Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL^–1 and 26 mgL^–1 ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MS Murashige and Skoog - NAA napthaleneacetic acid - Trp tryptophan - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from tissue cultures of Pokkali rice is promoted by optimizing callus to medium volume ratio and by a medium-conditioning factor produced by embryogenic callus

The frequency of plant regeneration from seed-derived Pokkali rice callus has been substantially increased. Four conclusions were drawn from the study: (1) Non-embryogenic callus consisting of elongated, highly-vacuolated cells did not produce regenerated plants. Embryogenic callus consisting of small, non-vacuolated cells produced somatic embryos and regenerated plants. (2) The numbers of plants could be markedly increased by optimizing a medium for embryogenic callus production and a second medium for plant regeneration from embryogenic callus. (3) The optimization of callus to medium volume ratio of 6.5 mg embryogenic callus per 1.0 ml of medium significantly increase plant production on regeneration medium. (4) A further significant increase was obtained by using regeneration medium previously conditioned for one or two weeks by optimal amounts of embryogenic callus. At present, the callus derived from a single seed in six months could theoretically be used in the seventh month to produce 127500 plants.This research was supported by the Agency for International Development under Contract No. AID/DSAN-C-0273     

Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

Long-duration,high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 m in diameter; NE callus consists of long tubular cells averaging 52 m in width and 355 m in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - Trp L-tryptophan - E embryogenic - NE non-embryogenic     

Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

In Vitro Selection for Salt Tolerance in Rice

In six cultivars of rice (Oryza sativa L.), Pusa Basmati 1, Basmati 370, Type III, Pant Dhan 4, CSR 10 and Pokkali, embryogenic callus growth, plant regeneration, and proline and total protein contents were studied under salt stress (on agar solidified media containing 0, 0.5, 1.0, 1.5 and 2.0 % NaCl). Four weeks after inoculation the callus fresh mass decreased with increasing salt concentration in all the six cultivars. The regeneration frequency in salt stressed callus was also lower as compared to control. 15 d and 30 d after inoculation proline content increased several fold whereas total protein content decreased markedly with increase in salt concentration.     

Isolation and Identification of a Senescence-promoting Substance from Wormwood (Artemisia absinthium L.)

The senescence-promoting substance of wormwood (Artemisia absinthium L.) as detected by the oat (Avena sativa L. cv “Victory”) leaf assay has been identified as (−)-methyl jasmonate, methyl (1S, 2R)-3-oxo-2-(2′-cis-pentenyl)-cyclopentane-1-acetate, by gas-liquid chromatography-mass spectrometry and optical rotatory dispersion. Its senescence-promoting effect was much stronger than that of abscisic acid, and even at such a low concentration as 1 to 2.5 micrograms per milliliter, it could completely eliminate the anti-senescence action of 2 micrograms per milliliter kinetin. Comparing the biological activity of the (−)- with the (±)-forms of methyl jasmonate, it seemed that only the (−)-form was biologically active.     

Improvement of rice callus regeneration in the presence of NaCl

The effects of abscisic acid (37.8 μM), polyethylene glycol (5%), proline (10 mM), tryptophan (490 μM) and indoleacetic acid (5.7 μM) on rice callus regeneration were studied at various doses of NaCl (0, 50 and 100 mM) on three month-old mature embryo-derived callus of two japonica (I Kong Pao and Aiwu) and two indica (IR 2153 and Nona Bokra) rice cultivars differing in salinity tolerance. NaCl strongly decreased the regeneration frequency of all cultivars but slightly increased the survival of regenerated plantlets. Tryptophan stimulated regeneration and increased subsequent survival rates of regenerated plantlets in all cultivars at all NaCl doses. Abscisic acid and polyethylene glycol, though not affecting the final regeneration percentages, delayed regeneration and reduced the mean number of plantlets produced per regenerating callus in all cultivars, as well as rooting ability and survival of regenerated plantlets in indica genotypes. Proline had no marked effect on regeneration, whatever the NaCl dose or cultivar, while indoleacetic acid reduced shoot regeneration and increased root regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

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.     

