Improved shoot regeneration system through leaf derived callus and nodule culture of Sansevieria cylindrica

Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cylindrica Bojer ex Hook was developed using leaf derived callus and nodule culture. Profuse callus induction on leaf discs was achieved on Murashige and Skoog (MS) medium supplemented with 10 μM indole-3-butyric acid (IBA), while a high frequency of nodulation was induced on 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) containing media. Shoot regeneration ability from cultured tissues occurred at varying degrees on all media. Through callus culture a maximum of 17.6 ± 0.14 shoots per culture was formed on medium containing 5μM 6-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA). Among nodule cultures, the 2,4-D generated nodules were more proliferative and regenerative as compared to 2,4,5-T induced nodules and a maximum of 25 ± 0.16 shoots per culture was produced on a medium containing 5 μM BA plus 1 μM NAA. The regenerated shoots were successfully rooted on a semi-solid half strength MS medium containing 5 μM IBA with an average root number 3.5 ± 0.18 and root length 6.5 ± 0.14 cm. The regenerative ability of callus tissues was steady upto one year, while the nodules retained the totipotency to regenerate on optimal medium even after 3 years of subculturing. The histological sections of nodules confirm the typical anatomy exhibiting the vascular elements in bundles with well demarcated cortex and epidermal covering.     

Effect of synthetic auxins on callus induction from tea stem tissue

A study was initiated to establish an in vitro culture protocol for tea (Camellia sinensis). Explant sources, disinfestation methods and culture media were examined. Segments (divots) were dissected from greenwood stem (current year growth) internodes of field grown plants. Disinfestation was achieved by separate treatments of 3.75% sodium hypochlorite and 7.5% CaCl₂. MS medium with sucrose (30 g/L), inositol (100 mg/L) and thiamine-HCl (1.3 mg/L) and kinetin was used with combinations of the auxins: (2,4-dichlorophenoxy) acetic acid (2,4-D), (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T), (naphthalene) acetic acid (NAA) and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram). Picloram (10^-7M) induced the most callus proliferation without kinetin. At a constant level of kinetin (10^-5M), the concentrations inducing the most callus growth were 10^-7M for 2,4-D, 10^-6M for 2,4,5-T, 10^-7M for Picloram and 10^-8M for NAA. A factorial test of 2,4,5-T and kinetin concentrations showed the optimum for callus growth was 10^-7M and 10^-5M, respectively.Technical Contribution No. 2532 of the South Carolina Agricultural Experiment Station, Clemson University.Graduate Research Assistant and Professor, respectively.     

Application of the disk method: responses to growth regulators

The paper disk method of screening several plant growth regulators was evaluated. Leaf explants ofVigna unguiculata (L) Walp. were placed on solidified Murashige and Skoog's minimal organics medium containing 0.5 mg/l nicotinic acid. Hormones were tested, singly and in combinations, on paper disks in large Petri plates (150×20 mm). Hormones tested were 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), picloram (4-amino-3,5,6-trichloropicolinic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid), BA (6-benzyladenine), 2iP (2-isopentenyl adenine), and kinetin [6-(furfurylamino)-purine]. Root formation was stimulated by IAA and IBA; dicamba, picloram, 2,4-D, and 2,4,5-T stimulated callus formation. All cytokinins tested suppressed root formation. Dicamba in combination with either 2iP or kinetin induced the greatest callus formation. Root formation was optimal with kinetin and either IAA or IBA. The disk method provided a rapid, nonquantitative evaluation of callus and root formation from leaf disks.     

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Summary Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - CA casamino acids - B5CA B5 with 0.2% casamino acids - IBA indolebutryric acid; Picloram (4-amino-3,5,6-tricnloro-2-pyridinecarboxylic acid) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - BA 6-benzylaminopurine     

Plant regeneration from in vitro cultures of anthers and mature seeds of rice (Oryza sativa L.) cv. Basmati-370

Pollen plants were obtained from anther-derived calli of the indica rice variety Basmati-370. Anther-response (anthers producing pollen derived calli) and plant regeneration frequency from the pollen derived calli. was very low. Donor plants which flowered at the average max/min. temperature of 34.2°/23.3°C gave a significantly higher anther-response to in vitro techniques, than did those which flowered at 29.1°/16.4°C. Somatic callus induction and subsequent plant regeneration was readily obtained from mature seed embryos. While 2,4-D or 2,4,5-T (1 or 2 mg/l) proved highly efficient for callus induction, tryptophan (50 or 100 mg/l) induced a high frequency of green plants from the calli.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - K kinetin - BA benzyladenine - Trp tryptophan - CW coconut water     

