Induction of mitosis in polytene nuclei and hormonal effect on nuclear changes during callus initiation in diploid Urginea indica Kunth. (liliaceae)

Bulb scale and inflorescence explants of Urginea indica Kunth. (2n=20) were cultured in vitro on modified Murashige & Skoog's medium with different hormonal composition. Media containing 2,4-dichlorophenoxy-acetic acid (2,4-D) (2 and 4 mgl^–1) and -naphthalene-acetic acid (NAA) (2 mgl^–1) could induce callus in inflorescence explants. Combination of 2,4-D (4 mgl^–1) + NAA (2 mgl^–1) + Kinetin (2 mgl^–1) only could induce callus formation in scale explants. The bulb scale explants contained mostly diploid cells while the inflorescence explants contained cells with nuclear DNA content ranging from 2C to 64C. The lowest karyological heterogeneity was recorded in callus derived from bulb scale and in callus derived from inflorescence induced with NAA. The highest variability was recorded on media with 2,4-D alone. Induction of division, probably of the pre-existing polytenic nuclei in the inflorescence explant, has been suggested to be the cause of origin of polyploid cells in such cases.     

SERK Gene Homolog Expression,Polyamines and Amino Acids Associated with Somatic Embryogenic Competence of Ocotea catharinensis Mez. (Lauraceae)

In the present work, we investigate the association of SERK gene homolog expression, polyamines (PAs) and amino acids related to putrescine synthesis (arginine and ornithine) and polyamines degradation (-aminobutiric acid) or S-adenosylmethionine synthesis (methionine), with the embryogenic competence in cell aggregates of Ocotea catharinensis Mez. (Lauraceae). Cell aggregates were cultivated during 7 days in woody plant medium (WPM) supplemented with 20 g l^–1 sucrose, 22 g l^–1 sorbitol, 400 mg l^–1 glutamine and 2 g l^–1 phytagel, and in Murashige and Skoog medium (MS) supplemented 20 g l^–1 sucrose, 3 g l^–1 activated charcoal, 2 g l^–1Phytagel with and without 40 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The cell aggregates cultivated in MS plus 2,4-D and in the WPM medium showed hybridization with a SERK gene homolog both in northern and in situ hybridization experiments. Cell aggregates cultivated in an MS basal medium, without 2,4-D, did not exhibit any hybridization signal to the SERK probe used, thus they were considered potentially non-embryogenic cells. In all three media only free polyamines were detected. The higher putrescine levels occurring in WPM callus were associated with a higher arginine and ornithine content, lower -aminobutiric acid level, and SERK homolog expression. Putrescine was also the major polyamine in the MS medium. In the MS plus 2,4-D medium, the levels of putrescine, spermidine and spermine were similar. Spermine exhibited similar and the lowest levels in all media. Spermidine intermediary levels occurred in the WPM and MS media. In cell aggregates methionine level was lowest in the MS plus 2,4-D medium, but similar in the MS and WPM media.     

Achillea millefolium (yarrow) cell suspension cultures: Establishment and growth conditions

Summary Friable calli were obtained fromAchillea millefolium L. hypocotyls, in Gamborg B5 medium, supplemented with 1.5mg.1^–1 2,4-D / 0.1mg.1^–1 Kin, and used for the production of cell suspension cultures in the same liquid medium. The growth pattern of the cultures was determined in permanent light or dark conditions and with different inoculum densities, basal media, growth regulators and sucrose concentrations. Different sources and nitrogen amounts were assayed to study the effect on yarrow cell growth. The conditions found to be optimal for growth of yarrow cell suspension cultures were: 70g (f.w.).1^–1 of initial inoculum in Gamborg B5 medium, supplemented with 1.5mg. 1^–1 2,4-D / 0.1mg.1^–1 Kin, NO₃ ^–/NH₄ ⁺ (30/lmM), and 2% sucrose, in darkness. In these culture conditions the cell suspensions showed a doubling time of 35–40h.Abbreviations 2,4-D dichlorophenoxyacetic acid - NAA naphtalenacetic acid - BA benzyladenine - Kin Kinetin     

Potassium influx and efflux of 2,4-D- and MCPA-treated rice plants

Summary A study was made of the effects of the herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (4-chloro-2-methyl-phenoxyacetic acid) on ion uptake, leakage and growth of rice seedlings. Using isotopically-labelled solutions containing different concentrations of 2,4-D or MCPA, it was established that 10^–4 M 2,4-D or MCPA effectively inhibited potassium ion uptake, while K-ion leakage from the roots occurred only at 10^–3 M. The growth of the rice seedlings was markedly retarded even at low (10^–6 M) concentrations, and the roots and shoots showed different tolerances to the herbicide. At 10^–8 M herbicide, the effects were not injurious, but rather favourable. Reduction in root length by herbicides was not in accordance with dry-matter production.     

Effect of 2,4-dichlorophenoxyacetic acid on the dark- and light-induced pulvinar movements in Cassia fasciculata Michx

2,4-dichlorophenoxyacetic (2,4-D) applied to excised leaves of Cassia fasciculata modified the dark-induced (scotonasty) and light-induced (photonasty) leaflet movements, showing that this compound acts on rapid turgor variation and the concomitant ion migrations, in particular K⁺. 2,4-D inhibited the scotonastic closure in a dose-dependent manner from 10^–8 M to 10^–5 M and promoted the photonastic opening in the same range of concentrations. The compound acted rapidly since a treatment as short as 5 min gave an obvious effect on the motile reaction; however, a lag period of 45–60 min was needed to observe its effect. Although 2,4-D is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. The physiological results are discussed in relation to the chemical properties and the characteristics of transport of the molecule.Abbreviations ABA abscisic acid - 6-BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - MES 2-(N-morpholino)-ethanesulphonic acid     

Somatic embryogenesis from mesophyll protoplasts of Trigonella corniculata (leguminosae)

Mesophyll protoplasts isolated enzymatically from Trigonella corniculata divided to form callus, with a plating efficiency of 49% in Kao (1977) medium. Protoplast-derived tissues formed somatic embryoids at high frequency on MS medium with 2.0 mg L^–7 NAA and 0.5 mg L^–7 BAP. Embryoids developed into plants on MS medium lacking hormones.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - MES 2-(N-morpholine) ethanesulphonic acid - NAA -naphthaleneacetic acid On leave from Genetics Institute, Academia Sinica, Beijing, China     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

Somatic embryogenesis and plant regeneration in callus culture derived from immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson

Callus was induced on the wounded immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson (Berberidaceae) on a medium based on Murashige and Skoog's (1962) formula supplemented with 1 mg/l 2,4-dichlorophenoxy-acetic acid (2,4-D). Spontaneous embryoid formation occurred on the media containing low concentrations of 2,4-D (0.1–0.5 mg/l). These embryoids germinated in either MS or B5 medium containing 1 mg/l N⁶-benzyladenine and 1 mg/l gibberellic acid. The regenerated plantlets were successfully transferred to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GA₃ gibberellic acid - MS medium Murashige and Skoog's (1962) medium     

Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium

Summary The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp. Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h^–1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methyphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions. 2,4-DP-Grown cells (10⁸) of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h. Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol. Offprint requests to: F. Streichsbier     

Effects of auxins and cytokinins on tissue culture of Grateloupia dichotoma (Gigartinales,Rhodophyta)

The role played by plant growth regulators in algae is poorly known. In order to increase the knowledge about the function of auxins and cytokinins in seaweeds, explants such as apical and intercalary segments and callus-like structures (CLS) of Grateloupia dichotoma were cultured in semi-solid or liquid artificial media ASP 12-NTA. Two auxins, indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and one cytokinin, 6-benzylaminopurine (BA), at concentrations of 0.5 and 5.0 mg l^–1 were tested. Moreover, IAA and BA were tested together at concentrations of 1:5 and 5:1 mg l^–1. All treatments promoted the growth of CLS in intercalary segments; CLS from apical segments were significantly higher in treatments with 2,4-D or IAA:BA (1:5 mg 1^–1). The morphogenetic responses for auxins and BA were opposite, auxins inhibited while BA promoted the formation of lateral branches; however, auxins promoted the elongation of such branches. The process of plant regeneration observed on CLS was stimulated significantly by treatment with high concentration of BA or IAA:BA (1:5 mg 1^–1) in semi-solid and liquid media. The growth of upright axes was stimulated significantly by treatment with 2,4-D in semi-solid medium, and IAA:BA (1:5 mg l^–1) in liquid medium. These results show the importance that plant growth regulators could have in the control of growth, morphogenetic processes and micropropagation in red algae.This paper is part of the PhD thesis of NSY.     

In vitro regeneration from excised leaves of Flaveria trinervia (Sprengel) C. Mohr

Flaveria trinervia (Compositae) leaves are used for the treatment of jaundice and fever. From the leaf callus cultures regeneration of plantlets has been achieved. The results showed that BAP greatly stimulated the bud formation in concentrations ranging from 2–5 mg l^–1 than at very low concentrations (0.2–1.0 mg l^–1). Roots developed on the regenerated shoots, over a range of treatments, but were most prolific in the medium containing 1 mg l^–1 IAA. Histological observations revealed that cultured spongy cells of the mesophyll were greatly enlarged and underwent repeated cell divisions leading to the formation of hard nodular callus from which shoot buds differentiated. The shoots obtained were readily rooted and transplanted into glass houses. Cytological studies of the callus showed abnormalities such as bridges, endomitosis and multinucleolate conditions. Root tip squashes of the regenerated plants showed no variations and were diploid in chromosome number.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA napthalene acetic acid - IAA indole acetic acid - BAP 6-benzyl aminopurine - Kn kinetin     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45–50% were obtained in liquid medium and 55–60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1–2 mm in size) were obtained in 5–6 weeks, which upon transfer to MSB medium with 0.5 mg 1^–1 each of 2,4-D, BA, Kn and 500 mg 1^–1 CH produced compact green calli in 4–6 weeks. After 3–4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1^–1 each of BA, Kn, ZT, 0.1 mg 1^–1 NAA and 500 mg 1^–1 CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA₃ enhanced shoot regeneration up to 30%. Shoots of 0.5–1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1^–1 TH and 0.5 mg 1^–1 GA₃ for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2–3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1^–1 IBA or 0.5 mg 1^–1 NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.Abbreviations BA 6-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellic acid - IBA indole-3-butyric acid - Kn kinetin - MES 2[N-morpholino] ethane sulfonic acid - NAA naphthaleneacetic acid - TH thidiazuron - ZT zeatin     

Somatic embryogenesis in cell cultures of Thapsia garganica

Cell cultures from different species of the genus Thapsia (Apiaceae) have been investigated. In one 4-yearold line of T. garganica L. spontaneous somatic embryogenesis up to the globular stage occurred in a suspension culture containing 1 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D). Also callus cultures of this line, previously maintained on a medium containing 1 mg l^–1 2,4-D, when transferred to various media deprived of 2,4-D, produced somatic embryos that developed into plantlets. Cell culture, embryos and regenerated organs were analysed for their content of thapsigargins. The undifferentiated cell culture did not synthezise thapsigargins, but was found to produce a yet unidentified compound not present in planta. White embryos in the pre-cotyledonary stage did not synthezise thapsigargins either, but when the embryos developed to the cotyledonary stage and became green, the synthesis started. Regenerated roots and shoots also contained thapsigargins.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EtOAc ethyl acetate - FDA fluorescein diacetate - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - 2-iP 2-isopentenyladenine - NAA 1-Napthaleneacetic acid     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

Comparison of a gene probe with classical methods for detecting 2,4-dichlorophenoxyacetic acid (2,4-D)-biodegrading bacteria in natural waters

Colony hybridizations with a gene probe for enumeration of 2,4-dichlorophenoxy-acetic acid (2,4-D)-degrading bacteria were compared with classical enrichment and radiolabel most-probable-number (MPN) assay methods. Two natural water samples (rivers) and raw sewage were tested by each method. UV scans of enrichment cultures revealed 2,4-D degradation with raw sewage occurred in 4–11 days, 4–>22 days with Mary's River water, and 5–>22 days with Willamette River water. [¹⁴C]-2,4-D MPN analysis, measuring release of¹⁴CO₂, yielded estimates of bacteria per milliliter able to degrade 2,4-D. Raw sewage estimates were 1.4 × 10⁵ 2,4-D degraders/ml, Mary's River >1.6 × 10⁵/ml, and Willamette River water 1.6 × 10⁴/ml. Activities noted by UV scan enrichment data supported these results.Autoradiograms of colony blots were also used to estimate numbers of 2,4-D-degrading bacteria. These estimates were also supported by the UV scan data from enrichment cultures. Raw sewage gave counts between 5 × 10⁴ and 2.9 × 10⁵ 2,4-D-degrading bacteria/ml, which correlates well with the estimates obtained by¹⁴C-MPN analyses. River waters, both much lower in total bacterial counts and organic carbon than raw sewage, yielded fewer 2,4-D-degrading bacteria than estimated by¹⁴C-MPN. Media composition and cometabolism may account for discrepancies in estimates for 2,4-D-degrading bacteria observed when colony blot and¹⁴C-MPN analyses were compared.Replica plating made it possible to test for 2,4-D biodegradation from colonies reactive in autoradiograms. Five of 12 (42%) colonies reacting in the colony hybridization exhibited biodegradation activities. Nonreactive colonies failed to degrade 2,4-D.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency