Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl^–1 BAP, 0.05 mgl^–1 IBA and 50 gml^–1 kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IBA indole-butyric acid - PAGE polyacrylamide gel electrophoresis - NPTII neomycin phosphotransferase II - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecylsulphate     

Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l⁻¹ indole-3-acetic acid and 1.0 mg l⁻¹ 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g⁻¹ dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.     

Induction of hairy roots and plant regeneration from the medicinal plant <Emphasis Type="Italic">Pogostemon Cablin</Emphasis>

An efficient transformation system for the medicinal and aromatic plant, Pogostemon cablin Benth was developed by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots formed directly from the cut edges of leaf explants or via callus stage 8 days after inoculation with the bacterium. The highest frequency of leaf explant transformation by Agrobacterium rhizogenes ATCC15834 was about 80% after infection for 25 days. Hairy roots grew rapidly on plant growth regulators (PGRs)-free Murashige and Skoog (MS) or 6,7-V medium and had characteristics of transformed roots such as fast growth and high lateral branching. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. The hairy root line, PL6, grew very slowly in the first 8 days, then grew very quickly between day 8 and day 24. The optimum medium for callus induction of hairy roots consisted of 2.0 mg l⁻¹ benzyladenine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA); while optimum medium for adventitious shoot regeneration from these cultures consisted of 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Adventitious shoots could be rooted on 1/2MS. Southern blot analysis confirmed that rol genes of TL-DNA of Ri plasmid was integrated with at least three copies into the genome of hairy roots- regenerated P. cablin plants. The results presented provide a solid foundation for production of patchouli essential oil from hairy roots or its regenerated plants and also provide possibilities for utilization of artifical polyploidization or chemical mutation of hairy roots for improving germplasm and breeding of a new cultivar of P. cablin.     

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g⁻¹ of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L⁻¹ naphthaleneacetic acid and 2 mg L⁻¹ 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.     

Secoiridoid content of Blackstonia perfoliata in vivo and in vitro

Summary This study reports the analysis of secondary metabolites of gentiopicrin, swertiamarin, and sweroside in shoot and root cultures of yellow wort (Blackstonia perfoliata), which were initiated from seeds, grown on Murashige and Skoog (MS) medium. Shoot cultures of B. perfoliata were inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS and hairy roots appeared at the infected sites after 3 wk of inoculation. Tips of adventitious roots of B. perfoliata were grown on hormone-free MS medium and three clones of the transformed roots regenerated shoots spontaneously. Gentiopicrin, swertiamarin, and sweroside were detected in both roots and shoots of B. perfoliata in vitro and in vivo, but gentiopicrin was found to be the major compound. The concentration of growth regulator in the medium affected the production of secoiridoids in B. perfoliata in vitro, where the level of gentiopicrin was higher in plants grown in the presence of indole-3-butyric acid, but the presence of 6-benzylaminopurine was inhibitory to secoiridoid production.     

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l^–1 agar, 7.5 mg ⋅ l^–1 6-benzylaminopurine, 1.0 mg ⋅ l^–1 -naphthaleneacetic acid, 300 mg ⋅ l^–1 cefotaxime and 80 mg ⋅ l^–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil. Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T_L-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l^–1.     

Agrobacterium rhizogenes-mediated transformation of Echinacea purpurea

Summary Echinacea purpurea seedlings were inoculated with several Agrobacterium rhizogenes strains in order to obtain hairy roots. Infection with A. rhizogenes strains LMG63 and LMG150 resulted in callus formation. Upon infection with strains ATCC 15834 and R1601 hairy roots were obtained. Opine detection confirmed transformation of E. purpurea. Comparative HPLC fingerprint analysis of the alkamides from natural plant source, control tissues, and transformed callus and roots indicated that transformed callus and hairy roots might be a promising source for continuous and standardized production of the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide and related amides.Abbreviations HPLC high-pressure liquid chromatography - MS Murashige and Skoog culture medium     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct shoot regeneration from nodal explants of <Emphasis Type="Italic">Sida cordifolia</Emphasis> Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 6-benzylaminopurine, 0.5 mg l⁻¹ α-naphthalene acidic acid, 1.0 mg l⁻¹ adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.     

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.     

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10¹⁴ plantlets could be produced in a year from one expiant following the proposed protocol.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

The effects of exogenous auxin and rol genes on root formation in Rosa hybrida L. ‘Moneyway’

The effect of indole-3-butyric acid (IBA) on the formation of non-transformed and rol gene transformed roots on stem slices of in vitro cultured shoots of Rosa hybrida L. Moneyway was examined. Formation of adventitious roots on this rootstock was dependent on the IBA dose; it was not affected by the presence of other root primordia on the same explant. Application of 0.32 to 1 M IBA during 5 days, followed by transfer to medium without hormones resulted in maximum root formation (90%) after three weeks. The formation of such untransformed roots was completely inhibited by transfer to medium with 5 mg 1^–1 kanamycin two days after excision. Ri roots were formed upon inoculation with A. rhizogenes LBA9402 harbouring two plasmids: pRi1855, comprising the rol genes and the binary plasmid p 35Sgusintron with the nptII gene for kanamycin resistance and the CaMV 35Sgusintron gene. The formation of these Ri roots on kanamycin-containing medium was independent of the presence of IBA. Stem slices inoculated with a disarmed A. tumefaciens GV3101, harbouring only the nptII gene, formed callus and subsequently roots in the presence of kanamycin exclusively on medium with high IBA concentrations (10 or 100 M). Root formation at 100 M IBA was considerably improved by transformation with the rolB gene under the influence of the strong CaMV 35S promoter. In addition, low IBA (0.1 and 1 M) stimulated the formation of roots only on stem slices transformed with A. tumefaciens harbouring the rolA+rolB+rolC genes; the rooting response at 10 M IBA was much improved. It was concluded that the 35SrolB gene and especially a combination of rolA, B and C genes promote the rooting response.Abbreviations BAP 6-benzylaminopurine - FeEDDHA ferric ethylenediamine di(o-hydroxyphenylacetate) - IBA indole-3-butyric acid - LSD least significant difference - RIM root induction medium - SE standard error - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.