in vitro culture period showed the formation of numerous meristematic zones (precursors of adventitious bud and shoot development) in the parenchyma mass; these meristems were present only in the expiants subjected to the colchicine and to the other treatments able to stimulate shoot differentiation. Nuclei of different sizes were observed in the explants treated with colchicine at low dose, the largest, probably, polyploids. Polyploid nuclei, in fact, were found in the regenerated plants.

In vitro morphogenetic capacity of Nicotiana tabacum L. var. BEL W3 upon cyclic culture and regeneration

SummaryVariation in the capacity to regenerate shoots and morphogenetic potential were investigated by cyclic culture and regeneration of Nicotiana tabacum var. BEL W3. Explants were excised from different shoots formed by the same callus line. Differences in the organogenetic in vitro response among regenerated plantlets was observed in two subsequent regenerate ‘generations’. A progressive decrease of the regenerative capacity was observed across callus line ‘generations’.

Morphological and cytological observations of wild species and hybrids of Gypsophila

Gypsophilapaniculata L. is among the 10 best selling cut flower species globally and is considered a “target” plant for breeding varieties. The aim of this work was to make the genetic improvement of Gypsophila more rational and focused. The study of basic chromosome number and the reproductive system (potential barriers to intraspecific and interspecific incompatibilities) of the wild species as well as the commercial hybrids forms the basis of crossbreeding and selection of variety. A significant difference in ploidy was found between the species paniculata, its cultivar Flocon de Neige doubles, the species libanotica Boiss. (n = 34) and Pacifica Kom. (n = 68). A significant difference in the chromosome number was found among the commercial hybrids of Gypsophila under investigation, and this difference was observed among them as well as when compared with the wild type species. The flower structure of commercial hybrids can be remarkably complex. Some hybrids such as Biancanieves® and MeryG® showed empty pollen sacs, whereas others such as “Mirabella” and New Love® showed pollen grains trapped into anthers. However, the pollen, when present, exhibits immature or degenerated appearance. The examined commercial hybrids showed a gynoecium morphologically and anatomically conforming to that of wild species and it was potentially fertile. Therefore, the fertile pollen collected from the wild species could be used to fertilize the compatible eggs of commercial hybrids, thereby speeding the process of breeding varieties.