Effect of plant growth regulators on morphogenesis and forskolin production in Plectranthus barbatus Andrews

Plectranthus barbatus (syn. Coleus forskohlii) is the only known source of forskolin, a compound with a wide range of pharmacological activities. Here, an efficient protocol for adventitious root regeneration from leaf explants of P. barbatus was developed. Different concentrations of plant growth regulators individually and in combination were used to induce roots in vitro. Morphogenic responses and forskolin production varied depending on the concentrations of plant growth regulators added to the medium. Lower concentrations of auxins trigger callus proliferation while higher concentrations induced adventitious root regeneration. Of all the auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2 (2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP), and 4-amino-3,5,6-trichloropicolinic acid (picloram) induced callus, whereas α-naphthaleneacetic acid (NAA), indole-3-acetic acid, and indole-3-butyric acid induced rhizogenesis. Use of picloram at 1.0 and 0.5 mg l⁻¹ resulted in the formation of friable callus, and when combined with 0.5 mg l⁻¹ 6-benzylamino purine (BA), rhizogenic callus was produced. The cytokinins BA and kinetin produced a mixed response of multiple shoot regeneration, callus proliferation, and rhizogenesis. The maximum forskolin content of 1,178 mg kg⁻¹ dry weight was found in root cultures initiated on Gamborg’s B₅ medium supplemented with 0.5 mg l⁻¹ NAA. The biosynthesis of forskolin was differentiation dependent, and rhizogenic cultures exhibited the maximum biosynthetic potential for forskolin.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

Direct somatic embryogenesis and plantlet regeneration in oil palm

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.     

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 mesocotyl callus of Hordeum vulgare L.

Callus tissue was induced on barley mesocotyl explants of germinated seven-day-old seedlings on MS medium supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically different tissue cultures were maintained in vitro for a long time: a callus tissue without organogenesis and a culture with high rhizogenic capacity. Shoots and plantlets were generated when the auxin-media induced callus was transferred to medium supplemented with 3 M TIBA. In 62% of cultures, during the first five subcultures, four to twentyeight plants per single mesocotyl were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one mesocotyl was forty. Plantlets rooted well on MS with 5.7 M IAA and survived transplantation to soil in high percentages.Abbreviations IAA indole-3yl-acetic acid - BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog (1962) medium     

Effect of BA,NAA, and 2,4-D on red maple callus growth

Established red maple (Acer rubrum L.) callus was cultured on media varying in auxin (NAA or 2,4-D) and cytokinin (BA) concentrations. Callus growth was positively affected by the presence of both an auxin and cytokinin in the medium. Optimal growth depended on the ratio of cytokinin/auxin as well as the total amount of plant growth regulators in the medium.Abbreviations (NAA) naphthaleneacetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (BA) and 6-benzylaminopurine     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

Toward the production of artemisinin through tissue culture: Determining nutrient-hormone combinations suitable for cell suspension cultures

Summary Artemisia annua L. is the source of a potent antimalarial, artemisinin. As part of a program to produce artemisinin through tissue culture, a series of 14 multifactorial experiments were conducted to determine suitable conditions for initiating and maintaining friable callus fromA. annua. In the first six experiments, three different nutrient formulations [Gamborg B5 (B5), Murashige and Skoog (MS), and Whetmore and Rier (WR)], each with 32 combinations of auxins and cytokinins [2,4-dichlorophenoxyacetic acid (2,4-D) with benzyladenine (BA), or 1-naphthaleneacetic acid (NAA) with 6-furfurylaminopurine (kinetin)], were tested. Both B5 and WR nutrients supported friable callus formation from leaf explants with some combinations of auxin and cytokinin. Inasmuch as friable callus seemed to be produced over a wider range of auxin and cytokinin concentrations in combination with B5, the remaining experiments were conducted solely with this nutrient formulation. In the remaining eight experiments, it was determined that friable callus formed when combinations of NAA with kinetin or 2,4-D and BA were used with B5 medium. Lighter colored, more friable callus formed in response to 2,4-D and BA than with NAA and kinetin. No single combination of concentrations of auxin and cytokinin seemed to be “ideal” for producing friable callus. Ranges of 2,4-D from 0.5 to 2.0 with BA between 0.025 and 0.1, or NAA between 0.5 and 2.0 with kinetin between 0.5 and 1.0 mg/liter, produced acceptable results.     

Induction of somatic embryos in cotyledonary tissue of soybean,Glycine max L. Merr

A method for the induction of somatic embryos in soybean tissue cultures is described. Cotyledons from immature embryos were utilized as explant source. Supplementing the culture medium with auxins (2,4-D, MCPA, 2,4,5-T, NAA, IAA, IBA) caused formation of meristematic tissue on cotyledon explants. The extent of meristematic tissue formed depended on the kind and concentration of auxin in the culture medium. With 2,4-D and MCPA, embryoids originated from meristematic tissue. Embryoid formation rates were influenced by the developmental stage of the embryos serving as explant source and auxin concentration. Addition of cytokinins to the medium containing 2,4-D or supplementing it with high sugar concentrations inhibited the formation of meristematic tissue and of embryoids on cotyledon explants.Abbreviations BA 6-benzyladenin - 2,4-D 2,4-dichlorphenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - L2 Phillips and Collins (1979) medium Present and correspondence address: Akademie der Landwirtschaftswissenschaften der DDR, Institut für Pflanzenernährung, DDR-6909, Jena     

Influence of type of explant,plant growth regulators,salt composition of basal medium,and light on callogenesis and regeneration in <Emphasis Type="Italic">Passiflora suberosa</Emphasis> L. (Passifloraceae)

Passiflora suberosa is used in popular medicine, improvement programs, and as an ornamental plant. The goal of this study was to establish efficient protocols for plant regeneration and callus induction from nodal, internodal and leaf segments excised from in vitro-grown plants. The different morphogenetic responses were modulated by the type and concentration of plant growth regulators, according to the basal medium and light conditions. Shoot formation occurred through three pathways: (1) development of preexisting meristems, (2) direct organogenesis, and (3) indirect organogenesis. Development of preexisting meristems was observed from nodal segments (1 shoot/explant) in response to α-naphthaleneacetic acid (NAA), picloram (PIC), and 2,4-dichlorophenoxyacetic acid (2,4-D), using two basal media (MS and MSM). Direct organogenesis in this species was obtained for the first time in this work, through shoot development from internodal segments in the presence of 6-benzyladenine (BA). The highest regeneration rates were achieved on MSM medium, regardless of the BA concentration. Indirect organogenesis was achieved from all explant types on media supplemented with BA, used alone or in combination with NAA. The highest regeneration efficiency was obtained from internodal segments cultured on MSM medium plus 44.4 μM BA. Compact, friable, or mucilaginous non-morphogenic calluses were induced by thidiazuron, PIC, 2,4-D, and NAA. High-yielding friable calluses obtained on MSM medium supplemented with 28.9 μM PIC are being used for the establishment of suspension cultures and further analysis of the production of bioactive compounds.     

Effects of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Triticum aestivum L.)

Summary In order to know the mutagenic effects of synthetic auxins (NAA, 2,4-D, and 2,4,5-T) and a cytokinin (kinetin) in vitro, sister chromatid exchanges (SCEs) were analyzed in cultured cells of a hexaploid wheat (Triticum aestivum L.). In the MS medium supplemented with 2.0 mg/l 2,4-D, the mean number of SCEs per cell was 15.2, and per pg of DNA, 0.42. No significant effect was found in the treatments of NAA or 2,4-D at concentrations of 0.5–10.0 mg/l, whereas more than 2.0 mg/l of 2,4,5-T induced dramatic increases of SCEs. Kinetin itself had no significant effect on SCE induction, but there was a tendency that SCEs induced by 2,4,5-T were suppressed by kinetin.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Growth and net photosynthetic rates of Eucalyptus tereticornis Smith under photomixotrophic and various photoautotrophic micropropagation conditions

Calli were induced from leaf explants of seedling in Citrus grandis (L.) Osbeck (pummelo) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA), 2,4,5-trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic, 4-chlorophenoxyacetic acid, 4-methoxy-3,6-dichlorobenzoic acid or 4-amino-3,5,6-trichloropicolinic acid. 2,4-D was most effective. Only green, compact calli induced by 2,4-D at low concentrations (0.9 and 4.5 M) were capable of shoot formation and regenerated more than 13 shoots per callus on MS medium containing at least 6.66 M benzyladenine (BA). Calli induced by other auxins did not regenerate shoots on MS medium containing BA at all concentrations studied. A multiplication rate of 5–7 shoots was achieved from shoot tip culture on MS medium with 0.89 M BA. Roots developed when regenerated shoots were cultured on MS medium with 9.84 M indole-3-butyric acid and 5.37 M NAA. No response was obtained on mature leaves cultured on MS medium supplemented with the above mentioned auxins at various concentrations.