Embryogenic Suspension Culture and Plant Regeneration from Suspension-Derived Proto-plasts of Cucumber (Cucumis sativus L.)

Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog     

Growth interactions between calli and explants of rose plants in vitro

Growth of explants or calli of two rose cultivars ‘Sonia’ and ‘Golden Times’, was extensively promoted when they were grown on agar together with calli of rose rootstocks Rosa indica or Rosa canina, while growth of callus of a miniature rose cultivar was either not affected or inhibited. The growth of R. indica callus was inhibited when accompanied by explants of ‘Sonia’ or ‘Golden Times’. Promotion or inhibition of explants or callus growth was also observed when the agar medium was supplemented with conditioned liquid medium from cell suspension cultures of cv. Sonia or R. indica. Autoclaved conditioned medium from R. indica lost its promoting effect, while that from Sonia lost its inhibiting effect after autoclaving. The possible interaction between the rootstock and scion tissues is discussed.     

Effects of ABA on secondary embryogenesis from somatic embryos induced from inflorescence culture ofAralia cordata Thunb

In order to investigate the effect of ABA on secondary embryogenesis from somatic embryos inAralia cordata Thunb., embryogenic callus and somatic embryos were induced from inflorescence on solid MS basal medium supplemented with 1.5 mg/L 2,4-D after eight weeks without subculture. For mass production of somatic embryos, embryogenic cell clumps were maintained in liquid MS medium supplemented with 1.0 mg/L 2,4-D, and then transferred to 2, 4-D-free medium. When developing embryos at various stages were cultured separately in liquid medium with ABA (0 to 2.0 mg/L) for three weeks, and then cultured in ABA-free liquid medium for two weeks, torpedo-shaped embryos exhibited secondary embryogenesis of 65.9% in only 0.2 mg/L ABA pretreatment. Cotyledonary embryos in cultures by 0.2, 0.5 and 1.0 mg/L ABA pretreatment also exhibited secondary embryogenesis (73%, 9.4% and 6.0%, respectively). However, globular and heart-shaped somatic embryos treated with ABA did not form secondary embryos on their hypocotyl surfaces. When cotyledonary embryos were cultured in ABA-free medium or 0.2 mg/L ABA treated medium for three weeks, and then in ABA-free liquid medium for 6 weeks, the germination frequency was lower in medium with 0.2 mg/L ABA (45.9%) than in hormone-free medium (56.8%). This result seems to be related to the high frequency of secondary embryogenesis. It is suggested that secondary embryogenesis by ABA application depends upon the stage of embryo cultured and the ABA concentration.     

Effects of Activated Charcoal on Growth and Morphogenesis in Cell Cultures

The effects of activated charcoal on growth and morphogenesis in plate cultures of different plant cells have been studied. It was shown that medium containing charcoal induced embryogenesis in cultures of Daucus carota in which embryo formation could not be brought about by omitting auxin from the medium. Charcoal-medium also induced abundant root formation in older cultures of Allium cepa, which normally did not produce roots. The growth of cultures of Glycine max and Haplopappus gracilis was totally inhibited by charcoal. It is thought that activated charcoal removes substances from the medium, one of which might be auxin.     

Improved somatic embryogenesis in wheat by partial simulation of the in-ovulo oxygen,growth-regulator and desiccation environments

The effects of O₂, growth-regulators and desiccation on callus growth and somatic embryo (embryoid) development were investigated in cultures of immature embryos of two lines of Triticum aestivum L. Callus and embryoid formation were induced on media that contained N⁶-furfurylamin-opurine (kinetin) and either 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-o-anisic acid, either with or without abscisic acid (ABA). Cultures containing differentiated embryoids were then exposed to high concentrations of both ABA and indole-3-acetic acid, after which samples were desiccated to approx. 10% tissue moisture. Incubating cultures in 3.2 mmol·l^-1 O₂ (approx. 9%, low-O₂) increased embryoid formation sixfold in one wheat line and nearly threefold in another. In the former line low-O₂ caused the formation of mostly embryogenic callus. Low-O₂ also decreased precocious germination of immature embryos, decreased callus growth, and improved development and viability of the resultant embryoids. Including 1.9 mol·l^-1 ABA in the callus-induction medium reduced germination of immature embryos and reduced the incidence of embryoids with visible abnormalities. Despite the improved morphology, significantly fewer of the embryoids produced on ABA-containing medium germinated. Desiccation significantly enhanced germination of these embryoids as well as those produced on ABA-free medium.Abbreviations ABA abscisic acid - DPA days post-anthesis - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophen-oxyacetic acid - FW fresh weight - IAA indole-3-acetic acid - Kin kinetin (N⁶-furfurylaminopurine) - MS Murashige and Skoog (1962) medium Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3565     

Efficient induction of microspore embryogenesis using abscisic acid,jasmonic acid and salicylic acid in Brassica napus L

The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l^?1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l^?1 for 24 h. Highest embryo yield (286.0 embryos Petri dish^?1) was achieved using 1.0 mg l^?1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l^?1 JA for 12 h. JA (5.0 mg l^?1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l^?1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish^?1) relative to the control (136.2 embryos Petri dish^?1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.     

