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.     

An efficient regeneration system via direct and indirect organogenesis for the medicinal plant <Emphasis Type="Italic">Dysosma versipellis</Emphasis> (Hance) M. Cheng and its potential as a podophyllotoxin source

Dysosma versipellis (Hance) M. Cheng is an endangered plant due to overharvesting for the extraction of podophyllotoxin. Thus, the in vitro technique is valuable for the propagation of this species. When the explants of rhizome buds were cultured on Murashige and Skoog’s (MS) medium with 6-benzyladenine (BA) (1.0 mg l⁻¹), gibberellic acid (GA₃) (0.5 mg l⁻¹) and zeatin (Zea) (0.5 mg l⁻¹), multiple buds were regenerated directly on the explants without callusing within 6 weeks. Callus was induced from the leaf segment cultures on MS basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg l⁻¹) and BA (0.2 mg l⁻¹) within 4 weeks. The adventitious buds were differentiated when the calli were subcultured on MS medium supplemented with BA (1.0 mg l⁻¹) and thidiazuron (TDZ) (0.2 mg l⁻¹) within 6 weeks. The adventitious buds obtained from callus and the rhizome-buds rooted with a frequency of 100% on half strength MS medium fortified with indole-3-butyric acid (IBA) 0.5 mg l⁻¹ and activated charcoal (AC) 0.5 g l⁻¹ for 4 weeks. The rooted shoots were successfully transplanted from a mixture of vermiculite:soil (1:1 v/v) to the field with a survival rate of 85%. Podophyllotoxin production in calli, cultured rhizomes, rhizomes of transplanting plants from the garden and rhizomes in the wild field was confirmed by high-performance liquid chromatography (HPLC) analysis. Our results suggest that calli, cultured rhizomes and rhizomes of transplanting plants would be the potential sources of podophyllotoxin.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

Shoot induction and regeneration using internodal transverse thin cell layer culture in <Emphasis Type="Italic">Sesamum indicum</Emphasis> L.

An efficient protocol for shoot regeneration was developed for sesame (Sesamum indicum L.) internodes using the transverse thin cell layer (tTCL) culture method. The frequency of shoot regeneration and the number of adventitious buds produced from regenerated shoots depend significantly on explant age, thickness of the tTCL sections, and the phytohormones supplemented to the culture medium. A combination of 6-benzyladenine (2.0 mg l⁻¹) and α-naphthaleneacetic acid (0.5 mg l⁻¹) was found to be the best phytohormone combination for shoot bud induction, with the maximum number of shoots obtained when the tTCL sections were 0.5–1.0 mm thick and derived from 4- to 6-week-old seedlings of sesame. Well-developed shoots were rooted on MS medium without phytohormones, and 80% of the regenerated plantlets were successfully established in 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.     

Shoot regeneration and free-radical scavenging activity in <Emphasis Type="Italic">Silybum marianum</Emphasis> L.

The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with 2.0 mg l⁻¹ gibberellic acid (GA₃) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues and wild-grown plants.     

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.     

Direct Adventitious Shoot Formation from Apical Shoot Explants of Euphorbia tirucalli

The induction of adventitious buds from apical shoot explants of Euphorbia tirucalli was studied. On average, 10.5 adventitious buds were efficiently induced in a ring on the segment from one apical explant on MS (Murashige and Skoog) medium supplemented with 0.5 mg l⁻¹ thidiazuron and 0.5 mg l⁻¹ benzylaminopurine. The adventitious buds could develop into adventitious shoots during subsequent cultures on hormone-free MS medium. For rooting, shoot clumps were cultured on half-strength MS medium containing 0.2 mg l⁻¹ α-naphthaleneacetic acid or indole-3-butyric acid. All the rooted plants survived establishment in soil within 2 months.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 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, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. 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. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.     

In vitro regeneration from cotyledon explants in figleaf gourd (<Emphasis Type="Italic">Cucurbita ficifolia</Emphasis> Bouché), a rootstock for Cucurbitaceae

An efficient plant regeneration system has been developed for figleaf gourd (Cucurbita ficifolia Bouché), which is exclusively used as a rootstock for cucumber. The protocol is based on results obtained from a series of culture experiments involving different parts of the cotyledons and various media. The culture of cotyledon explants was critical for the enhancement of shoot regeneration frequency. The lower parts of the cotyledon excised at the plumule base were found to display a markedly enhanced production of adventitious shoots compared to other cotyledon regions. Culture in silver nitrate-supplemented Murashige and Skoog (MS) medium was not beneficial for shoot regeneration and suppressed root regeneration. Efficient shoot regeneration was obtained on MS medium containing 1.0 mg l⁻¹ zeatin and 0.1 mg l⁻¹ indole-3-acetic acid. Regenerated shoots successfully elongated and rooted in medium containing 0.1 mg l⁻¹ 1-naphthaleneacetic acid after 10–15 days of subculturing. The plantlets were satisfactorily acclimatized in a greenhouse and grew into normal plants without any morphological alterations.     

