Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

The estimation of phenylalanine ammonia-lyase (PAL) activity in intact cells of higher plant tissue

A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33–40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments ³H is stereospecifically released from the pro-3S-position of L-[2,3-³H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, ³H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.Abbreviations AOA -aminooxyacetic acid - L-AOD L-aminoacid oxidase (EC 1.4.3.2) - D-AOD D-amino-acid oxidase (EC 1.4.3.3) - L-AOPP L--aminooxy--phenylpropionic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TAL tyrosine ammonia-lyase     

Plant regeneration and genetic transformation of Lotus angustissimus

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid with kinetin, N⁶ – ² or benzyladenine in different combinations and concentrations. Only those calli induced in presence of -naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - 1AA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2iP N⁶ – ² - PA proanthocyanidins - NOS nopaline synthase - NI TII neomycin phosphotransferase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Summary Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy     

Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.     

Effects of culture conditions on isolated microspore response of barley cultivar Igri

Pretreatment of anthers in mannitol prior to isolation of microspores by glass rod homogenization was effective for in vitro induction of embryogenesis in barley cv. Igri. A procedure for separation of viable microspores using centrifugation on 20% maltose was developed. The concentration of microspores was important and greatly increased the number of developing structures. Initial culture of microspores on FHG medium containing 62 g l^-1 maltose, 4.4 M (1 mg l^-1) BA and 200 g l^-1 Ficoll-400 resulted in high frequencies of plant regeneration. Albino plant frequency was correlated to length of time in culture. Stock plant condition appeared to be a major factor influencing induction frequency. From 868 to 1738 green plants per 100 anthers were produced. The number of calli and embryos obtained and the number of green plantlets regenerated were improved by increasing the Ficoll concentration from 100 g l^-1 to 400 g l^-1 during the culture period compared to continuous culture on FHG Ficoll 200 g l^-1.Abbreviations BA benzyladenine     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Regeneration of peppermint and orange mint from leaf disks

Leaf disks from peppermint, spearmint, orange mint, lavender mint and Scotch spearmint were cultured on various Murashige-Skoog-based media in order to regenerate shoots. A significantly larger average number of orange mint leaf disks regenerated shoots on basal medium containing 44.4 M benzyladenine (BA) and 250 ml l^-1 coconut water (CW). Shoots regenerated from peppermint leaf disks cultured on basal medium containing 44.4 M BA and 250 ml or 450 ml l^-1 CW. The most shoots regenerated from orange mint leaf disks cultured on medium containing 10 g l^-1 washed Difco Bacto-agar. Disks excised from the bases of the first expanding pair of orange mint leaves cultured under dark conditions regenerated a significantly larger average number of shoots. Histological studies suggested that shoots regenerated from the palisade parenchyma cells associated with vascular tissue.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TIBA 2,3,5-triiodobenzoic acid - CW coconut water     

The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue

During the first 24 h of in vitro incubation of excised potato tuber (Solanum tuberosum L.) discs, the appearance of phenylalanine ammonia-lyase (PAL; EC 3.4.1.5) and the accumulation of chlorogenic acid are both stimulated by infection with Phytophthora infestans (Mont.) de Bary. Whereas in control tissue the level of PAL reached a stable plateau value after 40 h, in infected tissue it subsequently rose again, in one experiment, as the fungal mycelium developed. In the infected but not the control tissue, the level of chlorogenic acid subsequently fell to about to about 20% of its maximum after 50 h. The time courses of increases in cinnamic acid 4-hydroxylase (CA4H; EC 1.14.13.11; 0–60 h) and of caffeic acid acid o-methyltransferase (COMT; EC 2.1.1.42; 0–160 h) are not altered by fungal infection. If the discs are restored to the tuber environment immediately after excision, by placing them inside a host tuber, the activity of PAL as well as those of CA4H and COMT remained at the constant low endogenous level for at least 60 h, irrespective of whether the discs had first been inoculated with P. infestans. The increase in PAL may not be an obligatory feature of the P. infestans/potato compatible interaction but dependent on an underlying wound response. The experiments provide further evidence that PAL is the rate limiting step of chlorogenic acid biosynthesis in potato tuber discs.Abbreviations PAL phenylalanine ammonia-lyase - CA4H cinnamic acid 4-hydroxylase - COMT caffeic acid o-methyltransferase - CGA chlrogenic acid (5-o-caffeoylquinic acid) - gfwt gram fresh weight     

A simplified medium for in vitro tracheary element differentiation in mesophyll suspension cultures from Zinnia elegans L.

A simplified medium has been developed for the differentiation of tracheary elements in suspension cultures of mesophyll cells of Zinnia elegans L. All inorganic salts contained in media used previously were retained in the simplified medium, but most were reduced in concentration. The only organic supplements required for optimum differentiation were thiamine and nicotinic acid, in addition to the plant growth regulators N6-benzylaminopurine and -naphthyleneacetic acid, and sucrose as a carbon source. Mannitol, an osmoticum, was necessary for rapid, synchronous differentiation. This simplified medium is particularly suitable for studies of the role of Ca²⁺ in tracheary element differentiation due to the elimination of myo-inositol, an intermediate in the phosphatidyl inositol signal transduction pathway and reduction in the concentrations of Mg²⁺ and Mn²⁺, which block calcium channels. It is also possible to eliminate EDTA from the medium, enabling studies using specific calcium chelators. Additional culture variables for the optimization of differentiation are discussed.Abbreviation TE tracheary element     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Rapid differentiation of tracheary elements in cultured explants of Jerusalem artichoke

the culture of Jerusalem artichoke (Helianthus tuberosus L.) tuber explants on filter paper discs moistened with liquid medium resulted in rapid and consistent xylem differentiation. The number of tracheary elements increased in discrete steps, the first at 48 h with a second at 56–58 h, following partially synchronous mitoses at 20 and 30 h. Factors favouring xylem cell differentiation were optimum levels of both an auxin and a cytokinin, low medium nitrogen concentrations, small volumes of medium, and high culture temperatures. A cell counting method employing Feulgen-stained nuclei and suitable for quantifyings small numbers of immature tracheary elements is described.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - BAP benzylaminopurine - GA₃ gibberellic acid     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

Studies of protoplast culture types and plant regeneration from callusderived protoplasts of Asparagus officinalis L. cv. Lucullus 234

Plating efficiency and colony formation of callus-derived protoplasts of Asparagus officinalis L. cv. Lucullus 234 differed significantly with different protoplast culture media and types of culture. Osmotic conditions and hormone concentrations of liquid media produced the greatest influence on plating efficiency and colony formation in bead culture. Protoplasts grew best in bead culture with a solid modified Kao & Michayluk protoplast culture medium (KM) supplemented with 0.5 mg l^–1 -naphthaleneacetic acid (NAA), 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l^–1 kinetin, and 0.6% agarose (KM6) and a liquid modified KM medium differing from KM6 medium in sugar content, having 0.18 M sucrose and 0.18 M mannitol (A8). An average plating efficiency of 19.1% and colony formation of 15.5% was obtained one week after isolation in bead culture with the KM6 and A8 media. The highest average shoot regeneration of 92.3% was obtained with a Murashige & Skoog medium (MS) containing 0.125 mg l^–1 NAA, 0.125 mg l^–1 2,4-D, 0.25 mg l^–1 6-benzylaminopurine (6-BAP) and 3% sucrose. Plants have been regenerated and transferred to the greenhouse.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine