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     

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     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid     

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica

Summary InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 M 4-PU and 1 M NAA at a cell density of 5 × 10⁴/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1–1 M 4-PU and 1 M NAA, and 10 M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 M 4-PU or 10 M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 M IBA and 0.1 M NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IBA indole-3-butyric acid - 2ip N ⁶-(2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - 4-PU N-(2-chloro-4-pyridyl)-N–phenylurea - TDZ thidiazuron     

Factors that effect leaf regeneration efficiency in apple,and effect of antibiotics in morphogenesis

Several factors that affect the frequency of organogenesis in apple leaf explants were examined for the scion cultivars Empire, Freedom, Golden Delicious, Liberty, McIntosh, and Mutsu and for the rootstocks Malling 7A and Malling 26. The main factors affecting morphogenesis were BA concentration, basal medium, leaf explant origin and maturity, explant orientation, and photosynthetic photon flux. Depending on the genotype, optimal regeneration was obtained using either 22.2 or 31.1 M BA and the N6 basal medium, with the exception of Golden Delicious which regenerated better on MS medium. After 6 weeks, the average number of shoots per segment varied from 5 to 16, and the percentage of regeneration between 70 and 100%, depending on the genotype tested and the maturity of the explant. Regeneration capacity increased dramatically from the tip towards the base of the leaf, and was higher from the middle to the proximal end.Cefotaxime and carbenicillin, two antibiotics commonly used during transformation studies to eliminate Agrobacterium tumefaciens from plant tissue, were tested to determine their effect on morphogenesis. Cefotaxime at a dose of 250 mg 1^-1 enhanced regeneration and shoot development, whereas carbenicillin at a dose of 500 mg l^-1 induced abundant callus formation and inhibited regeneration. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. Schemes for selection and recovery of transgenic apple plants when kanamycin is used as the selection agent are discussed.Abbreviations BA benzyladenine - Cef cefotaxime - Crb carbenicillin - IBA indolebutyric acid - Kan kanamycin - LS Linsmaier and Skoog (1965) medium - M Malling - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - N6 medium (Chu et al. 1975) as modified by Welander (1988) - PPF photosynthetic photon flux     

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     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from leaf mesophyll protoplasts of the tropical woody plant,passionfruit (Passiflora edulis fv flavicarpa Degener.): the importance of the antibiotic cefotaxime in the culture medium

Enzymatic digestion of newly expanded leaves of glasshouse-grown seedlings of passionfruit released protoplasts which exhibited highest division frequency (38.6%) when plated at a density of 1.5×10⁵ ppts ml^–1 in agarose-solidified droplets of KM8P medium containing the antibiotic cefotaxime (250 g ml^–1). Cefotaxime was essential for sustained cell division. Protoplast-derived calli were cultured on agarsolidified MS medium with 5.0 mg H NAA, 0.25 mg l^–1 BAP and additional vitamins. These calli regenerated shoots on transfer to MS medium with 1.0 mg l^–1 BAP. Regenerated shoots were rooted in half-strength MS medium with 3.0 mg l^–1 IBA and 0.5 mg l^–1 NAA (7 d), followed by sub-culture to MS medium lacking growth regulators. The ability to regenerate plants from protoplasts of passionfruit is discussed in relation to the application of somatic cell techniques for the genetic improvement of this economically important tropical woody plant.Abbreviations B5 medium after Gamborg et al. (1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - d day - FDA fluorescein diacetate - FPE final plating efficiency - f. wt fresh weight - h hour - 1BA 4-indole-3yl-butyric acid - IPE initial plating efficiency - MES 2-N-morpholinoethane sulphonic acid - MS medium after Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (M. Wt. 10,000) - rpm rotations per minute     

Plant regeneration from callus and protoplasts in Medicago polymorpha

Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l^-1 2iP and 0.1 mg l^-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l^-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.Abbreviations NAA -naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - 2iP N⁶-²-isopentenyl-adenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - GFMS growth regulator free MS medium - Prol proline - Malt maltose     

Eradication of mosaic disease and rapid clonal multiplication of bananas and plantains through meristem tip culture

Heat therapy and meristem tip culturing were used in various cultivars of banana (Musa acuminata AAA cvs Grande Naine and Valary), and plantain (Musa acuminata x M. balbisiana AAB cvs Maricongo, Common Dwarf and Super Plantain) for rapid clonal propagation of mosaic disease-free plants. Suckers were subjected to heat therapy at 38–40°C for 14 days prior to the culture of their meristem tips (1.5–2.0 mm long having 6–8 vertical incisions) on modified MS medium containing 1.0 mg l^-1 thiamine HCl, 0.5 mg l^-1 nicotinic acid, 0.5 mg l^-1 pyridoxine HCl, 25 mg l^-1 ascorbic acid (filter sterilized), 0.7 mg l^-1 BA and 0.7 mg l^-1 kinetin. This culture medium alone was effective in preventing the oxidation of phenolic compounds present in explants, and in producing up to 13 rooted plantlets from a single meristem within 10 to 12 weeks. Plants derived from heat-treated meristems of infected plants were free from the disease, as determined by visual inspection, mechanical inoculation to Cucumis sativus, and electron microscopy.     

Fertile Indica rice plants regenerated from protoplasts isolated from scutellar tissue of immature embryos

Summary We report on the regeneration of fertile Indica rice (Oryza sativa L.) plants from protoplasts isolated from scutellar tissue of immature embryos. The average yields of protoplasts after purification ranged from 2.8 × 10⁵ to 3.5 × 10⁵ protoplasts per fifty embryos. Protoplasts developed rapidly to colonies when cultured in maltose containing medium using the nurse culture method. Upto 146 or 39 visible colonies per 10⁶ protoplasts were obtained for the varieties Basmati 370 and IR43 respectively. Of two basal culture media compared, R2 medium containing 3 mg l^–1 kinetin, 1 mg l^–1 naphthalene acetic acid (NAA), 30 g l^–1 maltose and 3.0 g l^–1 agarose was found to be more effective in producing green plants. All scutellum protoplast-derived plants that were transferred to the greenhouse survived and were fertile.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Plant regeneration from protoplasts of peppermint (Mentha piperita L.)

Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - KIN kinetin - ZEA zeatin - CPW cell and protoplast wash solution - B5 Gamborg et al. (1968) mineral elements - MS Murashige and Skoog (1962) mineral elements     

Media optimization for maximum biomass production in cell cultures of pacific yew

Three cell lines of Taxus brevifolia Nutt. with differing growth rates were used to assess the effects of basal salt mixtures, carbohydrates, organic nitrogen additives, vitamin formulations, and plant growth regulators on callus growth. Gamborg's B5 major salts provided significantly better growth than all other salt formulations tested. The greatest biomass was obtained with 1% total carbohydrate. The best carbohydrate combination, 0.5% fructose + 0.5% sucrose, was significantly better than all other combinations of carbohydrates tested. A complex vitamin mixture was significantly better than any one previously published vitamin formulation. Greatest rates of callus growth were obtained with 4.14 M (1 mg l^-1 picloram, 0.46 M (0.1 mg l^-1 kinetin, and 0.38 M (0.1 mg l^-1) abscisic acid or 0.29 M (0.1 mg l^-1 gibberellic acid. Our final medium, TM5, is superior to published methods for the general callus culture of T. brevifolia. This medium has improved growth in three tested cell lines to provide doubling times of 3.5 to 5.6 days, an average 5.3-fold increase over our previously published medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid, 2ip-6-(,-dimethylamino)-purine - ABA abscisic acid - BA 6-benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Plant regeneration from protoplasts isolated from cell suspension cultures of the wild tomato,Lycopersicon chilense Dun.

A protocol has been established for rapid, high frequency plant regeneration from protoplasts of the wild tomato species Lycopersicon chilense Dun. Cell suspension cultures were obtained from calli initiated from seedling stem explants. Protoplasts were isolated from cell suspensions by an overnight one-step enzyme digestion, purified by washing in salts solution and cultured in liquid medium. Dilution of liquid medium every 3 days, with medium containing low levels of growth regulators and sucrose, was critical for sustained colony formation. Up to 70% of protoplast-derived calli regenerated shoots when cultured on agar-solidified medium with Murashige & Skoog (1962) salts and vitamins, 2.0 mg l^-1 zeatin and 0.1 mg l^-1 indole acetic acid for 21 days, followed by transfer to the same medium lacking indole acetic acid.Abbreviations BAP 6-benzylaminopurine - IAA indole acetic acid - IBA indole butyric acid - MES-2 (N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method

Factors influencing in vitro growth rates of soybean embryos were investigated using embryos isolated in the cotyledon stage. The influence of these factors on final plant recovery from the embryos cultured was tested. Sucrose and glucose could serve as carbon sources with final plant yields being higher with sucrose than with glucose. A culture medium containing only KNO₃ (25 mM) as the nitrogen source supported embryo growth. Adding glutamine (10 mM) to the medium containing KNO₃ increased final plant recovery to 25%. Of several vitamin supplements tested a combination of pyridoxine-HCl, nicotinic acid and pantothenic acid (0.5; 1.0; 0.5 mg l^-1) provided the best growth and plant yield. Of the plant growth regulators tested IAA, BAP and GA₃ stimulated embryo growth and plant development when added to the medium at a low concentration (0.1 M). The optimal temperature for in vitro growth of cotyledon stage embryos was 27°C. Temperatures above 30°C caused growth retardation and reduced plant yield. A protocol for culturing soybean cotyledon stage embryos under conditions ensuring high plant recovery is proposed.Abbreviations IBA indole-3-butyric acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Direct somatic embryogenesis and plant regeneration from leaf, petiole, and stem explants of Golden Pothos

Somatic embryos directly formed at cut edges or on the surface of leaf explants, around cut ends or along side surfaces of petiole and stem explants of Golden Pothos [Epipremnum aureum (Linden & Andre) Bunt.] on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) or N-phenyl-N-1, 2, 3-thiadiazol-5-ylurea (TDZ) with -naphthalene acetic acid (NAA) and a medium called MK containing MS salts with Kaos vitamins, supplemented with 2.0 mg/l TDZ and 0.2 mg/l NAA. Somatic embryos were also produced on MS medium containing 2.0 mg/l kinetin (KN) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) from leaf and petiole explants, MS medium supplemented with 2.0 mg/l CPPU and 0.5 mg/l 2,4-D from petiole and stem explants, and 2.0 mg/l TDZ and 0.2 mg/l or 0.5 mg/l 2,4-D from stem explants. In addition, somatic embryos occurred from stem explants on Chus N₆ medium containing 2.0 mg/l CPPU and 0.2 mg/l NAA. Somatic embryos matured and grew into multiple buds, shoots, or even plantlets after 2–3 months on the initial culture medium. Germination was optimal on MS medium containing either 2 mg/l 6-benzylaminopurine (BA) and 0.2 mg/l NAA or 2 mg/l zeatin and 0.2 mg/l NAA. Shoots elongated better and roots developed well on MS medium with no growth regulators. Approximately 30–100 plantlets were regenerated from each explant. The regenerated plants grew vigorously after transplanting to a soil-less container substrate in a shaded greenhouse.