Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.     

Inhibition of flower induction on in vitro chicory root explants by hydroponic forcing pretreatment of roots

Fragments of vernalized chicory roots (Cichorium intybus L.) cultured in vitro under continuous light flower almost 100%. When chicory roots were placed in hydroponic forcing before in vitro culture, flowering percentage was reduced by half. The build-up of inhibition during 3 weeks of hydroponic forcing was studied in detail. The third week, in which growth of the chicory head is the strongest, was especially important in the inhibition process. When the root apex was eliminated during hydroponic forcing, flowering inhibition in vitro was weaker. The same observation was made when adventitious roots, developed during hydroponic forcing, were removed. The photoperiodic conditions during hydroponic forcing had no influence on the build-up of inhibition. It is suggested that activity of the apex and, possibly, of the adventitious roots during hydroponic forcing cause the flowering inhibition on chicory root fragments in vitro.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) from leaf explants and accumulation of esculin

Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl⁻¹ casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants.     

Nuove ricerche sulla coltura in vitro di Aucuba japonica Thunb

Abstract

New researches on the culture « in vitro » of Aucuba japonica Thunb, – Caulinar fragments grown in vitro in nutrient media solidified with agar are compared with fragments grown on a filter paper support on unshaken liquid media. Tissue cultures of Aucuba are established putting down caulinar fragments just taken from the plant with isolated callus; subcultures are obtained more easily in July. We have observed that during the months of March or April, caulinar fragments, taken from the plant at various months of the year, of different ages and cultivated on various media, unexpectedly produce many roots.     

Morfogenesi in espianti caulinari di Aucuba japonica Thunb. coltivati in vitro

Abstract

Morphogenesis in caulinar fragments of Aucuba japonica Thunb. in vitro. — Caulinar fragments of Aucuba japonica Thunb. are grown in vitro in different media. They show an obvious polarity; in fact only the basal section surface shows a vigorous proliferation and is able to form roots. When this surface emerges it gives rise to a green luxuriant callus; when the same surface is immersed it forms a protuberance in the shape of crateriform invaginations. Our histological studies indicate not only the connection between new proliferations and the pre-existent vascular tissues, but also the areas where the pseudothalli originate. The changes in the cortex and the central cylinder which are caused by the in vitro cultures were observed.     

A phytochrome-mediated alteration from stem to rosette growth on chicory root explants in vitro

Chicory root explants (Cichorium intybus L. var. foliosum) of two cultivars, taken before and after hydroponic forcing, were cultured in vitro in complete darkness supplemented with red and far-red light treatments. Using 5 min red light per day, the strong stem elongation occurring in complete darkness was converted to rosette formation. This reaction was reversed to stem elongation (accompanied by leaf formation) adding 15 min far-red light after the red light. Fifteen min far-red light per day alone caused the same reaction as 5 min red/15 min far-red light. Far-red light followed by red light caused rosette formation. In stems, formed under complete darkness in vitro, the presence of phytochrome was shown. No phytochrome was detected in the root fragment itself.Abbreviations R red light - FR far-red light - GA gibberellinic acid - A absorbance - FW fresh weight     

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

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. Received: 31 August 1998 / Revision received: 27 October 1998 / Accepted: 10 November 1998     

Immature embryos culture in Italian red chicory

Chicory (Cichorium intybus L.) embryo maturation in vitro was achieved in B5 medium without growth regulators. Immature embryos were collected at different developmental stages 4–168 h after pollination. Only a few plantlets were obtained from ovules collected shortly after pollination since the embryos at these early stages developed abnormal leaves and roots which regenerated shoots ‘via’ organogenesis. Zygotic embryos collected 24–72 h after pollination needed one month of culture in vitro before acclimation. Embryos collected at the heart or early torpedo stage of development (three days after pollination) produced green plantlets after two weeks of in vitro culture and thus they could be transferred to the greenhouse for acclimation. Culture of zygotic embryos in vitro has shown to be a suitable technique to accelerate the breeding process in biennial cultivated Italian red chicory. In fact it is possible to obtain plantlets ready for performing the selection for the desired agronomic traits one month after pollination. Since flowering occurs after vernalization, the selection can be performed in the correct season before flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Furofuran lignans from a callus culture of Cichorium intybus

Three new and one known furofuran lignans—syringaresinol derivatives—along with the known phenylpropanoids cichoriin and syringin were isolated from a callus tissue of Cichorium intybus. The compounds were characterised by spectral methods. This is the first report on the presence of furofuran lignans in Cichorium species.     

Evolution des Teneurs en Acides Chlorogénique et "Isochlorogénique' au Cours du Traitement de Vernalisation des Racines de Cichorium intybus

In a cold-requiring variety of endive (chicory, Cichorium intybus L. ev. Witloof) we could demonstrate a relationship between the variations in the content of chlorogenic acid during the cold treatment of the roots and their disposition to produce flowers in vitro. An increase in the level of chlorogenic acid in the roots preceded the development of their aptitude to form flower buds. In addition, the three main forms of “isochlorogenic’ acid are present in the roots of this cultivar: 3,5-dicaffeoyl-quinic acid, 3,4-dicaffeoyl-quinic acid, and traces of 4,5-dicaffeoyl-quinic acid. The variations in the levels of chlorogenic and “isochlorogenic’ acids indicate that part of the chlorogenic acid (3-caffeoyl-quinic acid) is converted to 3,5-dicaffeoyl-quinic acid.     

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

Summary A transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d⁻¹) and esculin content (18.5gl⁻¹), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.     

Mycorrhization alleviates benzo[a]pyrene-induced oxidative stress in an in vitro chicory root model

Among chemicals that are widely spread both in terrestrial and aquatic ecosystems, benzo[a]pyrene is a major source of concern. However, little is known about its adverse effects on plants, as well as about the role of mycorrhization in protection of plant grown in benzo[a]pyrene-polluted conditions. Hence, to contribute to a better understanding of the adverse effects of polycyclic aromatic hydrocarbons on the partners of mycorrhizal symbiotic association, benzo[a]pyrene-induced oxidative stress was studied in transformed Cichorium intybus roots grown in vitro and colonized or not by Glomus intraradices. The arbuscular mycorrhizal fungus development (colonization, extraradical hyphae length, and spore formation) was significantly reduced in response to increasing concentrations of benzo[a]pyrene (35–280 μM). The higher length of arbuscular mycorrhizal roots, compared to non-arbuscular mycorrhizal roots following benzo[a]pyrene exposure, pointed out a lower toxicity of benzo[a]pyrene in arbuscular mycorrhizal roots, thereby suggesting protection of the roots by mycorrhization. Accordingly, in benzo[a]pyrene-exposed arbuscular mycorrhizal roots, statistically significant decreases were observed in malondialdehyde concentration and 8-hydroxy-2′-desoxyguanosine formation. The higher superoxide dismutase activity detected in mycorrhizal chicory roots could explain the benzo[a]pyrene tolerance of the colonized roots. Taken together, these results support an essential role of mycorrhizal fungi in protecting plants submitted to polycyclic aromatic hydrocarbon, notably by reducing polycyclic aromatic hydrocarbon-induced oxidative stress damage.     

Frammenti cotiledonari di Aesculus hippocastanum L. coltivati in vitro. Primi dati sul comportamento dell'amido e dell'escina

Abstract

Fragments of Aesculus hippocastanum L. cotyledons grown in vitro. First results about starch and aescin characteristic features.—Cotyledon fragments of Aesculus hippocastanum grown in vitro in different media have been able to form callus and roots. The starch granules in the new cells are compound in structure and morphologically different from the simple cotyledon granules, whereas they are similar to the granules of the other parts of the plant in toto. Moreover, the callus has no aescin even though it originates from the cotyledor tissues.     

VARIATIONS OF MORPHOGENETIC BEHAVIOR IN PLANT TISSUE CULTURES I. CICHORIUM ENDIVIA

Several-years-old callus tissue derived from mature embryos of endive (Cichorium endivia Linn., Compositae) was grown on synthetic liquid and/or agar nutrient media. Incorporation of yeast extract or high concentrations of inositol, kinetin, casein hydrolysates (pancreatic and acid hydrolysates), etc., improved growth and organ formation. Rosettes of leaves, shoots and roots were differentiated on synthetic media. On agar media shoots arose first and were from marginal meristematic areas, while the roots arose later and were from pockets of meristematic tissue located in the deeper regions of the callus. In liquid media embryoids from single cells were formed which first developed roots and then shoots.     

Variations du taux en composés sulfhydrilés acidosolubles de tissus cultivés in vitro

Variations of the Level of the Nitrogen and Thiol Groups in Tissues Culture.—Distribution of nitrogen and thiol compounds was analysed in the fragments of carrot roots cultivated in vitro. A typical gradient of –SH groups was observed: high level in the callus, low level in the base of the fragment (in contact with the medium). In discontinuous light, callus—which have a great amount of chlorophylls—contain less sulfhydric compounds than in continuous dark. With increasing age of the tissues, the concentration of the endogenous –SH groups was decreasing. The probable biochemical relations between –SH level, auxin content and auxin-oxidase activity are discussed.     

Direct embryo formation in leaves of Camillia japonica L.

Summary The culture conditions for direct embryo formation in leaves of Camellia japonica L. were established. An auxin treatment followed by incubation during 11 days in darkness on diluted Murashige and Skoog modified basal medium induced direct morphogenesis. The number of subcultures, subculture interval and leaf age affected in vitro leaf response. The results showed that the cells from a cultured leaf respond differently to the same culture conditions by forming embryos, roots, and non-morphogenic as well as organogenic callus. Direct embryo formation occurred only in the marginal leaf regions. Direct root formation only occurred in a well-defined region of the midrib whereas callus was preferentially formed on the leaf basis. The results suggest the existence of differences in morphogenic competence according to leaf regions. Plantlet regeneration was successfully achieved from somatic embryos and from leaf basisderived callus, via shoot bud induction.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.Abbreviations TGase transglutaminase - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

In vitro studies on Pea (Pisum sativum L.): I. Callus formation,regeneration and rooting

Abstract

Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.