Adventitious bud regeneration from leaf explants of <Emphasis Type="Italic">Platanus occidentalis</Emphasis> L. and genetic stability assessment

The aim of this work is to develop a method of plant regeneration from leaf explants of Platanus occidentalis L. successfully. Woody plant medium (HortScience 16:453–459, 1981) and Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium were used as induced and rooted basal medium, respectively. The effects of combinations of 6-BA, IBA, NAA and KT with different concentrations on adventitious bud regeneration from P. occidentalis leaf explants were compared. The results showed that the highest shoot regeneration frequency (90%) and maximum number (13.72 ± 0.44) of shoots per explant was recorded on WPM medium supplemented with 22.20 mmol l⁻¹ 6-BA and 0.49 mmol l⁻¹ IBA. A 40-day-old explants were much more productive for shoot formation than others in this study. The regenerated shoots were cultured on MS medium supplemented with 1.33 mmol l⁻¹ 6-BA, 0.16 mmol l⁻¹ NAA and 2% (w/v) adenine, after 2-week shoots were transferred to 1/2 MS medium supplemented with 0.49 mmol l⁻¹ IBA for rooting. Hardened plantlets via acclimatization were transferred to pots and transplanted to the soil finally. To ascertain whether tissue culture had effects on the genetic stability of plantlets regenerated, the genetic diversity was assessed using RAPD marker. A total of 96 bands ranging from 0.5 to 2.2 kb with an average of 6.4 bands per primer, were obtained using 15 primers. Amplified products exhibited few of polymorphic patterns across all the plants of P. occidentalis and the overall frequency of detection of somaclonal polymorphisms was lower than 0.0104%. Yuehua Sun, Yanling Zhao, and Xiaojuan Wang contributed equally to this work.     

Adventitious shoot regeneration of scotch spearmint (menthaxgracilis Sole)

Summary The effect of different cytokinins on in vitro adventitious shoot regeneration from internodal explants of Menthaxgracilis Sole (scoth spearmint) was investigated. Murashige and Skoog (MS) medium containing 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% (w/v) sucrose, 10% (v/v) coconut water and supplemented with 4.5 μM thidiazuron (TDZ) was effective in inducing adventitious shoot formation from callus. The greatest percentage of explants with shoots (85%) with the highest mean number of shoots per explant (29) was obtained with explants from the 1st and the 2nd internodes from 2-wk-old stock plants growing on a medium containing MS basal salts, 2% sucrose, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, at TDZ 4.5 μM and 10% (v/v) coconut water and solidified with 0.2% (w/v) phytagel. The regenerated shoots rooted on a medium containing MS basal salts, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% sucrose, and 0.054 μM naphthalene acetic acid (NAA). Micropropagated plantlets were transplanted into soil and acclimated to greenhouse conditions. This is the first report describing adventitious shoot regeneration of scotch spearmint.     

An efficient protocol for large-scale plantlet production from male floral meristems of <Emphasis Type="Italic">Musa</Emphasis> spp. cultivars Virupakshi and Sirumalai

In vitro propagation has played a key role for obtaining large numbers of virus free, homogenous plants, and for breeding of plantains and bananas (Musa spp.). Explant sources utilized for banana micropropagation include suckers, shoot tips, and floral buds. The present study employed male floral meristems as explant material for micropropagation of hill banana ecotypes (AAB) ‘Virupakshi’ and ‘Sirumalai.’ Immature male floral buds were collected from healthy plants from hill banana growing areas. Exposure of explants to ethyl alcohol (70%, v/v) for 30 s, then mercuric chloride (0.1%, w/v) for 30 s, followed by three independent rinses of 5 min each in autoclaved, double-distilled water satisfactorily reduced the contamination. Male floral bud explants were cultured on Murashige and Skoog (MS) basal medium supplemented with different combinations of 6-benzylaminopurine (BAP), coconut water, naphthaleneacetic acid, gibberellic acid, and additional supplements. MS medium supplemented with 5 mg l⁻¹ BAP and coconut water (15%) was the most efficient media for shoot initiation and multiple shoot formation (15 shoots from a single part of a floral bud). The best response for shoot elongation was obtained using the combination of basal MS, 5 mg l⁻¹ BAP, 1 mg l⁻¹ naphthaleneacetic acid and 1.5 mg l⁻¹ gibberellic acid. Regenerated shoots were rooted in basal MS medium within 15–20 d. The rooted plantlets were transferred to a soil mixture and maintained at a temperature of 25 ± 2°C for 10 d and then at room temperature (30–32°C) for 2 wk, before transferring to a greenhouse. The regenerated plantlets showed 100% survival.     

<Emphasis Type="Italic">Crambe tataria</Emphasis>: actions for ex situ conservation

In vitro micropropagation by direct organogenesis and somatic embryogenesis via callus was developed for Crambe tataria (Brassicaceae). C. tataria is an endemic species of the Pontic-Pannonic region, but it is also present in Italy, where it is localized in Friuli on a characteristic grassland formation, called “magredi”. C. tataria is regarded as an endangered species. Leaf and root explants were subjected to plant regulator treatments, which invoked different morphogenic responses. Leaf explants produced more callus than root explants and a higher amount of callus was obtained with 1 mg l⁻¹ 2,4-D in combination with 2 mg l⁻¹ Kin. Somatic embryogenesis was obtained in calli maintained in a delayed subculture regime on media containing BAP in combination with NAA. Root explants cultured with BAP combined with NAA developed adventitious rosette shoots. Shoots rooted on half-strength MS media, and the number of roots per plantlet and their length were heavily dependent on sucrose content. The in vitro regenerated plantlets were acclimatized ex vitro and a mean of 50% of the plantlets survived and showed a true-to-type growth habit. This study describes the development of two in vitro micropropagation protocols, via direct organogenesis and via embryogenesis from callus, that are the basis for the application of in vitro tools for the establishment of basal collections with representative genetic diversity and for the long-term storage of plant genetic material.     

Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis

An efficient in vitro propagation is described for Spondias mangifera Willd., a medicinally important tree, using nodal explants obtained from 4-week-old seedlings. The frequency of shoot regeneration from seedling node was affected by various concentrations of BAP and successive transfer of mother explant. MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP) was optimal for shoot multiplication. Upon this medium, highest number of shoots (about 10.6) per explants was obtained after fourth subculture of mother explants. Half-strength MS medium containing IAA (1.0 mg l⁻¹) was most effective for rooting of shoots. Regenerated plantlets were successfully acclimatized and transferred into soil with 80–90% survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants. This is the first report on micropropagation of S. mangifera, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

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 regeneration and morphogenesis studies in common bean

An efficient protocol for high frequency in vitro regeneration of multiple shoots and somatic embryos from the embryonic axis of common bean (Phaseolus vulgaris) was developed. Ten common bean cultivars representing a wide range of diversity among current commercial market classes were used for in vitro regeneration evaluation in our study. These cultivars were tested on 63 different media formulations consisting of combinations of cytokinins, namely benzyladenine (BA) and thidiazuron (TDZ) at concentration levels of 0.0, 1.0, 2.5, 5.0 and 10.0 mg l⁻¹ and auxin, namely naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA) at concentration levels of 0.0, 0.05, 0.1 and 1.0 mg l⁻¹. P. vulgaris cv. Olathe pinto bean performed the best producing over 20 multiple shoots per explant while cv. Condor black bean was the poorest with nine multiple shoots per explant. The optimum media for regeneration of multiple shoots was 4.4 mg l⁻¹ Murashige and Skoog (MS) containing 2.5 mg l⁻¹ BA and 0.1 mg l⁻¹ IAA supplemented with 30 mg l⁻¹ silver nitrate. Adventitious shoots and somatic embryos were regenerated on 4.4 mg l⁻¹ MS medium containing 1 mg l⁻¹ TDZ and 0.05 mg l⁻¹ NAA supplemented with 30 mg l⁻¹ silver nitrate or activated charcoal. Efficient and effective rooting of plantlets was achieved by dipping the cut end base of in vitro regenerated shoots in 1.0 mg l⁻¹ indole-3-butyric acid (IBA) solution and culturing on media containing 4.4 mg l⁻¹ MS supplemented by 0.1 mg l⁻¹ IAA, NAA or IBA.