M.P. De Proft

Degree of DNA methylation in chicory (Cichorium intybus L.): influence of plant age and vernalization

Chicory plants (Cichorium intybus L. var. foliosum cv. Flash) were harvested every 14 days from 43 to 168 days after sowing. The tissue was either immediately analyzed or after a 4 week post-harvest cold treatment at 5°C. DNA methylation was measured, using high pressure liquid chromatography (HPLC), in the root tissue as well as in the shoot apices. During the growing season, the degree of methylation ranged between 10 and 16% for root tissue as well as for shoot apices. The reaction (methylation/demethylation) to cold treatment depended on the tissue type and the plant age. For root tissue, demethylation was observed after cold treatment in the beginning of the growing season followed by methylation in the middle part and strong demethylation at the end of the season. Except for two harvest dates, a demethylation of DNA in the shoot apex tissue occurred during cold treatment. Demethylation was strongest for the two last harvest dates. This is the first report on changes in degree of DNA methylation during a whole growing season and differences in the effect of cold depending on the age of a biennial plant.

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation

Abstract It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones.

Changes in sugar content and fatty acid composition of in vitro sugar beet shoots after cold acclimation: influence on survival after cryopreservation

To cryopreserve sugar beet shoot tips using an encapsulation-dehydration technique, cold hardening of in vitro plants was needed to obtain high survival rates after freezing. Cold acclimation not only enhanced dehydration and freezing tolerance, but also induced several changes in sugar beet shoots. Plants contained greater amounts of sucrose, D-glucose and D-fructose and the fatty acid composition of lipids changed. Furthermore, the unsaturation level of membrane lipids, estimated by the (C18:2 + C18:1)/C16:0 ratio, increased after cold hardening. These changes were correlated with better survival rates after cryopreservation.

Incompatibility reactions and genotypic identity status of five commercial chicory (Cichorium intybus L.) hybrids

Incompatibility reactions of chicory plants cv. Flash, cv. Carolus, cv. Pax, cv. Focus and cv. Sigma were determined. Different levels of incompatibility were found. The cv. Carolus was highly self-incompatible, cv. Flash and cv. Pax could be self-compatible or self-incompatible and the cv. Sigma and cv. Focus are self-compatible. All the tested cultivars were intra-compatible (pollinations within the same cultivar) and cross-compatible among them. There was difference in percentage of viable seeds when cultivars were self-pollinated and intra-pollinated. This difference in percentages of viable seeds can be used as a numerical criterion to determine genotypic identity of a cultivar. The higher this difference value, the less uniform the behaviour of the cultivars in the field and during hydroponic forcing for production of the chicory head was observed. The results led to the conclusion that some of the commercial cultivars are not genotypic identical which has unfavorable implications in Belgian endive production and commercialization. There was equal reaction to incompatibility in any sense of crossing (plants used as females compared with the result when plants were used as males in cross-pollinations).

In vivo and in vitro flowering response of chicory (Cichorium intybus L.) : Influence of plant age and vernalization

Abstract Chicory plants (Cichorium intybus L. var foliosum cv Flash) were tested with and without a 4-week-long cold treatment for in vivo and in vitro flowering potential every 2 weeks during the growing season. One hundred percent of the plants harvested 112 days or later after sowing and then vernalized flowered in vivo. In vitro, no vernalization was needed to initiate flowering-stems on chicory explants taken from roots of 100 days old and older. 5-Azacytidine, a DNA demethylation agent, increased the flowering percentage on explants from young, vernalized roots but could not induce more than 15% flowering on young, nonvernalized roots. The greater flowering potential of chicory root explants in vitro when compared to plants of the same age tested in vivo was clearly established. This result suggests that some negative control on flowering was removed when root explants were excised and the main plant body discarded.

Influence of gibberellin biosynthesis inhibitors on stem elongation and floral initiation on in vitro chicory root explants under dark and light conditions

Root explants of chicory (Cichorium intybus L.) were cultured in vitro under continuous light or darkness. On a standard medium (no plant growth regulators added), flowering-stems were initiated under continuous light while under continuous dark, vegetative-stems were formed. Different types of GA (gibberellin) biosynthesis inhibitors were added to the culture medium. Paclobutrazol and compounds belonging to the group of cyclohexanetriones clearly reduced flowering-stem growth under light conditions and vegetative-stem growth under dark conditions. Under light conditions, flower bud initiation was not affected. These and other results suggest that GA1 may be synthesized during the in vitro culture period and that it controls flowering-stem growth but not floral initiation.

Stem elongation and floral initiation on in vitro chicory root explants: influence of photoperiod

Chicory root explants (Cichorium intybus L.) were cultured in vitro under different photoperiods. In complete darkness, strong stem elongation, but no flowering induction was observed. We suggest that this stem elongation could be homologous to the pit growth in chicory heads in vivo. Under a photoperiod of 12 h (LI=±40 μE m−2 s−1), only vegetative growth was observed. Photoperiods of 16 h or more light a day induced the in vitro explants to develop stems bearing flower buds. When the in vitro cultures were kept in the dark for different durations starting from the first day of culture and afterwards transferred to long-day conditions, 4 days dark were sufficient to cause a decrease in flowering induction. We suggest that during the dark culture, a flowering inhibitory process was started.

Induction of stem elongation on in vitro chicory root explants under unfavourable photoperiodic conditions by gibberellin A3

Flowering stems are formed under long-day conditions on root explants of chicory (Cichorium intybus L.) cultured in vitro while under short days, only vegetative growth is observed. Under short-day conditions (12 h), stem elongation is induced by treating newly formed shoot apices with GA3 (1 μl, 10−3M). No flower buds were formed on the GA3-induced stems. Long days seem to be indispensable for the induction of flower buds on the elongated stem.

Frost sensitiveness of chicory roots (Cichorium intybus L.)

Abstract This study was conducted to determine if chicory roots ( Cichorium intybus L. var. foliosum ) can be stored at temperatures below −1°C. Roots from cultivar Focus, produced in hydroponics on two different artificial substrates, were stored at −1, −2.5, −5 and −7°C. After storage at sub-zero temperatures, roots with highest dry weight percentage showed highest chicon production and highest organogenesis capacity. Moreover, the electrical resistance of those roots never decreased significantly during storage, while roots with a lower dry weight percentage had reduced electrical resistance after storage at −7°C. Decrease in electrical resistance was detected before frost damage became visible. Thus, measuring the electrical resistance of root tissue can be used to predict frost damage. The most distinct frost damage symptoms were ‘water soaking’ and browning of the vascular bundles. According to these results, it can be concluded that storage temperatures below −1°C, but above −7°C, are not necessarily fatal for chicory roots: roots with relatively high dry weight percentage are best able to withstand low storage temperatures.

An in vitro model for the study of hydroponic forcing in chicory (Cichorium intybus)

An in vitro model for studying the influence of different factors on chicon formation during hydroponic forcing has been developed. The shoot apex was isolated from the chicory roots and cultured on a gelled nutrient medium. This medium was considered as a replacement of the root. Small chicons (5 g) were produced. Water and, more importantly, sucrose availability had important influences on the outgrowth of the chicons. When sucrose was added to the medium the chicon-weight increased two-fold. On a medium with low agar concentration (0.3% (w/v)), heavier chicons were produced compared with a medium with agar at 1.2% (w/v). Browning of the pith tissue (= flowering stem) decreased with increased agar concentration. The results presented indicate that the in vitro system can be used as a research model to study chicon development in relation to root functioning and composition.