Factors influencing plant regeneration from protoplasts isolated from long-term cell suspension culture of recalcitrant Indica rice cultivar IR36

Summary Factors influencing successful establishment of embryogenic cell-suspension cultures and plant regeneration from longterm cell suspension-derived protoplasts of the recalcitrant Indica rice cultivar IR36 were studied. The factors included cell and protoplast culture medium, protoplast culture procedure, the source of nurse cells, and the regeneration procedure. Embryogenic cell suspension cultures could only be established from mature seed-derived callus of IR36 in AA-based medium (Müller and Grafe, 1978). Protoplast-derived colonies could be obtained only using the filter-membrane nurse-culture procedure when Lolium multiflorum suspension cells served as nurse, rather than wild rice (Oryza ridleyi) and Japonica rice (Oryza sativa cv. Taipei 309) cells. The utilization of a two-step regeneration procedure led to regeneration of fertile plants from protoplasts isolated from 2-yr-old cell suspensions of IR36, one of the most important but recalcitrant rice cultivars.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum

Tissue culture methods were developed for the induction, maintenance, and regeneration of embryogenic callus in sweet sorghum (Sorghum bicolor) cultivars Keller, Rio, and Wray. No significant differences were observed in production of embryogenic callus in cultures established from developmentally immature or mature embryo explants cultured on LS medium with 2 mg/1 2,4-D plus 0.5 mg/1 kinetin. Prolific callus production did not occur until the third four-week culture period. Long-term maintenance of embryogenic callus was dependent upon the selective transfer of embryogenic callus, with other callus types discarded. High-frequency plant regeneration was achieved and quantified on a fresh weight basis of embryogenic callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - LS Linsmaier and Skoog basal medium (Linsmaier and Skoog, 1965)     

High frequency somatic embryogenesis from coffee leaves

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called high frequency embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1^-1 in a modified MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid, under 3 mol m^-2 s^-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 m in diameter) at an inoculum density of 5 g 1^-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1^-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×10⁵ somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.Abbreviations BA N⁶-benzyladenine - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - rpm rotations per minute - LFSE low frequency somatic embryogenesis - MS Murashige & Skoog medium - PPF photosynthetic photon flux - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Effects of different combinations of benzyl adenine and indole acetic acid concentrations on in vitro plant regeneration in hexaploid wheat

Development of a reliable in vitro plant regeneration procedure for hexaploid bread wheat (Triticum aestivum L.) is a prerequisite for its improvement by genetic transformation. Here, we report the effects of two growth regulators, benzyl adenine (BA) and indole acetic acid (IAA) on callus induction and plant regeneration from scutellum cultures of two commercial bread wheat cultivars: Giza 164 and Sakha 69. Callus induction was obtained from isolated embryos cultured on modified Murashige and Skoog (MS) basal medium. After four weeks of callus induction, all calli were plated on MS basal medium for regeneration. Wheat genotype and callus induction medium played a dominant role in plantlet regeneration. 2.0 mg/L BA and 0.2 mg/L IAA were the best combinations for inducing callus and let to highest regeneration frequency (81.67%) across the cultivars. Overall, based on our medium conditions, Giza 164 displayed higher regeneration frequency (81.11%) than Sakha 69. These results will facilitate genetic transformation for the economic varieties Giza 164 and Sakha 69.     

Significance of different carbon sources and sterilization methods on callus induction and plant regeneration of Miscanthus x ogiformis Honda `Giganteus'

Different carbon sources, sterilized by autoclaving or filter-sterilization, were tested during induction, maintenance, and plant regeneration of embryogenic Miscanthus x ogiformis Honda `Giganteus' callus, derived from various explant types. Explants from small immature inflorescences, between 2.5 and 8 mm, produced more embryogenic callus than explants from shorter or longer inflorescences, shoot apices or leaf explants. On medium containing mannitol or sorbitol, only small amounts of callus were induced and no embryogenic callus was formed. Callus induction and embryogenic callus formation on shoot apices and immature inflorescences did not differ significantly between media containing sucrose, glucose, fructose, maltose or a mixture of glucose and fructose. However, callus induction and embryogenic callus formation from leaf explants were best on glucose. A higher percentage of leaf explants formed callus on autoclaved sucrose, as opposed to the other carbon sources where filter-sterilization in general resulted in a higher callus percentage. The growth rate of embryogenic callus was influenced both by carbon source and sterilization method when less than 1 g of callus was inoculated. None of the tested carbon sources could considerably improve plant regeneration from M. `Giganteus' callus, but a higher number of plants tended to be regenerated per callus piece from filter-sterilized carbon sources. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sorbitol as the Primary Carbon Source for the Growth of Embryogenic Callus of Maize

The effects of various carbon sources on initiation and maintenance of embryogenic callus of maize (Zea mays L.) and on the regeneration of plants from embryogenic callus were studied. Growth of embryogenic callus tissue on media containing sucrose was typified by the subsequent growth of both embryogenic (regenerable) and nonembryogenic (nonregenerable) callus. Growth of embryogenic callus on sorbitol was unique among the carbon sources tested in that sorbitol supported the subsequent growth of only embryogenic callus. Further experiments demonstrated that embryogenic callus grown on sorbitol had a greater regenerative capacity (more plants produced per gram fresh weight of callus) than callus grown on sucrose. Sorbitol dehydrogenase was detected in embryogenic callus of maize at a specific activity roughly equivalent to that found in zygotic embryos of developing seeds. Nonembryogenic callus did not contain significant levels of sorbitol dehydrogenase activity.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

The Effect of Rye Chromosomes on Callus Induction and Regeneration in Callus Cultures of Immature Embryos of Wheat–Rye Substitution Lines,Triticum aestivum L. Cultivar Saratovskaya 29–Secale cereale L. Cultivar Onokhoiskaya

The effect of individual rye chromosomes on the induction of callus and the character of its regenerating capacity was studied with cultured immature embryos of wheat–rye (Triticum aestivum L. cv. Saratovskaya 29–Secale cereale L. cv. Onokhoiskaya) substitution lines. The genotypic diversity of the substitution lines proved to significantly affect variation of parameters characterizing the major types of callus cultures, that is, frequencies of embryogenic calli, which are capable of shoot regeneration, and of morphogenic calli, which produce root structures. Functioning in the genotypic background of common wheat cultivar Saratovskaya 29, chromosomes 2R and 3R of rye cultivar Onokhoiskaya stimulated significantly the induction of embryogenic callus highly capable of shoot regeneration. Rye chromosome 2R present in place of chromosome 2D in the common wheat genome suppressed the induction of callus producing root structures. Rye chromosomes 1R and 6R suppressed the induction of embryogenic callus capable of shoot regeneration.     

Callus induction,plant regeneration,and long-term maintenance of embryogenic cultures in <Emphasis Type="Italic">Zoysia matrella</Emphasis> [L.] Merr

An efficient protocol of callus induction, plant regeneration and long-term maintenance of embryogenic cultures for manilagrass was developed. Callus induction and embryogenic callus formation were influenced by cytokinins and nodal positions. Murashige and Skoog (MS) medium with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 0.02 mg l⁻¹ kinetin (KT) or 6-benzyladenine (BA) gave the highest frequency for both callus induction and embryogenic callus formation compared with 0.02 mg l⁻¹ thidiazuron (TDZ) or N⁶-(2-isopenteny) adenine (2iP). The frequency of callus induction of different nodes (from the first to the sixth node) varied from 22.5 to 92.1%, and the embryogenic callus formation frequencies ranged from 13.3 to 25.7%. The highest frequencies of callus induction and embryogenic callus formation (92.1 and 25.7%, respectively) were observed in the fourth node group. During subculture on callus induction and maintenance medium, somatic embryos formed on the surface of the embryogenic callus. On regeneration medium, the regeneration rates of embryogenic callus varied from 96.8 to 100% during the 4-year period of subculture. The results also indicate that preservation of manilagrass callus is stable at low-temperature (4°C) over a period of 11 months. No significant differences were found in the activities of superoxide dismutase (SOD), peroxidase (POD) and proline content of the plants regenerated from the 4-year subcultured callus on different regeneration media.