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.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Rapid and efficient regeneration in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill] from whole cotyledonary node explants

A rapid and efficient regeneration protocol was established for soybeans [Glycine max (L.) Merrill]. Whole cotyledonary node explants were collected from aseptic seedlings cultured on MSB₅ medium containing 0.4 mg l⁻¹ N⁶-benzyladenine (BA). The effects of the plant growth regulators BA, kinetin (KT), indole-3-butyric acid (IBA) as well as the explant genotype on shoot regeneration were evaluated by the orthogonal design [L₁₆(4⁵)]. The process of shoot development was also observed. The regenerated shoots were elongated on MSB₅ medium and sufficiently elongated shoots were rooted on MSB₅ medium containing 0.5 mg l⁻¹ IBA. The results showed that all three of the plant growth regulators significantly affected shoot regeneration, with BA exhibiting the greatest benefit. The best combination for shoot regeneration was MSB₅ medium supplemented with 3.0 mg l⁻¹ BA , 0.2 mg l⁻¹ IBA and 0.5 mg l⁻¹ KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30–35 shoots. Plantlets could be obtained within 2 months. The result of histocytological analysis indicated that protein accumulated gradually and reached to peak at late shoot bud formation.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

High-efficiency plant regeneration from leaf explants of male himalayan poplar (Populus ciliata wall.)

Summary This study reports a protocol for high-efficiency plant regeneration from leaf explants of male Himalayan poplar (Populus ciliata Wall.). Shoots were regenerated at high frequencies from explants grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ kinetin and 0.2 mg l⁻¹ indole-3-acetic acid (IAA). Regenerated shoots developed roots in MS medium supplemented with 0.1 mg l⁻¹ IAA. Himalayan poplar plantlets could be produced within 2 mo. after acclimatization in a sterile mixture of sand and soil.     

Callus induction and plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient plant regeneration from immature embryo cultures of <Emphasis Type="Italic">Jatropha curcas</Emphasis>, a biodiesel plant

Jatropha curcas L. (Physic nut) is a commercially important non-edible oil seed crop known for its use as an alternate source of biodiesel. In order to investigate the morphogenic potential of immature embryo, explants from four developmental stages were cultured on medium supplemented with combinations of auxins and cytokinins. It was found that the size of embryo is critical for the establishment of callus. Immature embryos (1.1–1.5 cm) obtained from the fruits 6 weeks after pollination showed a good response of morphogenic callus induction (85.7%) and subsequent plant regeneration (70%) with the maximum number of plantlets (4.7/explant) on Murashige and Skoog’s (MS) medium supplemented with IBA (0.5 mg l⁻¹) and BA (1.0 mg l⁻¹). The above medium when supplemented with growth adjuvants such as 100 mg l⁻¹ casein hydrolysate + 200 mg l⁻¹ l-glutamine + 8.0 mg l⁻¹ CuSO₄ resulted in an even higher frequency of callus induction (100%). Plant regeneration (90%) with the maximum number of plantlets (10/explant) was achieved on MS medium supplemented with 500 mg l⁻¹ polyvinyl pyrrolidone + 30 mg l⁻¹ citric acid + 1 mg l⁻¹ BA + 0.5 mg l⁻¹ Kn + 0.25 mg l⁻¹ IBA. It was observed that plantlet regeneration could occur either through organogenesis of morphogenic callus or via multiplication of pre-existing meristem in immature embryos. The age of immature embryos and addition of a combination of growth adjuvants to the culture medium appear to be critical for obtaining high regeneration rates. Well-developed shoots rooted on half-strength MS medium supplemented with 0.5 mg l⁻¹ IBA and 342 mg l⁻¹ trehalose. The rooted plants after acclimatization were successfully transferred to the field in different agro-climatic zones in India. This protocol has been successfully evaluated on five elite lines of J. curcas.     

An efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.     

Plant regeneration from phyllode explants of Acacia crassicarpa via organogenesis

In this paper we report the establishment of Acacia crassicarpa regeneration through organogenesis. We used phyllode (leaf) explants excised from 60-day-old in vitro seedlings for green compact nodule induction and, tested Murashige and Skoog (MS) media supplemented with various concentrations of 1-phenyl-3-(thiadiazol-5-yl) urea (thidiazuron) (TDZ) and α-naphthaleneacetic acid (NAA). Under the optimized condition, green compact nodules and adventitious shoots were induced in 10 and 40 days, respectively, on the medium containing a combination of 0.5 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA. This medium also yielded the highest rate (56%) of adventitious shoots forming from the nodules. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoots to medium containing 0.1 mg l⁻¹ TDZ within 2 months. The elongated adventitious shoots were rooted at a rate of 96.5% on half-strength MS medium with 0.5 mg l⁻¹ 3-indolebutyric acid (IBA) in 1 month. Rooted plantlets were hardened and successfully established in soil with an 80% survival rate. To our knowledge, this is the first report describing a detailed protocol for regeneration through organogenesis using phyllodes as explants for A. crassicarpa.     

In vitro plant regeneration from cotyledon explants of Swainsona salsula Taubert

An effective protocol has been developed for plant regeneration from cotyledon explants of Swainsona salsula Taubert (Saline swainsona), a medicinal and agronomic shrub. Adventitious shoots were obtained from 83.2% of cotyledon explants from 3-day seedlings cultured on Murashige and Skoog (MS) medium containing 2.0 mg l⁻¹ thidiazuron (TDZ), with an average of 9.3 shoots per explant. Individual elongated shoots were rooted on half strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid (IBA), with 59.3% success. Regenerated plants with well developed shoots and roots were successfully transferred to soil, without detectable variants. Histological observation revealed that shoots developed from cotyledon explants via organogenesis, with little callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

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.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

In vitro micropropagation of Gymnema sylvestre – A multipurpose medicinal plant

The nature of the explant, seedling age, medium type, plant growth regulators, complex extracts (casein hydrolysate, coconut milk, malt extract and yeast extract) and antioxidants (activated charcoal, ascorbic acid, citric acid and polyvinylpyrrolidone) markedly influenced in vitro propagation of Gymnema sylvestre. A maximum number of shoots (57.2) were induced from 30 day old seedling axillary node explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (1 mg l⁻¹), kinetin (0.5 mg l⁻¹), 1-napthalene acetic acid (0.1 mg l⁻¹), malt extract (100 mg l⁻¹) and citric acid (100 mg l⁻¹). High frequency of rooting was obtained in axillary node explant derived shoots (50%) on half strength MS medium supplemented with IBA (3 mg l⁻¹). The plantlets, thus developed, were hardened and successfully established in natural soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.