High-efficiency phosphinothricin-based selection for alfalfa transformation

Despite the significant advantages of using herbicide resistance for selection of genetically engineered plants, alfalfa transformation has relied primarily on selection for antibiotic resistance. In the few studies reporting the use of resistance to the herbicide phosphinothricin (PPT), transformation efficiencies were low. The present investigation describes a PPT-based selection system for alfalfa transformation that uses the phosphinothricin acetyl-transferase (pat) gene as a selectable marker and 5.0 mg l⁻¹ of bialaphos as the selective agent. The method achieves transformation efficiencies, measured as the percentage of explants giving rise to one or more transformed plantlets, greater than 50%. These plantlets accumulated detectable amounts of PAT at levels varying from 2 to 1367 pg μg⁻¹ total protein. Transformed plants transferred to soil in the greenhouse were phenotypically normal and exhibited resistance to bialaphos leaf painting at 5 g l⁻¹ and applications of PPT equivalent to field-level use (0.5 kg ha⁻¹).     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm^–3 6-benzyladenine and 0.5 mg dm^–3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm^–3 phosphinothricin (L-PPT) supplemented medium were confirmed by GUS assay and PCR analysis. The transformation rate was 58 % with AGL1/ pDM805 and 25 % with LB5-1. Rooted plantlets were acclimated and then again screened for Basta®-resistance by spraying with 15 – 60 mg dm^–3 L-PPT. Surviving plants were selfed and Basta®-resistance was demonstrated in T₁ progeny.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Optimizing plant regeneration from seed-derived callus cultures of Kentucky bluegrass. The effect of benzyladenine

The effect of benzyladenine (BA) on the production of shoot-forming callus from seeds of two Poa pratensis cultivars was studied. Addition of low concentrations (0.1–0.3 mg l^-1) of BA to Murashige & Skoog (MS) callus induction medium containing 1 or 2 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d) stimulated somatic embryogenesis and strongly increased the percentage of seeds producing shoot-forming callus in both cultivars.     

Herbicide-tolerant Transgenic Pineapple (Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple(Ananas comosus‘Phuket’) were produced followingmicroprojectile-mediated delivery of the plasmid AHC25, carryingthe ß-glucuronidase (gus) reporter gene and the bialaphosresistance (bar) gene for herbicide tolerance, into leaves ofmicropropagated shoots. Transformed plants were regeneratedfrom bombarded leaf bases on Murashige and Skoog-based mediumcontaining 0.5 mg l^-12,4-dichlorophenoxyacetic acid, 2.0 mgl^-16-benzylamino purine and 0.5 mg l^-1phosphinothricin. Integrationand expression of thebar gene in regenerated plants was confirmedby Southern analysis and RT-PCR, respectively. Regenerated plantswere assessed in vitro and under glasshouse conditions for theirtolerance to the commercial herbicide Basta^TM, containing glufosinateammonium as the active component. Plants sprayed with Basta^TMcontainingconcentrations of glufosinate ammonium up to 1400 mg l^-1remainedhealthy and retained their pigmentation. The generation of herbicide-tolerantpineapple will facilitate more efficient weed control in thiswidely cultivated tropical crop. Copyright 2001 Annals of BotanyCompany bar gene, Biolistics, herbicide tolerance, pineapple, phosphinothricin (PPT)     

Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator combination in japonica rice (Oryza sativa)

The influence of maltose and growth regulators on microspore culture response was investigated in japonica rice. High frequency of callus induction of isolated microspores was obtained with liquid medium containing MS salts, 100 mg l^–1 myo-inositol, 1 mg l^–1 thiamine-HCl, 500 mg l^–1 glutamine, 60 g l^–1 maltose, and several growth regulators. The effect of maltose on promoting callus formation was associated with keeping a high proportion of swollen microspores after 5 day preculture and increasing the microspore division rate on the 3rd day after culture initiation. No significant effect of maltose in place of sucrose on plantlet regeneration was seen in regeneration medium. Among the growth regulators tested, the combination of auxin 2,4-dichlorophenoxyacetic acid (1 mg l^–1), naphthaleneacetic acid (1 mg l^–1), and cytokinin (6-benzyl-aminopurine 1 mg l^–1) in the medium proved to be much better for callus formation than in the other media, and the percentage of callusing microspores of that medium reached 0.86%. Indole-3-acetic acid (0.5 mg l^–1) and kinetin (2 mg l^–1) in regeneration medium were beneficial for green plantlet differentiation. The results also showed that the frequencies of microspores initial division, callus formation and green plant regeneration varied among genotypes no matter what kind of growth regulator and sugar were used. Xiushui 117 was the best variety for callusing followed by 02428 & Taipei 309. Taipei 309 showed a good ability for green plantlet regeneration.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6-BA 6-benzylaminopurine - KT kinetin - IAA indole-3 acetic 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     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and development of herbicide-resistant sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> species hybrids) using axillary buds

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing -glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l^–1) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l^–1 6-benzyladenine (BA) and 5.0 mg l^–1 PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l^–1 BA, 1.0 mg l^–1 kinetin (Kin), 0.5 mg l^–1 -napthaleneacetic acid (NAA) and 5.0 mg l^–1 PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l^–1 NAA and 5.0 mg l^–1 PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical -glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50–60% of the transgenic plants sprayed with BASTA (60 g l^–1 glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.Abbreviations BA 6-Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kin Kinetin - NAA -Naphthaleneacetic acid - Nos Nopaline synthase - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PPT Phosphinothricin - YEP Yeast extract and peptone     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoog's or Gamborg's media supplemented with 1 to 2 mg l^–1 of 2,4-dichlorophenoxyacetic acid.Abbreviations B5 Gamborg's medium - 2,4-Dscd 2,4-dichlorophenoxyacetic acid - IAA indolacetic acid - MS Murashige & Skoog's medium - NAA naphtaleneacetic acid     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Somatic embryogenesis and plant regeneration of pepper in liquid media

A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 M 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 M 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l^-1 l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 M abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.