Somatic Embryogenesis in Sugarcane (Saccharum officinarum L.): Growth and Plant Regeneration from Embryogenic Cell Suspension Cultures

Embryogenic cell suspension cultures were established from calliderived from young leaves of sugarcane (Saccharum officinarumL.) by placing them in liquid medium containing 5 per cent coconutwater (CW), 2–3 mg 1^–1 2, 4-D and 500 mg 1^–1casein hydrolysate (CH). The cultures were maintained by transferring2.5–5.0 ml of the suspension to 35 ml of fresh mediumevery 4–5 days. Organized structures resembling the earlystages of embryogeny were formed when 2, 4-D in the medium waslowered (0.1–1.0 mg 1^–1) but these did not developbeyond the globular or early scutellar stages. High levels ofsucrose (6–10 per cent) promoted the formation of proembryoids.Plating of the suspension on MS agar medium supplemented with0.25–2.0 mg 1^–1 2, 4-D, 5 per cent CW, 500 mg 1^–1CH, with or without activated charcoal, resulted in the formationof embryogenic calli. A large number of embryoids were formedin media containing lower 2, 4-D concentrations. Transfer ofembryoids to half-strength MS medium with 6 per cent sucroseestablished plantlets which were successfully transferred tosoil. Saccharum officinarumL, sugarcane, suspension culture, embryogenesis, regeneration     

Control of Embryoid Development in Tissue Cultures of Celery

Scanning electron microscope photographs of the embryoids showedglobular embryoids attached to the surface of aggregates inliquid medium and also some free floating. The surface structureof the unattached embryoids was very irregular, but, with thechange to polarized growth in the heart and torpedo forms, thesurface of the embryoid became smoother. The stage of developmentof the embryoids could be controlled by modifying the compositionof the medium to the extent that the majority of the embryoidsin the culture were either globular or torpedo forms. One ofthe most effective compounds in controlling development was2,4–dichlorophenoxy acetic acid (2,4–D). At high2,4–D concentrations, embryogenesis in the callus wasrestricted to the globular stage and after two subcultures itwas totally repressed, while after ten subcultures the potentialfor embryogenesis was lost and could not be regained even aftersubculture on a normal medium. On the normal agar medium thecallus always continued to show embryogenesis, but when it wastransferred to liquid medium of the same composition, embryoidswere produced in the first subculture but the potential haddeclined by the third subculture, when only roots were produced,and after ten subcultures cell growth and all differentiationwas totally it hibited. However, in the first subculture inliquid medium, embryogenesis was sequential with the whole cultureprogressing from globular to torpedo forms. This was particularlyeffective when the callus inoculum had been maintained on ahigh 2,4–D concentration for the two subcultures priorto inoculation of the liquid medium. By making use of this sequentialchange in embryoid development, a large number of embryoidscould be obtained at any particular stage. Apium graveolens, celery, tissue culture, embryoids, 2,4–D     

Improved cryopreservation procedure for long term storage of synchronised culture of grapevine

Anther-derived pre-embryogenic masses (PEMs) of callus, established via suspension cultures, were encapsulated to form synthetic seeds suitable for cryopreservation. The synchronised suspension culture proliferation necessitated the optimisation of plant growth regulators for different cultivars. The growth phase and density of the culture were also important as well as the exposure of cells to vitrification solution containing 0.75 M sucrose with 0.1 M CaCl₂ and 2.0 % sodium alginate (pH 5.7). Pre-treatment of the encapsulated cells for 2 d with Nitsch and Nitsch (NN) medium containing 0.75 M sucrose solution followed by dehydration for 4 h in a laminar flow box provided maximum cell viability, which varied from 0 to 40 %. The embryo proliferation from the cryopreserved beads involved warming them and then transfer to NN medium containing glutamine (50 mg dm⁻³) and activated charcoal (2.5 %). The maximum number of embryos obtained was 31–53 per bead. Subculture into the same medium induced secondary embryogenesis, which was initiated from the meristematic region, radicle, and root cap. Proliferation and maturation of secondary embryos was faster than of primary embryos. No phenotypic variation or abnormal structures compared to the control were observed in the regenerated plantlets.     

Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze)

Addition of betaine to the inductionmedium significantly enhanced the rapid formation ofsomatic embryos directly without callusing from maturefresh seeds of tea within two weeks of cultureinitiation. The induction response was furtherenhanced when ABA (7.5 mgl^–1) was co-supplementedwith betaine in the induction medium. The rate ofinduction of somatic embryogenesis increased linearlywith external betaine concentration. Globular somaticembryo-like structures (embryoids) were observed in 4-week old cultures when inoculated on the inductionmedium without ABA and betaine. The positive effectof ABA on the induction process was found to bedependent on the presence of betaine in the medium. ABA alone in the medium could not bring the inductionstimulus in the explants; on the contrary, it provedinhibitory. The optimum response of ABA was observedwhen the medium was supplemented with 500 to1000 mgl^–1 of betaine. Primary somatic embryosobtained in the presence of ABA and betaine were ableto produce secondary embryos. A conversion rate of15–20% was achieved upon transfer of somatic embryosof size 3–5 mm in diameter to the basal medium consistof half strength of macro nutrients, full strength ofmicro nutrients and vitamins of MS. Medium wasfurther supplemented with 100 mg l^–1 each ofadenine hemisulfate sulphate and L-glutamine, 30 gl^–1 sucrose, gelled with 7 gl^–1 bitek agar. The plantlets regenerated by this procedure did notshow any visible abnormalities. This report for thefirst time details the potential use of betaine inplant tissue culture.     

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmano?lu’ and ‘Sar?a?lama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmano?lu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L)?+?kinetin (KIN; 2 mg/L)?+?indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L)?+?1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L)?+?IBA (0.01 mg/L), and (3) KIN (2 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sar?a?lama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L)?+?KIN (2 mg/L)?+?IBA (0.01 mg/L), (2) BA (1 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmano?lu’ and between 0 and 0.49 per ‘Sar?a?lama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA₃), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA?+?0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA₃ (3 mg/L).     

Somatic embryogenesis and plantlet formation from a rare and endangered tree species,Oplopanax elatus

We tested the possibility of plantlet formation via somatic embryogenesis with leaf segments and mature zygotic embryos from a rare and endangered tree species,Oplopanax elatus. To induce calli, expiants were cultured under darkness in a solid MS medium containing 3% sucrose, 1g L^-1 glutamine, and 0.3% gelrite. Treatment supplements included 2,4-D alone or in combination with thidiazuron. Generally, callus induction and growth were good from leaf expiants, whereas embryogenic calli could be induced only from zygotic embryos. These embryogenic calli were white or pale yellow and very friable. ABA and activated charcoal appeared to be important factors when inducing somatic embryos, with optimum levels being 0.1 mg L^-1 and 0.02%, respectively. Many somatic embryos showed abnormalities during their development on the germination medium, but 35% could be converted if placed on a medium containing gibberellic acid (GA₃). The germinating embryos sometimes formed secondary embryos at the lower portion of the hypocotyls. Normal or converted plantlets were acclimatized in an artificial soil mixture; their survival was about 60% after two months. This culturing system provides a feasible approach for regenerating plants, via somatic embryogenesis, from mature zygotic embryos.     

Endogenous ABA concentration and cytoplasmic membrane fluidity in microspores of oilseed rape (Brassica napus L.) genotypes differing in responsiveness to androgenesis induction

Key message

A better understanding of androgenesis with a focus on the changes in plasma membrane fluidity and endogenous ABA content affecting embryogenesis induction in microspore suspension of B. napus.

Abstract

Changes in plasma membrane fluidity (MF) and ABA content associated with androgenesis induction were under the study. Both parameters were monitored in microspores of two Brassica napus L. genotypes differing in their response to androgenic induction under heat (1 day at 32 °C). MF was assessed by DPH method. ABA content was evaluated by ELISA. Heat caused microspores’ plasma membrane to become more rigid. Lower MF in microspores of ‘DH 4079’ (of high androgenic potential) seems to maintain proper cell protection and leads to efficient embryogenesis induction. Plasma membrane remodelling coincided with changes of ABA content in microspores and in the culture medium in both genotypes. ABA concentration (μM) and ABA content (fmol per 10⁴ microspores or pmol g^?1 FW) were for the first time measured in microspores. ABA concentration (μM) in microspores and in the culture medium (nM) differed significantly for the genotype and the treatment. The interaction between both variables was also significant. In general, ABA content ranged from <3.5 to 87.1 fmol per 10⁴ microspores. The highest content of ABA was detected in ‘DH 4079’ microspores at 32 °C. Assuming a mean microspores’ radius of 10 μm, it corresponds to ABA concentration of 2.1 μM. Heat shock resulted in quantum of medium pH reduction (0.1–0.2) and increased levels of ABA in microspores and in the medium of both tested genotypes. However, heat induced increase of ABA content in microspores of non-responsive ‘Campino’ had no clear-cut impact, on androgenesis induction efficiency, which suggests a more complex mechanism of process initiation.     

