Showing papers on "Zeatin published in 1995"

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

Abstract: As known from literature lateral buds from pea (Pisum sativum) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists. The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Abstract: Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 μmol PPFD m-2 s-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 μmol PPFD m-2 s-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Abstract: Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 μM α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 μM inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 μM. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 μM and decreased thereafter up to a concentration 32 μM 2,4-d. A concentration of 8 μM BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.

Effects of auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis induction from shoot apices of pea

Abstract: Studies were conducted to test the effects of various auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis from shoot apices of pea (Pisum sativum L.) cultured on a sole medium. Picloram (4.5 μM) and 4-chlorophenoxyacetic acid (45 μM) were the most effective auxins. Addition of cytokinins (benzyladenine, zeatin, kinetin) to auxin-containing medium reduced embryo production. Amino acids (glutamine, alanine, proline) did not improve somatic embryogenesis. Carbohydrate seemed to be a critical factor. Embryogenic efficiency and embryo development were promoted by high carbohydrate concentration. The best results were obtained with fructose (252–504 mM); the number of somatic embryos per cultured explant was 3- to 4-fold higher compared to the control (84 mM sucrose). From these results, an optimized induction medium is proposed.

Somatic embryogenesis of Prunus subhirtella autumno rosa and regeneration of transgenic plants after Agrobacterium-mediated transformation.

Abstract: Embryogenic lines of Prunus subhirtella autumno rosa were established on a modified MS medium supplemented with 1 mg/l NAA, 0.06 mg/l IBA and 0.04 mg/l BA from petioles of axenically grown shoots of adult origin. To induce normal development of plantlets we compared a range of approaches on solid culture media as well as in suspension cultures including treatments with ABA, GA3, zeatin, darkness, and cold. A series of experiments were conducted to follow the temporal pattern of somatic embryo development.

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

Abstract: The responses of nitrate reductase (NR) activity and levels of NR-mRNA to environmental nitrate and exogenous cytokinins are characterised in roots and shoots of barley (Hordeum vulgare L., cv. Golf), using a chemostate-like culture system for controlling nitrate nutrition. Experiments were mainly performed with split root cultures where nitrate-N was supplied at a constant relative addition rate of 0.09 day −1 , and distributed between the subroots in a ratio of 20%:80%. The subroot NR-mRNA level and NR activity, as well as the endogenous level of zeatin riboside (ZR), increased when the local nitrate supply to one of the subroots was increased 4-fold by reversing the nitrate addition ratio (i.e. from 20%:80% to 80%:20%). Also shoot levels of ZR, NR-mRNA and NR activity increased in response to this treatment, even though the total nitrate supply remained unaltered. External supply of ZR at 0.1 μM caused an approximately 3-fold increase in root ZR levels within 6 h, which is comparable to the nitrate-induced increase in root ZR. External application of ZR, zeatin, isopentenyl adenine or isopentenyl adenosine at 0.1 μM caused from insignificant to 25% increases in NR-mRNA and activity in roots and up to 100% stimulation in shoots, whereas adenine or adenosine had no effect. No synergistic effects of perturbed nitrate supply and cytokinin application were detected in either roots or shoots. The translocation of nitrate from the root to the shoot was unaffected by application of ZR or switching the nitrate distribution ratio between subroots. The data give arguments for a physiological role of cytokinins in the response of root and shoot NR to environmental nitrate availability. The nature and limitations of the physiological role of cytokinins are discussed

Agrobacterium mediated genetic transformation and plant regeneration via organogenesis and somatic embryogenesis from cotyledon leaves in eggplant (Solanum melongena L. cv. 'Kecskeméti lila').

Abstract: Novel and efficient protocols for plant regeneration and genetic transformation from longitudinally-halved cotyledons ofin vitro raised seedlings in eggplant (Solanum melongena L.) are described. After co-cultivation withAgrobacterium vectors harboring neomycin phosphotransferase (nptll) as selectable marker, transgenic plantlets were regenerated on selective media containing 100 mg/l kanamycin. Transformants were recovered from embryogenic calli induced by 4 mg/lα-naphthaleneacetic acid (NAA), and from organogenic calli induced by the addition of 2 mg/l zeatin plus 0.01 mg/l NAA. Nineteen independent transgenic lines were grown to maturity. The structural integrity, expression and sexual transmission of the introduced genes for neomycin phosphotransferase and s-glucuronidase (gus) were investigated.

Simultaneous analysis of cytokinins, auxins and abscisic acid by combined immunoaffinity chromatography, high performance liquid chromatography and immunoassay

Abstract: A method has been developed for the rapid and simultaneous extraction and analysis from plant material of 3-indolylacetic acid (IAA), naphthalene acetic acid (NAA), abscisic acid (ABA) and the cytokinins benzyladenine (BA), zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine and isopentenyl adenosine. The method involves extraction with 80% (v/v) methanol, pre-purification of the extracts through reversed phase C18 Sep-Pak cartridges and immunopurification. The separation of the different compounds was accomplished by reverse-phase high performance liquid chromatography for cytokinins, and by partition with diethyl ether for IAA, NAA and ABA. After methylation of the IAA fraction with diazomethane, quantification of all plant growth regulators (PGRs) was made by immunoassay. The percentage recovery at each step was monitored following the addition of radioactive compounds at the beginning of the process. The final recovery was 68% for IAA, 92% for NAA, 76% for ABA and 75% for BA. The method was applied to the analysis of PGRs in tissues and callus of kiwifruit (Actinidia deliciosa Liang and Ferguson).

Callus induction and plant regeneration from gladiolus

Abstract: A method for the initiation of callus capable of plant regeneration from in vivo grown cormels of gladiolus (Gladiolus x grandiflorus Hort.) is described. Sliced cormels of the large-flowering hybrid, ‘Peter Pears’ were cultured in vitro on a modified Murashige and Skoog medium, supplemented with various auxins. Yellow callus, which was either friable or compact, could be induced on all media tested. Callus induced on media with naphthaleneacetic acid failed to proliferate. Callus induced on media with 9 mM 2,4-dichlorophenoxyacetic acid showed the best growth. Addition of micro-elements and vitamins increased the induction and growth of callus capable of plant regeneration. Explants taken from the middle part of the cormels had the highest competence for callus initiation. Callus was induced on several gladiolus hybrids and the South African species G. garnierii Klatt. Callus induction was genotype dependent and among the cultivars tested, ‘Peter Pears’ and ‘White Prosperity’ were superior with respect to callus production on the media with either 2,4-dichlorophenoxyacetic acid or picloram. Plants were regenerated from yellow compact callus of all genotypes on media containing zeatin and benzyladenine in various concentrations.

Differential response of the two cotyledons of Vigna radiata in vitro

Abstract: Cotyledons of mature seeds of Vigna radiata were found to be variable in their response to N 6-benzytadenine, kinet in and zeatin. The two cotyledons of a seed were designated as CE and C; where CE referred to the cotyledon that remained closely attached to the embryonal axis, and the other more loosely attached cotyledon was referred to as C. Shoots formed from the proximal end of both explants in all nine cultivars studied. Shoot regeneration was faster and regeneration efficiency was higher in CE explants than in C explants in these cultivars. BA was found to be the most suitable cytokinin for both multiple shoot induction and regeneration.

An evaluation of the role of cytokinins in the development of abnormal inflorescences in oil palms (Elaeis guineensis Jacq.) regenerated from tissue culture.

Abstract: Tissue cultures and regenerant plants from cell lines producing palms with normal and abnormal flowers were analyzed for cytokinin content and compared with zygotic embryos and seedlings. Immature inflorescences at the critical stage of flower development dissected from normal and abnormal palms were also analyzed. High performance liquid chromatography (HPLC)/radioimmunoassay and HPLC/enzyme-linked immunosorbent assay methods were used over a period of several years to measure the isoprenoid cytokinins. The results of analyses of endogenous aromatic cytokinins, present at comparable levels, will be reported separately. Oil palm cultures and regenerant plants contained relatively high concentrations of the 9-glucosides of isopentenyladenine ([9G]iP) and zeatin ([9G]Z). The predominant biologically active isoprenoid cytokinin present was zeatin riboside ([9R]Z), with lesser amounts of isopentenyladenine (iP) and isopentenyladenosine ([9R]iP). There was evidence of small amounts of dihydrozeatin compounds, but high concentrations (mainly as dihydrozeatin-9-glucoside ([9G]DHZ)) were confined to the haustorium of the zygotic embryo. Callus tissue contained very low concentrations of cytokinin. Frequently only [9G]iP could be detected, at about 1 pmol · g-1 fresh weight, with [9R]Z at less than 0.05 pmol · g-1. In comparison, nodular embryogenic tissues in vitro contained between 30 and 1,500 pmol · g-1 of [9G]iP, 5–50 pmol · g-1 of [9G]Z, and up to 12 pmol · g-1 of [9R]Z. Shoots of regenerant plantlets and seedlings contained lower concentrations of [9G]iP (3–30 pmol · g-1), although this was still the predominant cytokinin. [9R]Z and [9G]Z were present at between 2 and 15 pmol · g-1, with iP at 1–5 pmol · g-1 and [9R]iP at between 1 and 12 pmol · g-1. Seedlings contained similar amounts with the exception of a lower [9G]iP content (5–10 pmol · g-1) and more [9R]iP (10–20 pmol · g-1). Root tissues of ramets contained significantly higher concentrations of [9G]iP than shoots. Comparison of two isogenic lines of one clone giving rise to normal and abnormal palms showed significantly higher concentrations of [9R]Z and [9G]Z in the normal than in the abnormal line and, in embryoids only, higher [9G]iP in the normal line. In all other cases the between-done differences were greater than any normal/abnormal differences. There was a general tendency for increased concentrations of [9G]iP in abnormal lines and for this compound to be in a higher concentration in embryoids and plants derived from culture than in zygotic embryos and seedlings. Analysis of cytokinins in immature female inflorescences of normal and abnormal palms of a single clone showed the abnormal inflorescences to have higher concentrations of [9R]Z and [9R]DHZ and less [9G]Z than the normal inflorescences at comparable stages of development.

The effect of an elevated cytokinin level using the **ipt** gene and N^{6} -benzyladenine on single node and intact potato plant tuberization **in vitro**

Abstract: Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L-1 N 6-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje.

Abstract: A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv Bintje Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 00 or 90 μM, in combination with 228 μM kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR) Step II media were 2,4-D-free media containing 578 μM gibberellic acid (GA3) and growth regulators similar to those of step I media Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation Zeatin riboside containing step I and II media caused shoot regeneration in a high number (975±22) of explants Approximately, 337±84 shoots were regenerated from each leaf explant

Biological activity of galactoglucomannan-derived oligosaccharides

Abstract: A mixture of galactoglucomannan-derived oligosaccharides (GGMOs), degree of polymerization 4–8, (≈1.2 μM and ≈12 μM) stimulated the viability of spruce [Picea abies (L.) Karst] embryos predominately on media supplemented with indole-3-acetic acid: zeatin (0.01∶1, 1∶0.01 mg · 1-1), at pH 5.O. Their effects on the development and morphogenesis of embryos were dependent on the culture conditions used. These GGMOs also improved the viability of spruce protoplasts when applied at the same concentrations in combination with 1-naphthaleneacetic acid, and to a lesser extent with 2,4-dichlorophenoxyacetic acid at pH 3.8. Viability was also maintained in the presence of GGMOs when the growth hormones were absent; however, the efficiency of protoplast division was low.

Seeds, coated or impregnated with a PPFM

Abstract: Seeds coated or impregnated with at least one pink pigmented facultative methylotroph (PPFM) have improved germinability. PPFMs can be cultured to produce the cytokinin zeatin.

Identification of a major cytokinin in coconut milk

Abstract: A major cytokinin found in coconut milk was isolted by using the tobacco callus growth-promoting assay as a guide during purification. The structure of the factor was determined to be 14-O-{3-O-[β-d-galactopyranosyl-(1→2)-α-d--galactopyranosyl-(1→3)-α-L-arabinofuranosyl]-4-O-(α-L-arabinofuranosyl)-β-d-galactopyranosyl}-trans-zeatin riboside [G3A2-ZR] by various NMR techniques, including heteronuclear multiple bond connectivity by 2D multiple quantum NMR (HMBC), as well as mass spectroscopy and sugar analysis. The optimum concentration of G3A2-ZR for cytokinin activity in the tobacco callus assay was estimated to be 5×10−6 M, so that G3A2-ZR is one order of magnitude more potent than 1,3-diphenylurea and one order less potent than zeatin riboside. At least 20% of the cytokinin activity of coconut milk could be attributed to G3A2-ZR.

A procedure for quantification of cytokinins as free bases involving scintillation proximity immunoassay

Abstract: Cytokinins occur in a diversity of forms and determination of their individual levels requires extensive purification However, determination of the total level of each major base in free, riboside and nucleotide forms would often be adequate Hence, a methanolysis procedure which releases cytokinin bases from 9-ribosyl derivatives was developed and applied to plant extracts A simple procedure, involving low pressure column chromatography, for purification of the cytokinin bases in treated extracts, and a scintillation proximity immunoassay for their quantification, were developed The total level of each cytokinin base [N6-(2-isopentenyl)adenine, zeatin and dihydrozeatin] in free and ribosylated forms determined by these methods is reported for several plant tissues and the results are compared with those obtained after additional purification by HPLC Values for zeatin were not changed by HPLC but isopentenyl-adenine and dihydrozeatin levels were usually reduced indicating the presence of unknown compounds which cross-react in the immunoassay Modifications to the above purification method to quantify O-glucosyl cytokinins are also described The methods described facilitate the quantification of the total amount of each cytokinin base in forms closely associated with cytokinin action, and the detection of cytokinin biosynthesis by labelled precursor incorporation

Identification of an N-Glucoside of cis-Zeatin from Potato Tuber Sprouts.

Abstract: A compound was isolated from potato (Solanum tuberosum L. cv Bintje) tuber sprouts by immunoaffinity chromatography with antibodies against the cytokinins zeatin riboside and isopentenyladenosine. Analysis by ultraviolet spectroscopy and gas chromatography-mass spectrometry of derivatives identified the compound as a 9-glucoside of 6-[(Z)-4-hydroxy-3-methyl-2-butenylamino]purine (cis-zeatin). N-glucosides have often been reported as metabolites of other cytokinins, but to our knowledge, they have never before been found for cis-zeatin. The finding gives proof that cis-zeatin, a modified base in tRNA, also exists as a free substance in plants, since the glucoside, unlike other tRNA-free cis-zeatins described earlier by others, cannot arise by enzymatic degradation of tRNA during plant extraction.

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato.

Abstract: We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens Growth and development of several transformants were examined during in vitro culture Transformants exhibited phenotypes varying in severity and were divided into four classes A number of the ipt transformants had a normal phenotype, as non-transformed plants Others showed a mild to severe ‘cytokinin-like’ phenotype Transformants with a mild phenotype exhibited reduced internode length and reduced root development Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils Several adventitious meristems and shoots developed from below the surface of these structures It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation

Embryogenesis and plant regeneration from maize zygotes by in vitro culture of fertilized embryo sacs.

Abstract: Fertilized embryo sacs of Zea mays were isolated and cultured In vitro. Each explant contained one zygote and 2–4 endosperm nuclei which formed, respectively, embryo and cellular endosperm during the culture. In our double-layer/two-phase culture system, NBM medium (Mol et al. 1993) supplemented with 0.1–1.0 mg·l−1 zeatin and 12 % sucrose showed the best results. On this medium, embryos were isolated from 37–54 % of two-week-old explants. They were similar to maize embryos developing in vivo. We have shown that development of stage-2 embryos (according to Abbe and Stein 1954) with two leaf primordia and normally differentiated provascular tissue is possible from the maize zygote in an in vitro culture system. Some embryos with enlarged and deformed scutellum or whole apical parts were also found. Up to 62 % of the embryos germinating on a simple medium regenerated into mature and fertile plants; i.e. 23 % of explants yielded plants. This unproved culture method results in better embryo differentiation and 14-fold increase of regeneration frequency than previous protocol.

In vitro regeneration of Pacific silver fir (Abies amabilis) plantlets and histological analysis of shoot formation

Abstract: Summary A protocol is described for the in vitro propagation of Abies amabilis (Dougl.) Forbes. Over 60% of the cotyledonary explants from 5-day-old cotyledons formed shoots when cultured for 7 days on Schenk and Hildebrandt’s (SH) medium containing 10 µM N 6 -benzyladenine (BA) followed by another 7 days on SH medium containing 10 µM each of BA and zeatin. Shoot multiplication was unsuccessful. Seventeen percent rooting was obtained after pulsing fo r4hi n 1 mM indole-3-butyric acid and planting pulsed shoots in 1/1 (v/v) perlite/vermiculite. Cell clusters (promeristemoids) of five to seven cells were observed on the cotyledonary explants after 7 days of culture in the presence of cytokinin. These cells developed further into meristematic domes and apical meristems. In the absence of cytokinin, stomata and resin canals reached maturity, whereas cells within the cortex became vacuolated and developed into palisade and spongy mesophyll.

Stimulation of Shoot Regeneration from Cotyledon Segments of Tomato (Lycopersicon esculentum Mill.)

Abstract: Cotyledon segments of tomato (Lycopersicon esculentum Mill.) were cultured on the Murashige and Skoog (MS) medium containing 0.1 mg•liter-1 IAA, 1.0 mg•liter-1 zeatin, and 3% sucrose, and agar, agarose or gellan gum as a gelling agent and supported on a polyester material. Many adventitious buds were induced and the induced adventitious shoots grew normally on all the agars and gellan gum. Normal adventitious shoots were rarely obtained using agarose and polyester support.Shoot regeneration was stimulated by a water extract from agar when explants were cultured on the polyester support. Ethanol soluble and insoluble fractions of agar extract stimulated shoot regeneration; the former was less effective than the latter.These results indicate that the extractable, highly hydrophilic substances in agar are probably one of the factors that make them suitable for the regeneration of tomato plants.

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

Abstract: High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.

An efficient in vitro regeneration system for Australian-grown chickpea (Cicer arietinum) cultivars

Abstract: A comparison of methods for efficient in vitro regeneration of Australian-grown chickpea (Cicer arietinum L.) cultivars was undertaken. The most efficient regeneration system was one where immature cotyledon and embryonic axis explants, 14-21 days post-pollination, were cultivated on Murashige and Skoog's salts with Gamborg's vitamins, 1.0, 3.0 or 5.2 mg L-1 zeatin, 0 or 35 μg L-1 indole-3-acetic acid, 30 g L-1 sucrose and 8 g L-1 Phytagar. The first embryoid structures appeared after 2 weeks of culture at 25 ± 1°C in dim light (150 μmol m-2 s-1) and formed directly on the edges of the immature cotyledons or petiole stumps. Between 10 and 20 structures were produced on each cotyledon explant in two cultivars, however, the embryogenic structures which developed on cv. Narayen were more efficiently transformed into shoots than far cv. Amethyst. An efficient regeneration medium (2 mg L-1 naphthaleneacetic acid, 1/2 Murashige and Skoog's salts with Gamborg's vitamins, and 0.5 g L-1 activated charcoal) was used to develop a portion of the shoots into morphologically normal plants growing in a vermiculite and soil potting mix in a growth room. Less efficient in vitro regeneration was observed when hypocotyl and shoot sections, and shoot apices were induced to form callus and plants by organogenesis. These plants could not be established in a potting mix. The amount and type of callus produced varied between explant type and cultivar.

Regeneration of dihaploid chicory (Cichorium intybus L. var. foliosum Hegi) via microspore culture.

Abstract: In order to develop fully inbred chicory plants, dihaploid plants were raised from callus derived from microspores of three selected Witloof, Robin and Treviso types. Microspores were isolated from florets containing pollen at the uninuclear state and cultured in a modified MS medium plus 0.5 mg/l 2,4-D, 0.5 mg/l IAA and 2.0 mg/l zeatin. During culture periods of up to 6 months, gametoplasts emerged from pollen grains, divided and started to form colonies and calli. These were subcultured on the same basal medium supplemented with 0.5 mg/l BA and 0.5 mg/l IAA. Shoot growth was enhanced on a low salt-containing medium supplemented with 0.4mg/l kinetin and 0.2 mg/l IAA. Shoots were rooted on a half-strength Lepoivre medium plus 0.2mg/l IBA and finally transferred to soil. Florets were excised from 34 capitula, but only microspores from four of them developed into plants via callus. More than 450 plants were raised in the greenhouse and the field. Leaves from these plants were subjected to DNA fluorescence analysis via flow cytometry : a range of ploidy levels was detected. The cell composition of 44 of these plants was predominantly haploid, with a diploid background. Regenerant plant phenotypes were compared with the parent genotypes. The value of such haploids in commercial chicory breeding is discussed.