Effects of virus infection on plant growth,root development and phytohormone levels in in vitro-cultured pear plants

Virus infection is common in pear (Pyrus spp.) trees commercially cultivated in China. In this study, the growth speed, root development and phytohormone levels of virus-free and virus-infected pear cultures were comparatively investigated. Results showed that the co-infection of Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) significantly decreased the growth and proliferation of in vitro plants of P. communis cv. ‘Confenence’, but showed relatively less effect for P. pyrifolia cv. ‘Jinshui no. 2’, P. communis cv. ‘Red Clapp Favonite’ and P. sinkiangensis cv. ‘Korla’. Strong inhibition of ASGV infection to root development was observed for in vitro plants of ‘Jinshui no. 2’, but not for ‘Confenence’. Furthermore, during root induction, ASGV infection significantly increased CTKs/IAA ratio and decreased IAA/ABA ratio in the rooting region of in vitro plants of ‘Jinshui no. 2’. Nevertheless, for in vitro plants of ‘Confenence’, these values were rarely influenced by ASGV and ASPV infection. The result indicated that the changes of CTKs/IAA and IAA/ABA ratios might be responsible for rooting inhibition of in vitro plants of ‘Jinshui no. 2’.     

In vitro culture of the seagrass Halophila decipiens

The seagrass Halophila decipiens Ostenfeld was grown axenically in a culture medium consisting of 20% artificial seawater, f/4 nutrients (except that glutamic acid was the nitrogen source), and 1% sucrose (w:v). The culture medium was adjusted to pH 5.0. A root–rhizome layer was created by solidifying a portion of the medium with 0.9% agar (w:v) and 1% activated charcoal (w:v). The rhizome layer also contained the following vitamins: 0.5 mg l⁻¹ nicotinic acid, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ biotin, 0.5 mg l⁻¹ cyanocobalamin and 0.1 mg l⁻¹ of thiamine HCl. The liquid overlay (without vitamins or charcoal) was poured onto the agar-solidified root–rhizome layer. Growth of H. decipiens was not improved by the addition of the auxins indoleacetic acid (IAA), indolebutyric acid (IBA) or naphthaleneacetic acid (NAA) at either of the tested concentrations (10 and 50 μM). At a concentration of 10 μM, the cytokinins 6-(γ,γ-dimethylallylamino) purine (2iP) and benzylaminopurine (BA) stimulated shoot and branch production compared to controls with no cytokinins. Among the tested nitrogen sources, growth was best on 1.7 mM glutamic acid. Cultures grown on 1.7 mM NH₄Cl showed the same growth rates as those grown on glutamic acid, but the leaves were smaller and curled, suggesting incipient ammonium toxicity. Use of nitrate or urea led to mortality of the cultures. Long term axenic culture of H. decipiens appears to require the added vitamins. Hence, H. decipiens is the first seagrass known to need exogenous vitamins. Cultures of H. decipiens died when grown suspended in liquid cultures or in a biphasic medium system without activated charcoal in the root–rhizome layer. The use of more highly charged κ-carrageenan could not replace the use of activated charcoal and agar in the root–rhizome layer.     

CHARACTERIZATION OF AN EMBRYOGENIC CELL SUSPENSION CULTURE DERIVED FROM CULTURED INFLORESCENCES OF PENNISETUM AMERICANUM (PEARL MILLET,GRAMINEAE)

An embryogenic suspension culture was established from cultured inflorescence segments of Pennisetum americanum in Murashige and Skoog's medium supplemented with 2.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 5% coconut milk. The suspension was composed of two major cell types: 1) small, richly cytoplasmic and starch-containing cells, generally found in small, compact clumps, here termed embryogenic cells; and 2) elongated, thick-walled cells with large vacuoles. By manipulating the duration of culture and dilution ratios (cell suspension: fresh medium) at the time of subculture, suspensions consisting predominantly of embryogenic cells were obtained. Suspensions grown for 2-3 wks were transferred to agar media with reduced amounts of 2,4-D. This resulted in the production of hundreds of globular and early cotyledonary embryoids. Further development of the embryoids was promoted by their transfer to a medium containing abscisic acid. Many of the embryoids germinated and produced normal green plants. Atypical embryoids, some containing many shoot meristems and a leafy scutellum, were also observed. The relevance of such atypical embryoids in the interpretation of organogenesis and embryogenesis reported in tissue cultures of cereal species is discussed. It is also suggested that somatic embryogenesis occurs in tissue cultures of most, if not all, species of cereals and grasses.     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture